PT97011 DNA fingerprints and cryopreservation of potato ... · for improved quality assurance Final report for project PT97011 Principal Investigator James F. Hutchinson Agriculture

PT97011 DNA fingerprints and cryopreservation of potato cultivars for improved quality assurance

D Isengger and J Hutchinson Agriculture Victoria

danikah

Stamp

PT97011

This report is published by the Horticulture Australia Ltd to pass on information concerning horticultural research and development undertaken for the potato industry.

The research contained in this report was funded by Horticulture Australia Ltd with the financial support of the potato industry.

All expressions of opinion are not to be regarded as expressing the opinion of Horticulture Australia Ltd or any authority of the Australian Government.

The Corporation and the Australian Government accept no responsibility for any of the opinions or the accuracy of the information contained in this report and readers should rely upon their own enquiries in making decisions concerning their own interests.

Cover price: $22.00 (GST Inclusive) ISBN 0 7341 0187 2

Published and distributed by: Horticultural Australia Ltd Level 1 50 Carrington Street Sydney N S W 2000 Telephone: (02) 8295 2300 Fax: (02) 8295 2399 E-Mail: [email protected]

© Copyright 2001

Horticulture Australia Limited

mailto:[email protected]

DNA fingerprints and cryopreservation of potato

cultivars for improved quality assurance.

Final report for HRDC project PT97011

Prepared by

Daniel Isenegger and James F. Hutchinson

Agriculture Victoria Institute for Horticultural Development, Knoxfield

June 2000

DNA fingerprints and cryopreservation of potato cultivars for improved quality assurance

Final report for project PT97011

Principal Investigator James F. Hutchinson Agriculture Victoria Institute for Horticultural Development Private Bag 15 Scoresby Business Centre VIC. 3176 Ph. (03)9210 9222 Fax. (03) 9800 3521 Email: [email protected]

Purpose of the report This project (PT97011) was directed towards the compilation of: 1. a DNA database for the rapid identification and differentiation of important potato

cultivars, 2. the evaluation of cryopreservation for the long term storage of potato germplasm.

During the life of this project some additional research was conducted including: • examination of the genetic relationships between potato cultivars grown in

Australia, • an evaluation of additional molecular marker systems for cultivar identification, • an investigation of a bacterial contaminant in potato plant tissue cultures and the

development of a rapid diagnostic tool for its detection.

Funded by: The Horticultural Research & Development Corporation, the Australian Potato Industry Council and Agriculture Victoria.

Text by: Daniel Isenegger and James F. Hutchinson

Date of report: June 2000

Any recommendations contained in this publication do not necessarily represent current HRDC policy. No person should act on the basis of the contents of this publication, whether as to matters of fact or opinion or other content, without first obtaining specific, independent professional advice in respect of the matters set out in this publication.

mailto:[email protected]


1.0 Introduction

Methods for improving the quality standards and efficiency of the various potato seed schemes in Australia will benefit the whole industry. With any seed

scheme there are a number of concerns and potential problems. For a variety of reasons, cultivars can become mixed or incorrectly labelled at any part of the production chain from the tissue culture laboratory to the grower. The earlier that cultivars are mixed the greater the consequences of either economic loss or litigation to tissue culture laboratories, mangers of seed schemes, seed growers and end use growers.

This report has two main features: • A database of DNA fingerprints that provides a rapid method for cultivar

identification and differentiation; • An evaluation of cryopreservation of shoot tips in liquid nitrogen for long term

maintenance of potato cultivar collections.

Public good cultivars maintained in the various seed schemes were identified in consultation with each collaborator (Appendix 1). The cultivar lists were then divided into those that should have DNA fingerprints developed (Appendix 2), or for cryopreservation (Appendix 3).

The cultivar lists were prepared in consultation with: Victoria: Keith Blackmore (VicSPA)

Janet Tregenza, Andrew Henderson (Institute for Horticultural Development - Knoxfield) Corina Horstra, Graeme Wilson, Roger Kirkham (DTD - Toolangi)

NSW: Clarrie Beckingham (NSW Agriculture) Tasmania: Frank Mulcahy (ex. Department of Primary Industry and Fisheries) WA: Murray Hegney (ex. Western Potatoes)

Peter Dawson (WA Agriculture)

The authors acknowledge and thank APIC and HRDC for funding this research, Roger Kirkham and Graeme Wilson for providing plant material and helpful discussions and Peter Franz for help with statistical analysis. Special thanks to Lawrie Shaw and Jeff Peterson.

This report is divided into a number of chapters. The main two chapters report the development of a DNA fingerprint database (Chapter 2) and the evaluation for the cryopreservation (Chapter 3). Other chapters report additional research and experiments that were linked to the research findings in Chapters 2 and 3. A glossary of technical terms can be found in Appendix 4.

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1.1 Media Summary DNA fingerprinting and cryopreservation technology has been used and evaluated with potato for improved quality assurance in the industry. Cultivars can become mixed or incorrectly labelled at any stage of the production chain from the tissue culture laboratory to the commercial grower, which can take five to eight years. This can have major consequences for industry seed schemes and germplasm resources.

Traditional methods for cultivar identification are based on observing plant characteristics such as leaf shape, flower colour, tuber shape and colour, which can be unreliable and time consuming as it is influenced by environmental conditions, plant age and often require plants to reach maturity. Methods that can rapidly identify and distinguish cultivars are of considerable value. A DNA fingerprint database has been developed for 64 cultivars whereby, accurate and reliable test results can be generated within days. DNA fingerprints are also not influenced by environmental conditions and are a new and valuable tool now available to the industry.

There has been some exciting and potentially useful spin offs from developing the DNA fingerprints. For example, for the first time the genetic relationships have been studied between Australian grown potato cultivars. This can be particularly useful for conventional breeding programs as parental lines can be more efficiently selected. Another useful outcome has been DNA to identify an endogenous bacterial contaminant in tissue culture plants which can assist with quality assurance.

There are considerable financial costs to maintain cultivars in collections in tissue culture and the field. The evaluation of cryopreservation for long term storage using 37 potato cultivars has shown promise as a valuable adjunct for maintaining the industries germplasm collection. Cryopreservation has the advantage of lower maintenance requirements however, the improvement of recovery rates from future research would be beneficial.

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1.2 Technical Summary Potatoes are a major horticultural crop which would benefit by the appropriate use of molecular and biotechnological approaches.

For a number of reasons potato cultivars can become mixed at any stage of the production chain from the tissue culture laboratory to the commercial grower, which can take five to eight years. This can have major consequences for industry seed schemes and germplasm resources. Accurate and reliable cultivar identification is essential for crop certification and quality assurance. At present the industry mainly relies on the assessment of morphological markers which are often unreliable as they are subject to environmental influence and are also time consuming as plants often require assessment at maturity.

A database of DNA fingerprints, based on Random Amplified Polymorphic DNA (RAPD) analysis, were generated for 64 important cultivars grown in Australia. This database provides profiles that can rapidly identify and differentiate cultivars. Results can be obtained within days and are not influenced by environmental conditions and physiological age and status. Additional research using Simple Sequence Repeat (SSR) analysis has shown the ability to differentiate potato cultivars and is also a powerful approach for cultivar identification and differentiation. However, additional research using the SSR system is required for routine use in the future.

Clonal variants of cultivars Atlantic, Kennebec, Sebago, Delaware and Russet Burbank were found to be indistinguishable, however this does not necessarily mean that they are genetically identical. The ability to detect small genetic differences that may exist is often difficult and associated with chance. Screening additional primers or other molecular markers in conjunction with extensive field and glasshouse assessments may need to be conducted to confirm if genetic differences exist between clonal variants.

Additional outcomes have also been achieved from this research. For the first time, the genetic relationships between cultivars grown in Australia have been examined. This information is valuable for breeding programs and germplasm collections in Australia as it can assist in efficient selection of parent lines and provide a handle for maintaining genetic diversity.

The detection of a RAPD marker from endogenous bacterial contamination Bacillus circulans (commonly known as White Ghost) in tissue culture plants has also lead to the development of a PCR-based diagnostic tool which has potential use for quality assurance testing.

In Australia, majority of potato cultivars originate from tissue culture and are in long term storage which incurs considerable maintenance cost. A valuable and efficient adjunct for long term storage is to use cryopreservation techniques. The cryopreservation method used involves treating shoot tips with the cryoprotectant DMSO and freezing them in liquid nitrogen. Maintenance costs are reduced by the elimination of frequent subculturing.

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Preliminary research that evaluated cryopreservation has found that all 37 potato cultivars tested survived cryopreservation. Thirty cultivars regenerated into tissue culture plantlets. The survival and regeneration rates were found to be affected by cultivar type. Although further research can improve survival and regeneration rates, cryopreservation for long term storage of potato cultivars has shown considerable promise.


2.0 DNA fingerprinting of potato cultivars 2.1 Introduction

Potato {Solarium tuberosum L.) is clonally propagated and is a major vegetable crop grown in Australia. The majority of potatoes grown in Australia originate from

pathogen tested schemes that are maintained in tissue culture. Cultivars are then grown in accredited facilities to produce seed tubers before reaching the end use growers for the fresh and processing markets. This production chain can take 5 to 8 generations with each step requiring certification.

The maintenance of the genetic integrity of cultivars and breeding lines is essential for quality assurance. Mixed or incorrectly identified cultivars can lead to economic loss and litigation.

Historically, cultivar identification has relied on morphological descriptions, such as shape and colour of leaves, flowers and tubers. This approach is subjective and takes particular time if it is necessary to use information collected at different developmental stages of the plant. In addition, these characteristics can be affected by environmental conditions. Biochemical markers such as isozymes have been used for potato cultivar identification (Douches and Ludlam 1991), however, they are limited by the small number of polymorphisms generated.

DNA fingerprinting is a valuable tool for cultivar identification and differentiation, and can be used to assist in the application for registering Plant Breeders' Rights (Morell et al. 1995). Random Amplified Polymorphic DNA (RAPD) analysis (Welsh and McClelland 1990; Williams et al. 1990), have been successfully used in studies with potato, that include the differentiation and identification of cultivars and clonal variants (Demeke et al. 1993; Hosaka et al. 1994; Sosinski and Douches 1996; Ford and Taylor 1997), identification of somatic hybrids (Baird et al. 1992; Rasmussen and Rasmussen 1995) and in the assessment of genetic diversity (Quiros et al. 1993; del Rio et al. 1997'a; del Rio et al. 1997b; Paz and Veilleux 1997).

Once the cultivars for DNA fingerprinting were identified, we decided that the first task should be to compare cultivars maintained in the tissue culture collections (primarily IHD - Knoxfield) and the field/tuber collection (primarily IHD - Toolangi), to be certain that samples used were reliable.

The primary objectives of this chapters were to: • Compare the DNA fingerprints generated by RAPD of commonly grown potato

cultivars from the two collections maintained in tissue culture and field/tubers; • Generate DNA fingerprints using RAPDs for important potato cultivars.

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2.2 Methods and materials 2.2.1 Source of plant material Plant material was obtained from cultivar collections maintained in tissue culture and the field at IHD - Knoxfield and Toolangi. Material not available in Victoria was obtained from collaborators.

2.2.2 DNA isolation DNA was isolated from leaf tissue (Fulton et al. 1995), dissolved in TE buffer and RNAase treated at 37°C for 1 hour. DNA concentration was determined using a fluorometer (DynaQuant 200, Hoefer Pharmacia Biotech, USA) and was assessed on an agarose gel to ensure that high molecular weight DNA was present.

2.2.3 RAPD analysis One hundred primers were initially screened and 26 selected to generate DNA fingerprints (Table 2.1). All reactions were carried out in a final volume of 25 uL containing 10 mM Tris-HCl (pH 8.3), 3mM Mg2+, 50 mM KC1, 100 ^M dNTP, 0.4 uM primer, 25 ng DNA and 0.5 U of Taq polymerase (Boehringer Mannheim, Germany). Thermocycling was conducted in a Hybaid PCR Express thermocycler (Hybaid, UK) with 35 cycles of 94°C for 30 s, 45°C for 1 min and 72°C for 1 min. First denaturation cycle was 94°C for 1 min; last extension cycle was 72°C for 5 mins..

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Table 2.1. RAPD primer sequences used for DNA fingerprinting of potato cultivars. Primer(1) Sequence (5'-3') 2-05 CAA TCG CCG T 2-07 GAA CGG ACT C 2-10 GGT GAT CAG G 2-14 GTC CCG TGG T 2-17 CACAGGCGGA 2-19 GGA CGG CGT T OPD-03 GTC GCC GTC A OPD-08 GTG TGC CCC A OPD-11 AGC GCC ATT G OPI-06 AAG GCG GCA G OPI-07 CAG CGA CAA G OPI-11 ACA TGC CGT G OPI-14 TGA CGG CGG T OPI-18 TGC CCA GCC T OPI-20 AAA GTG CGG G OPJ-04 CCGAACACGG OPJ-06 TCG TTC CGC A OP J-10 AAG CCC GAG G OPJ-17 ACG CCA GTT C OPJ-18 TGG TCG CAG A OPJ-20 AAG CGG CCT C OPB-01 GTT TGG CTC C OPB-06 TGC TCT GCC C OPB-11 GTA GAC CCG T OPB-12 CCT TGA CGC A OPB-18 CCA CAG CAG T

( } Primers were obtained from Geneworks Pty. Ltd. (Sth. Aust.) or Operon Technologies Inc. (Alemeda, Ca. USA).

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2.2.4 Visualisation and analysis of PCR products Twenty uL of PCR product was separated in a 1.5% agarose gel with xl TAE buffer containing 0.5 fj,g mL"1 ethidium bromide and visualised under ultra violet light (305 nm). Gels were photographed and images stored and analysed using Kodak Digital Science™ ID Image Analysis Software.

2.3 Results 2.3.1 Comparison of tissue culture and field cultivar collections DNA fingerprints obtained from cultivars maintained as tissue culture and field collections were compared using RAPD analysis. A total of 65 cultivars and eight clonal variants (Appendix 2) were compared. For 63 cultivars there were no differences in the DNA fingerprints obtained. Discrepancies were found for the cultivars Kufri Jyoti and Redsen. With Kufri Jyoti, 14 of 19 primers screened showed polymorphisms between the tissue culture and field sourced sample. For example, primers 2-17, 2-19, OPD-03, OPD-11, OPI-06, OPI-07 and OPI-18 show polymorphic bands that distinguished between the tissue culture and field samples (Fig. 2.1). Further analysis using additional samples obtained from tissue culture and field, found all 4 tissue culture samples tested were indistinguishable to each other but could be distinguished from all 5 samples derived from the field, showing these two genotypes were different.


2-17 2-19 D3 Dl l 16 17 118

T F T F T F T F T F T F T F M

Figure 2.1. RAPD profiles of potato plants labelled Kufri Jyoti maintained in tissue culture (T) and field (F) with primers 2-17, 2-19, OPD-3, OPD-11, OPI-6, OPI-7 and OPI-18. Polymorphic bands are revealed with all primers shown. Lane M = Kilobase DNA ladder molecular weight marker (Amersham Pharmacia Biotech).

With cultivar Redsen, 12 of the 18 primers screened generated polymorphisms that could distinguish the two samples. Analysis of additional samples have shown that two of the five tissue cultures tested were distinguishable from each other and the six field samples tested. The three other samples were found to be indistinguishable from the six samples derived from the field (Fig. 2.2). It is likely that the nine samples that could not distinguished are true to type and correctly labelled.

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1 2 3 4 5 M 6 7 8 9 10 11

'./ 2.0 kb

— 1.5 kb

— 1.0 kb

Figure 2.2. RAPD profiles of 11 potato plants labelled Redsen maintained in tissue culture (lanes 1-5) and field (lanes 6-11) with primer 2-17. The arrows indicate polymorphic bands that distinguish the two tissue culture samples (lanes 1 and 2) from the other plants tested. Lane M = Kilobase DNA ladder molecular weight marker (Amersham Pharmacia Biotech).

2.3.2 Generation of DNA fingerprints for nominated cultivars The DNA fingerprints for the 64 cultivars and eight clonal variants are presented in Figure 2.3 (located at end of section 2.5 References).

2.4 Discussion As potatoes are clonally propagated, the DNA fingerprint for a given cultivar will generate profiles that are indistinguishable. During the initial stage of this work, PCR-based RAPD analysis was demonstrated to be useful in detecting incorrectly labelled tissue cultures in cultivars Kufri Jyoti and Redsen. Consequently, incorrectly labelled stocks were discarded and replaced with true-to-type cultures, except for Kufri Jyoti.

The genetic integrity of cultivars is of paramount importance for quality assurance in the potato industry seed schemes. Ramifications of incorrectly labelled cultivars can not only lead to wasteful work and economic loss, but also litigation.

Using RAPDs 64 potato cultivars could be identified with 17 primers which generated 150 markers. This compares favourably with other studies where 36 cultivars grown in Canada were identified with two primers (Demeke et al. 1993), and 46 cultivars grown in the USA were identified with 10 primers that produced 43 polymorphisms (Sosinki and Douches 1996).

Using Simple Sequence Repeat (SSR) analysis, 24 of 40 potato cultivars could be distinguished with 6 primer sets, but have failed to distinguish some of the potato cultivars we distinguished for example, Russet Burbank, Spunta and Red LaSoda (Schnieder and Douches 1997). Comparing three DNA fingerprinting systems (SSRs, RAPDs and Amplified Fragment Length Polymorphisms [AFLPs]), 16 potato cultivars could be distinguished, but more polymorphisms were produced from SSRs (Milbourne et al. 1997). Kawchuck et al. (1996) were able to distinguish 75 out of 95

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potato cultivars with three SSR primer sets. This suggests that SSR analysis could be a suitable method for potato cultivar identification, but high developmental costs and technical demand is usually associated with this system.

The number of primers required to identify incorrectly labelled cultivars depends on knowing if the material could be one of the few known cultivars or completely unknown. If the suspect material is one of a few known cultivars, than cultivar specific RAPD markers can be selected to identify the cultivar. If however, no knowledge of the cultivar identity is known, it is necessary to use considerably more primers (ca. 20) and compare the DNA fingerprint to the database. The number of primers required depends on the level of polymorphisms generated. Between the most genetically similar cultivars the level of polymorphism in this database is about 12 % (see Chapter 7).

2.5 References Baird, E., Cooper-Bland, S., Waugh, R , DeMaine, M. and Powell, W. (1992).

Molecular characterisation of inter- and intra-specific somatic hybrids of potato using randomly amplified polymorphic DNA (RAPD) markers. Molecular and General Genetics 233, 469-75.

del Rio, A.H., Bamberg, J.B. and Huaman, Z. (1997a). Assessing changes in the genetic diversity of potato gene banks. 1. Effects of seed increase. Theoretical and Applied Genetics 95,191-198.

del Rio, A.H., Bamberg, J.B. and Huaman, Z. (1997b). Assessing changes in the genetic diversity of potato gene banks. 2. In situ vs ex situ. Theoretical and Applied Genetics 95,199-204.

Demeke, T., Kawchuck, L.M. and Lynch, D.R. (1993). Identification of potato cultivars and clonal variants by random amplified polymorphic DNA analysis. American Potato Journal 70, 561-9.

Douches, D.S. and Ludlam, K. (1991). Electrophoretic characterization of North American potato cultivars. American Potato Journal 68, 767-780.

Fulton, T.M., Chunwongse, J. and Tanksley, S.D. (1995). Microprep protocol for extraction of DNA from tomato and other herbaceous plants. Plant Molecular Biology Reporter 13,207-9.

Hosaka, K., Mori., M., and Ogawa, K. (1994). Genetic relationships of Japanese potato cultivars assessed by RAPD analysis. American Potato Journal 71, 535-46.

Kawchuck, L.M., Lynch, D.R., Thomas, J., Penner, B., Sillito, D. and Kulscar, F. (1996). Characterization of Solanum tuberosum simple sequence repeats and application to potato cultivar identification. American Potato Journal 73, 325-35.

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Milbourne, D., Meyer, R., Bradshaw, J.E., Baird, E., Bonar, N., Provan, J., Powell, W. and Waugh, R. (1997). Comparison of PCR-based marker systems for the analysis of genetic relationships in cultivated potato. Molecular Breeding 3, 127-36.

Morell, M.K., Peakall, R, Appels, R, Preston, L.R. and Lloyd, H.L. (1995). DNA profiling techniques for plant variety identification. Australian Journal of Experimental Agriculture 35, 807-19.

Paz, M.M. and Veilleux, R.E. (1997). Genetic diversity based on randomly amplified polymorphic DNA (RAPD) and its relationship with the performance of diploid potato hybrids. Journal of the American Society for Horticultural Science 122, 740-47.

Quiros, C.F., Ceada, A., Georgescu, A, and Hu, J. (1993). Use of RAPD markers in potato genetics: segregations in diploid and tetraploid families. American Potato Journal 70, 35-42.

Rasmussen, J.O. and Rasmussen, O.S. (1995). Characterization of somatic hybrids of potato by use of RAPD markers and isozyme analysis. Physiologia Plantarum 93, 357-64.

Schneider, K. and Douches, D.S. (1997). Assessment of PCR-based simple sequence repeats to fingerprint North American potato cultivars. American Potato Journal 74,149-60.

Sosinski, B. and Douches, D.S. (1996). Using polymerase chain reaction-based DNA amplification to fingerprint North American potato cultivars. Hortscience 31, 130-3.

Welsh, J. and McClelland, M. (1990). Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Research 18, 7213-18.

Williams, J.G.K., Kubelik, A.R, Livak, K.J., Rafalski, J.A. and Tingey, V. (1990). DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Research 18, 6531-35.

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Figure 2.3. DNA fingerprints for the 64 cultivars and eight clonal variants.


Cultivar: 80-30-12

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Cultivar: 92-19-10

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Cultivar: Atlantic Line A

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Cultivar: Atlantic Line B

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18


Cultivar: Bismark

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Cultivar: Bremer

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22


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23


Cultivar: Coliban line B

a gel.

1.5 kb

800 bp

500 bp

CO 0 0 r—1

o o ^ 1 1 1

o o *—i ^t

IT) r-- O ^t r 0 \ Q Q Q i—i i—t i

h—1 1

t—I < — > o T-H T—H * — 1 r H P H f H P-i CI- Q- c± a_

£ <N <N 1 1 1

( N o o o o o o o ^

PgeL

1.5 kb

800 bp

500 bp

oo o ^H (SI o

t - 00 O O >0 '—i O —<

1 1

(N i—i

i

oo 1

I 1 1—1 t-H t—J > -> • - > » — s *—.> 1—9 PQ PQ PQ PQ PQ P H P H P L i Q H P H p H P H O H P M a H P H P - l P U ^

2 0 0 0 0 0 0 0 0 0 0 0 0 0 ^


24


Cultivar: Crystal

a gel.

1.5 kb

800 bp

500 bp

m co »—i O O -H

• i i o o ^ H

T - H

»T) l > O Tl- t ^ CT\ Q Q Q 1 1—1

1 H"H 1—1 M

o o T - H I — 1 >—1 < — I PH PL, PH PH PH P H PH

& CN <N <N Cs| ?N <N O O O O O O O k rp«*

PgeL

1.5 kb

800 bp

500 bp

CO o *t v© O r- CO O o o t—t Oi CO

* - H <N o o T — 1 —* 1—H ( N * i | i • 1

M 1

)—H »—1 i 1 i

i—» 1

t—» i PQ PQ PQ PQ PQ

PH PH PH PH PH PH PH PH PH P H PH PH PH

^ o o o o o o o o o o o o o ^

•Hpttf- ^ ^ -'¥* *f»;fK.-3!38 ? • * , -


25


Cultivar: Dalmore

a gel.

1.5 kb

800 bp

500 bp

r f O OO - H O O - H

I 1 I o r--o r—1 T-H

i n f - o ^t ! > • O Q Q Q 1 1—1 H H M

1 r—1

s o o 1—1 r_l T-H < — ' Pi P i P i P i P i P i P i s CN <N CN <N <N rs o o o o o o o k

Pgel.

1.5 kb

800 bp

500 bp

oo o -* o o

o l^- 00 © O -06

T-H 1

<N

-18

1 1—1

i r—( t—i >—i

1 1—J

1 1—»

i

*—> i PQ PQ PQ PQ PQ

P I P I P H P H P H P I P I P I P I P I P I P I P I , .

S o o o o o o o o o o o o o S .l4Hfr0&r 8P* <sr v!f> S W T P * ;

,>*T«y.

»* < r#' * f f


26


Cultivar: Delaware Line A

a gel.

1.5 kb

800 bp

500 bp

-03 CO r-4

O —' 1 1 O o

»—H r—I

"<fr IT) l">» O <=t r>- ON Q Q Q t -H I—1 1—1

1 1—1

s o 1 CN

r—t

<N

i—H 1

i—i i O O O O O O O ^

PgeL

1.5 kb

800 bp

500 bp

CO O o O

o r—1

CO O -01

O ^H 1 1

i—i i -1

8

I I 1—1 h—I

t - 5 Hr> >-> 1

• — > • i

•—» i—> PQ PQ PQ PQ PQ

S o o o o o o o o o o o o o ^

teMr* i.JU&JttB


27


Cultivar: Denali

a gel.

1.5 kb

800 bp

500 bp

m OO rH O O r-H

1 I 1 o o r - H ^

i n C- o ^ t ^ ON Q Q Q i—i i 1

1—1 t - H i_> <_> 1—H i — i i—< i—1 P i P I OH n, n, n, CL s i i

<N i i

<N O O O O O O O ^

Pgel.

1.5 kb

800 bp

500 bp

oo o o o o oo o -0

1 -0

6 T—( T-H

1 »—i

i

oo i—i

1 1—1

1 *—> 1—> t — s • — > H-> »—4 PQ PQ PQ PQ PQ

P H P L I P - I P H P L I P L I P - I P L I P H P L H P L I P - I P L I


• * # *

If ' 1


28


Cultivar: Desiree

a gel.

1.5 kb

800 bp

500 bp

ro oo T—H

© o 1

1 o r--o

i—H 1—H

«o r- O "* r - ON 6 Q Q •—1 • i—i

1 1—1

s i < — > •

CN 2-1 •

C O 2-1 P H P H P H P H P H P H P H , _

O O O O O O O ^

Pgel.

1.5 kb \ £

800 bp

500 bp

oo o ' * O

o OO o O -06 ^H <N OO

i—1 i—H I—1 1 1 1

s PL,

o OPI

-O

PJ-

PH

o 1—> PH

O

>—s PH O

A A pQ PQ PQ PQ PQ P H P H P H P H P H P H P H ^

o o o o o o o S p SS5

*5> ""i -^s .

A A pQ PQ PQ PQ PQ P H P H P H P H P H P H P H ^

o o o o o o o S

{ •

• %

_1V

J 1

j - 4 1 - •

- ' & fed

M =marker (100 base-pair ladder, ex. Amersham Pharmacia Biotech)

29


Cultivar: Donnelly (88-31-5)

a gel.

1.5 kb

800 bp

500 bp

m oo -—i O O r->

1 1 1 o O r—1 ^ t

U~) r-» O ^r r- ON Q Q Q H-1 I—1 1

(—1 i

O o < — ' < — ' > — • r—1 PH PH PH OH PH P i P i

L ^ CN <N <N cq <N <N O O O O O O O _^J

Pgel.

1.5 kb

800 bp

500 bp

CO o ^J- vo O r- 0 0 O r—> ^ H <N CO

»-H CN o o i—i *—i l - H <N i i | 1 1

1—1 1 1

i—» • I—» 1—» <—> m PQ PQ ffl CQ

PH PL, PH PH PH PH PH PH PH PH PH P H P H

^ o o o o o o o o o o o o o £


30


Cultivar: Dynamite (91-153-1)

a gel.

1.5 kb

800 bp

500 bp

m oo -—i O O - H

1 1 1 o O - H ^r

>o O O ^ r G\ Q Q Q i H H

1 1—1

c_> < — > * -~ i * - H r—I »-H P-t P < PH Q_ Ou a_ Cu s <N CJ i 1

<N 1 O O O O O O O ^

Pgel.

1.5 kb

800 bp

500 bp

oo o o o

o l > 00 o o 1 -0

6 »—1

T—1 1 -1

2 -1

8

1 I—1

1 H-> i—> t — » 1—J 1—» i—» -nq PQ PQ PQ PQ



31


Cultivar: Evans

a gel.

1.5 kb

800 bp

500 bp

*o i> o TJ- r~ o\ © © T - ' i—1 r-H _H

^ 1 1 1 1 1 1 << <N CM (N CN C-4 <N

m oo T—i © © T—i * 0 t"- i—i ^ -

1 1 1 © © r—1 i—1

P H P H P - I P H C H P H P H ^

O O O O O O O ^

Pgel.

1.5 kb

800 bp

500 bp

oo © © ©

© OO © T — 1

© -06

r—1 (N OO

i—1

1—H HH I—j H-> t — J 1

*—> i PQ PQ PQ PQ PQ

OH PL, PH PH fin OH PH PH cu a* CL CL. PH s o o o o o o o o d o d d o S

M - marker (100 base-pair ladder, ex. Amersham Pharmacia Biotech)

32

PT97011 fingerprints and cryopreservation of potato cultivars for improved quality assurance

Cultivar: Exton

a gel.

1.5 kb

800 bp

500 bp

o o —i ^d t-» i—i TJ-

< N ( N ( N C S ( N < N O O O O O O O ^

Pgel.

oo o <N

o I

O © O 00 o

i

ianiai iai lPH'alSSp-i

i—' Ô i—< CN OO O O «-H r-H ^

PH OH OH PH PH S o o o o o o o o o o o o o S

1.5 kb \ J

800 bp

500 bp

M : marker (100 base-pair ladder, ex. Amersham Pharmacia Biotech)

33


Cultivar: Foxton

a gel.

rn oo ^ O O -H 1 1 1

o o I — I ^

in f- o -sr O o\ Q Q Q •

i — i l-H i

1 — 1

o o > — 1 <—| > — i *—< OH PH PH PH PH PH PH

>5 CN CN CN CN (N CN o o o o o o o ^ 1 t

1.5 kb

800 bp

500 bp

PgeL

CO o "tf *o o r-- CO O T — 1 ,-H CN CO

I—H CN o o r—H T-H 1 — < CN • 1 i | i 1 1

h—1 *—> > — > 1—> > — > t — J i — > PQ m m PQ PQ PH PH PH PH PH PH PH PH P-i PH PH P^ PH ^ o o o o o o o o o o o o o &

500 bp


34


Cultivar: Kennebec Line 2

a gel. I t

1.5 kb

800 bp

500 bp

fn OO rH O O <-• 1 1 1 o O

T-H

T—<

IT) r-- O "3" t-» ON Q Q Q l-H i 1 H—1 1—H

o o '—i T—H <—• i—i PH P-t PH PH Pn PH PH

L CM CM CM CM CM CM O O O O O O O

Pgel.

1.5 kb

800 bp

500 bp

00 o "<fr VO o o OO o T-H © ©

T-H cs 00 1—1 CM o o T-H T-H T-H CM 1 i i

t-H H H > — » t-i t — s 1

1—> 1—4 PQ PQ PQ PQ PQ S o o o o o o o o o o o . o o S

- •?" » ' "-"?f *


35


Cultivar: Kennebec Line 7

a gel.

1.5 kb

800 bp

500 bp

m oo <—i o o —i

i i i o o 1—1

i n f - O <<t r- ( ^ Q Q Q 1 H H

1 >—1 i—i

o O *—i I—* i—i r—i OH P H PH PH PH P H PH ^ «N <N <N <N <N <N O O O O O O O ^

T.,.«.•». I*-™. « £

fwm

PgeL

CO o -<* o o r-- CO O i—i r—t <N CO

r—i <N o o *—H * - H * - H (N i 1 1

1—I 1—1 1—» t - » H-» t - » 1

1—9 i—> PQ PQ PQ PH PQ PH P H PH P H PH PH PH PH PH PH PH eu p^ ,

' ^ o o o o o o o o o o o o o ^

? * , 7 4 r * ; 4 i < K *- »sr *.* ** wsj

1.5 kb

800 bp

500 bp


36


Cultivar: Kennebec Line B

a gel.

1.5 kb

800 bp

500 bp

m oo ^H o o -H i i i o o

- H "Sf

i n f - o ' r r - ON Q Q Q 1 1—1

1 h—1 H—1 h—1

o O '-"* > — i > — i < — I P-i PH PM PH a* PH OH

^ <N CM Cvl (N CN <N) O O O O O O O ^

Pgel.

oo o ^ VO o t ^ OO o <~> o <N oo

I—H (N C_3 O * — < * - H T»-H <N • i 1

1—1 1 - 1 1—» • — » 1 1—»

1 i

*—> PQ PQ PQ PQ PQ P H P - I P - I P - I P H P - I P - I P - I P H P - I P - I P H P H ^

S o o o o o o o o o o o o o S ¥". * • * +V"

1.5 kb

800 bp

500 bp


37


Cultivar: King Edward

a gel.

1.5 kb

800 bp

500 bp

m oo r-< © © -H

• i i o © T — 1

^

»n 1^- O •^r r^ ON Q Q Q 1 1—1

i t—i H—1

1 h—1

<_j> <~> T-—1 T — 1 *—< i—t ft - Q,J Q ,J a., O d Q , a_ s i i <N <N

i I

o o o o b o o ^

Pgel.

1.5 kb

800 bp

500 bp

CO 1 — <

o o o

O i—i

CO T — 1

o -01

-06

i 1

CO < — 1

1 1—1

1 >—> t — s •—» *—> 1-5 i—» m ffi ffl PQ m S o o o o o o o o o o o o o S

4f* v-.'


38


Cultivar: Kipfler

a gel.

1.5 kb

800 bp

500 bp

m oo H O O -H

i i i o o ^ H ^

IT) t-« O Tf O o Q Q Q t—t 1

• — 1 I — 1 i

o O i—i T — 1 i — 1 i — i PH PH P-I PM CU PH PH ^ <N <N <N <N ra <N o o o o o o o ^

*** • - •#«};*

pgeL

1.5 kb

800 bp

500 bp

CO o "xf VO o l> 00 O r-> © r—1 <N OO

•-H <N o o * — t *-H 1-H <N i i 1 | • 1 — 1

1 >—> 1

•—> 1—> »-> 1

t—> i

i—> PQ PQ PQ PQ PQ PH PH PH PH PH PH PH PH PH PH PH PH PH

>J o o o o o o o o o o o o o ^

i i i » . « « S i #J * - . * i*ji sK« JURE]BwKJry*


39


Cultivar: Knox

a gel.

1.5 kb

800 bp

500 bp

r o oo i—i O O i—i Ô f-- i—i -sh

i i i O O i—i i—*

o o ^ - H i - ^ - H f i H p L ( p L ( p L ( p L | p H p H

< ( S ( S N M ( S ( S O O O O O O O ^

P : ^ r hi

1 \"3." *%*/sw

Pgel.

1.5 kb

800 bp

500 bp

oo o CN O o

O i - H

oo O <N o -0

6 <N i

oo

1 — < • — i >—3 •—» •—» •—> >-> i—» PQ PQ PQ PQ PQ PH fin P H PH OH Cu OH CU cu o_ cu cu cu s o o o o o d d d d d d d d S

: •% ';MA £ •!&&


40


Cultivar: MacRusset

a gel.

500 bp

co oo ^H O O r-4

1 1 1 o o t - H • *

i n r- o - * t ^ o\ Q Q Q H H i

1—1 h-H

o o I—1 > — ' ' — i r—1 P-I 0-i PH PH PH PH

^ C-l CN <N «N CM <N O O O O O O O ^

?**' w

f -

i S

Pgel.

1.5 kb

800 bp

500 bp

oo o ^ t vo © t~- oo o r-> T - H C4 oo

* — 1 <N o o 1 — < T—1 » — t <N i | 1 1

1 t—1

i HH t - J

1 *—>

1 1—J

1 i—>

1 •—1

i >—> PQ « CQ m OH

P H P H P H P H P H P H P H P H P H P H P H P H P H , ^



41


Cultivar: Mondial

a gel.

1.5 kb

800 bp

500 bp

CO 0 0 <—I O O - H

1 1 1 o o ^ <<t

i n t-- o -3- t-- o\ Q Q Q 1 •—i

1 I—1 l - H

o o *—i »—i i—i T—1 0H PL, p_ OH PH PH PH

2 cs rq cs <N CS rs| o o o o o o o ;>

(3 gel.

CO o ^ vo o r^ 00 o c—> o ^ H oo

. - H rs o o l - H l - H l - H <N 1 1 1 i 1

1 l-H l - H

1 1—4

1 I—) 1—»

1 1—J 1—» I—> ffl CQ « PQ ffl

Q_i Q_i Q_i P_i Cu CLi Q-I Qu O H P H P H O H O H


1.5 kb

800 bp

500 bp

4Bpk&¥v'dr ii...w» ^ f?*^ !

g • y *F— >'

. * j j . - "


42


Cultivar: Mortifry (88-55-6)

a gel.

1.5 kb

800 bp

500 bp

ro oo i—i

© o 1

V o o i—1 ^ >o l-x O TJ- t^ ON Q Q Q r—1 t - H h—1

1 1—1

s 1 o 1

i r—i

i 2-1

O O O O O O O ^

Pgel.

1.5 kb

800 bp

500 bp

CO O o o

o T-H

OO O 1—H

O i

O —i 1 1

<N 00

i i i

HH 1—1 •—» t—j 1 — > »—> 1—> >—> PQ PQ PQ PQ PQ



43


Cultivar: My Fry (89-27-6)

a gel.

1.5 kb

800 bp

500 bp

i n t-» o TJ- t^ o\ C ? ^ 5 "—I '—* »—I i—I

^ I I I I I I

41 fN M (N fN (N N

O O -H i£ £; —' ^

o o o o o o o ^

Pgel.

1.5 kb

800 bp

500 bp

oo o ^ VO o [-» OO o r-^ o ^ H <N oo

1—H (N <_> o r—I y—t * — < <N 1 | i 1 i

1—4 1—1 t - » 1—5 1 » — > i

> — > PQ PQ PQ PQ PQ

P n P L i C L i C L i P H P i P L i P L i q L i P L i P H q i O H



44


Cultivar: Nadine

a gel.

1.5 kb

800 bp

500 bp

ro oo -H O O r-H

1 1 1 o o T—H

l O t ^ o "* r Os Q Q Q H H i

t—1 H H

o o I—1 » — ' t—H <—< PH PH PH PH PH PH PH ^ <N CN <N (N CM C4 O O O O O O O £

J**1^*- ^'h Jw* » S 3 i k ^ l ^ ^ E JB3C^< T C , 5S * •*** *«?•. TBI " T W - t ' i S 1 s - ' '••- *>3ia. » * -tit ,JW

^ • ^ ^ • : ' *• ' ' ' '"• ' * " " v s

Pgel.

1.5 kb

800 bp

500 bp

CO © TT ^D o r-> CO o T—1 T-H CN oo 1—1 CN o O t - H »-H — • < CN 1 1 1

H-H H H H-s i 1—3 1—>

1 1—5

1

> — > • PQ m PQ PQ PQ


S o o o o o o o o o o o o o S J a g •wy ^ B .

• • 'trow


45


Cultivar: Nicola

a gel.

-03

o o r-H TT i—l r—l

i n t> o ^r t ^ ON Q Q Q 1 HH

1 1 I—1 t—1

o O l-H i—i i—i I—H PH P H OH PH PH P H PH , S «N «N t N <N (N CN o o o o o o o £

' i < 1 - -, 1 1 . 3* IT

1 %W$' , >. • * I j

1.5 kb \ s j

800 bp

500 bp

Pgel.

OO o ^ *o O r^ oo O i—i <—> r->

^ H <N oo 1—4 <N o o l - H l - H i - H (N • i | i

l-H 1 l-H

1—5 »-» >—9 1 i

•—> i — > ffl P H PQ PQ PH , PH PH PH PH PH PH PH PH PH PH PH P-i PH

& o o o o o o o o o o o o o ^

t~. --.*,«B5

1.5 kb

800 bp

500 bp * asftjt-j-^ . ' , . !« ••»» •jat-.jjfe


46


Cultivar: Nooksack

a gel.

1.5 kb

800 bp

500 bp

O o ,_, \D f-- —i "sT • i i O O •—i <—•i

< < N ( N < N < N ( N < N O O O O O O O

Pgel.

1.5 kb

800 bp

500 bp

00 O —1 (N) o

M3 O CO O o 1

O r - i 1 1 i

00

1 1 t - » • - > i — , 1—J *—3 1—> PQ PQ PQ PQ PQ


. - . ' » . F ^ ^ <

" . . « . .


47


Cultivar: Norchip Line A

a gel.

1.5 kb

800 bp

500 bp

rn oo i—i

© i o

1

T—1 1 © o ^ ^T

u-> r O <* t - o\ a n Q 1 M

1 H H

i

< — > o > — i T—H » - H i — i PH Cu Cu CU Q_ CL CLr '& cs (N CN CS • <N o o o o o o o ^

*-LW

k*S4

PgeL

o o © ^ - v o © r - o o © z ; i £ z ! £ J 2 2 —* <N © © —< —' —' <N <? i V V V


1.5 kb

800 bp

500 bp

\ d


48


Cultivar: Onka

a gel.

-03 CO ^

o *-* O O ^ *=t

»/-> r» O "* r o\ Q Q Q i—i H-l i

H-1

o o > — i • — i > — i i—i P-t PH CL, OH PH PH PH

s CN CN <N <N rs CN o o o o o o o > R^V

1.5 kb

800 bp

500 bp

Pgel.

CO o J- ^D © r- CO O I — i o CN CO

1—1 fN o o T—H i — < 1—1 fN 1 1 1

1—1 1

1 — 1 1 1

1—J 1

1—J • — > •—s >—s PQ m PQ PQ PQ PH PH PH PH PH PH PH PH PH PH PH PH PU > i o o o o o o o o o o o o o ^

1.5 kb

800 bp

500 bp


49


Cultivar: Patrones

a gel.

1.5 kb

800 bp

500 bp

m oo r i © O ^H

© O r-H ,-H r-H i i i i i

Q ^ O H PLH

r-o •

i—i ,—, ,—, P i P-< P-i I I I I | , 5 » ! 5 5 5 H > " * H I - M I - H H M t a _

< N < N f N ( N < N ( N O O O O O O O ^

Pgel.

1.5 kb

800 bp

500 bp

0 0 o o o O I s oo o --H ' O - H rN OO O O —I - H r-H

S O O O O O O O O O O O O O S


50


Cultivar: Pinkeye

a gel.

1.5 kb

800 bp

500 bp

m oo © o • O o i — i

^ IT) t - O ^t r <3\ Q Q Q i

h—1 1 i

•—1

o O i—1 i—i i—1 i—i Pn a, Q^ CL Q j Q^ Q^ s <N i

<N i • i

O O O O O O O ^ — - »-1* •"- •*. ••" t r i f t fS

Pgel.

1.5 kb

800 bp

500 bp

oo o "Sf vo o r- OO o 1—1

o ra oo

1—I <N < w > o 1—1 1 " H 1—1 CN • 1 1 1 1 1

1—H l - H 1—» 1

H-» t-T> t — > • — • > PQ PQ PQ PQ PQ



51


Cultivar: Pontiac Line B

a gel.

1.5 kb

800 bp

500 bp

m oo o

i o i o O 1—1 ^r «n r- O ^t l > Q\ Q Q Q 1

1—1 H H 1

> — 1 i

>—1

^ 9 o <—' *—' > — < y—< PH PH PL, P H PH P H PH _ 2 (N «N <N <N <s\ (N O O O O O O O ^

* « v h • * • - * „"~ 3.SI •9 *

1. *

Pgel.

00 o *t VO O t ^ OO O * r-> r—1 <N OO

1—1 CM o o i—t i — i 1—t <N i i 1 | 1 1

M 1

h—1 1 • • • 1 1

• — > PQ PQ PQ PQ PQ PH f^4 PH PH PH PH PH PH PH P H PH P H PH

^ o o o o o o o o o o o o o ^

tSt. '&t~\

1.5 kb

800 bp

500 bp


52

PT97011 DNA fingerprints and cryopreservation of potato cuitivars for improved quality assurance

Cultivar: Purple Congo

a gel.

1.5 kb

800 bp

500 bp

i n

o r- O "3- r-

n oo - H O O ^H VO £ - r-H

i i i O O <—i

ON Q Q Q ^ ^ ^ *-< PH PH PL, PH PH PL( S ( N ( N < N C N < N ( N O O O O O O

Pgel.

00 O o o

o 1—H

oo o o -06

t — i -12

-18

HH 1—1 1-5 t - » •—» i—> t - » • PQ PQ PQ OQ PQ P H P H P - I P H P H P - I P H P H P H P H P H P H P H ^

S 0 0 0 0 0 0 0 0 0 . 0 0 0 0 S PHnfean* sfs p^ «*

1.5 kb

800 bp

3r

Jfc El"'

500 bp

^JW

g " ^ . ^ ^ !


53


Cultivar: Ranger Russet

a gel.

1.5 kb

800 bp

500 bp

m oo »—i _ _ o o ^ MD £- <-H TJ-

1 1 1 O O 1-H T—1

m r-- o TJ- r-- o\ Q Q Q HH ^ ^ HH O O y—< *—« r—t i—t

S r l i rsj rsj <N <N r l P H P H P H P - I P - I O - I P - I . O O y—< *—« r—t i—t

S r l i rsj rsj <N <N r l O O O O O O O ^

Pgel.

1.5 kb

800 bp

500 bp

CO o "3" v© o r CO o (—i o ^ H <N oo

T — t (N o O * — < * - H *—1 <N I 1 1 i 1 1

h—1 •—J I—» 1—» 1 1

t—J i PQ PQ PQ PQ PQ



54


Cultivar: Red Craigs Royal

a gel.

1.5 kb

800 bp

500 bp

en oo —i O © i—i ^ C-- <—i ' T

i i i O © >—' "—' i n t-« © " * C^ CT\ Q Q Q ^ ^ ^ ^

S CN © <—H I—1 T—1 J—1 P H P H P L H P H P - I P H P - I ^

S CN M M (N (N D O O O O O O O ^

(3 gel.

1.5 kb

800 bp

500 bp

oo © © ©

© t - CO © -01

-06

i -12 oo

i 1

1 — * h - 1 1—> •—s 1

1—> •—j 1

• — > PQ PQ PQ PQ PQ



55


Cultivar: Red La Soda

a gel.

1.5 kb

800 bp

500 bp

-03

-08 -

o O ^ ^r

w> f - O ^ r as « « Q 1 l - H

i HH

• HH

s o O » — ' i—i 1—H < — i PH PH PH PH PL, PH PH s <N <N <N CN <N <N o o o o o o o £

Pgel.

1.5 kb

800 bp

500 bp

oo o Tf vo O t-« OO O i—i c — i

^ (N 00 * — 1 CN o O * - H f — t »—1 «N i | i 1

H H 1

*—i >—» 1

*—> 1

t — J • — » pq pq PQ PQ pq OH PU PH PH PH PH PH PH PH PH PH PH PH ,

'& o o o o o o o o o o o o o &


56


Cultivar: Redsen

a gel.

1.5 kb

800 bp

500 bp

rO 00 r-H O O rH ^

O O - H — i ^ -H OH OH (X OH

1^ O

i

Pgel.

1.5 kb

800 bp

500 bp

oo o © o

o l > 00 o © \0 —' O —'

1 1 i

00

1 I—1 >—> 1—» 1 -5

I 1 *—> m PQ PQ ffl m S o o o o o o o o o o o o o S


57


Cultivar: Rideau

a gel.

1.5 kb

800 bp

500 bp

-03 oo ^H

o t-n i i o o

- H "* ir> t ^ O "3" l > OS Q Q Q 1

HH l - H l - H H H

O O r—1 » — i *—' y—i PH PH CL, PH P H OH PH % CN CN (N <N <N <N o o o o o o o >

PgeL

1.5 kb

800 bp

500 bp

00 o ^ VO o t ^ OO o (—1 i—H <N oo

T - H <N o o T—I r—1 »-H CS 1 I • l - H h—1 1—>

1 1—» 1—i H-5

1 1—J t—J PH PH PQ PQ PQ


S o o o o o o o o o o o o o S "MjOCta- <** wp *•. ft* ,'44; *^3?<


58


Cultivar: Riverina Russet

a gel.

1.5 kb

800 bp

500 bp

rn oo rn O O -H 1 1 1

VO

o o ^ "S|-

in r- o "*fr r G\ Q Q Q H H 1—1 i

1—H i

h—1

s o i—' 1—H 1—1 r—1 i — i PH PH PH Ou Ou CL CL s cq CN <N <N CN <N O O O O O O O ^

Pgel.

1.5 kb

800 bp

500 bp

OO O " J" Ô O I > OO *—I CN O O ^H ^H _

I I I I I I I I—H H H »—j I—j (—5 >—, • - «

^ 9^ Gb Pi P-t P-<

S O O O O " PH PH

T-H VO i—i o } 00 O O - ^ - H ^

I I I I I

. PQ PQ PQ PQ PH PH PH PH PH

O i

o o o o o o o PQ

o


59


Cultivar: Ruby Lou (90-40-1)

a gel.

1.5 kb

800 bp

500 bp

-03 CO r-H

O ^ H o o T — <

T-H l - H

ir> r-» O "<fr t > o\ Q Q Q h-H i—i 1

h—1 1

h—1 o © 1—H i—i >-H "—i cu PL. PL, PH a* PH PH

^ <N «N CN <N <N ?N o o o o o o o > V* .«•* *»* HR

-ijap

pgel.

1.5 kb

800 bp

500 bp

CO o •sr *o o r CO o r~> r - i ^ (N CO

1—H CN <_> o l - H l - H l - H CN i | I i 1

l-H H H t — J 1—» >-» 1—> t — s i — > PQ PH PQ PQ PQ P H P H P H P H P H P H P H P H P H P H P H P H P H ^


•J? -.''--* S ^ * j *

' i. '


60


Cultivar: Russet Burbank British Columbia

a gel.

1.5 kb

800 bp

500 bp

CO OO -—i O O —'

1 1 1 o o T — 4 "=t

i n f - O •sf l > 0\ Q Q Q »—i 1

l - H 1

1—t H—t

s o <_> '—' 1—1 I—H '—' OH PH PH PL, PH PH PH s <N CN <N CN CN CN o o o o o o o k

PgeL

1.5 kb

800 bp

500 bp

oo o CN © o

o t^ oo o ^H CN

© 1 -0

6 ^ CN 1

oo 1—1

I—1 l - H > — > i—> 1—» i—» i i PQ PQ PQ PQ PQ



61


Cultivar: Russet Burbank ex. Tas. '95

a gel.

1.5 kb

CO CO "—i © © - H

1 1 1 o o ^ t-

ir> f - o ^r r ON Q Q Q i i i—i h-H

o O * - H r - H *—H T — 1 P-i P H OH Cu Cu Cb CL % CN CN CN CN

1

CN <N O O O O O O O ^

(3 gel.

1.5 kb

800 bp

500 bp

oo o CN o o

o f - 00 O H CN

o O —< I 1 -1

2 00 I—H

1 1

1—I 1—1 I—> •-» H - j H i »-» I—s PQ PQ PQ PQ PQ

S o o o o o o o o o o o o o S ssrarpvsw WJJ v ,r


62


Cultivar: Russet Burbank Line A Ruen

a gel.

1.5 kb

800 bp

500 bp

m oo o o

1 1

r-H 1 o o

r - ( ^T

»n r- O ^ r- o\ Q Q Q 1 r—1

i h—1 (—1

s C_5 1 2-

1 T—4 1 i 2-

1 PM.PH OH OH PH OH P I

O O O O O O O ^

Pgel.

1.5 kb

800 bp

500 bp

oo I—H o

o r-« oo O i-H

o 1

O r - l 1 1

r - H OO I—1

l - H I—I 1—j t—» t—» > — > 1 1 OH PQ. PQ PQ PQ



63


Cultivar: Russet Burbank Netted Gem

a gel.

1.5 kb

800 bp

500 bp

C*"> CO r—1

O O r-> o O T—H "*

ir> t > O ^J- r>- o\ Q Q Q (-H >—i 1 t-H

1—1 o O r~l "—' i — i i—i P-i PH P-( OH Oi PU, OH

^ <N CN <N (N <N cq o o o o o o o ^

PgeL

1.5 kb

800 bp

500 bp

oo o ^ t Ô o f - OO o <—•> o ^ H <N CO

*—1 <N t_> c_> 1—H * - H * — 1 (N 1 | 1 h—1

HH t — j t—» K-L, 1—> 1—> i—» m PQ PQ PQ PQ



64


Cultivar: Russet Burbank Tooheys

a gel.

1.5 kb

800 bp

500 bp

? S5 *"? © © ^ ^

O O O O O O O ^ © r-H r-H

(N ( S CS ^ M (N

pgel.

oo I—1

o •<fr

o o o

I—1 oo o O -0

6

i

<N oo 1

l-H HH H-5 t - J »—5 • " -» 1 •—) m PQ m CQ ffl

S o o o o o o o o o o o o o S £ * * 4f» - * sKK"

1.5 kb

800 bp :. f

500 bp E H K


65


Cultivar: Sebago Line C

a gel.

1.5 kb

800 bp

500 bp

m oo y—i _ _ _ O O *—i Ô t-» y—< st

1 1 1 © © r—1 1—H

in i> o ^r r-- ON Q Q Q H H ^ H H © O - H r-i r-> r-, P H P H P H P H P H P H P H , _

S (S (N (N N (N fS O O O O O O O ^

trf&"'<fc:

Pgel.

1.5 kb

800 bp

500 bp

OO © ^r VO © r-- 0 0 © r-> o <N OO

1—1 <N o o *—t !—1 T-H (N i 1 1 1 1

HH 1—1 i

> — > t - » r-> 1

1—S i PQ PQ PH PQ PH

PH PH PL, PH PH PH PH PH PH PH PH PH PH '& o o o o o o o o o o o o o &

NB: Primer OPI-20 has been confirmed to produce identical profiles with Sebago New Brunswick


66


Cultivar: Sebago New Brunswick

a gel.

1.5 kb

800 bp

500 bp

m oo --< . ^ __._ Q Q _ , Ô t~- "—' "3"

i i i © O >—< >—< >n h o ^ M ^ Q Q P ^ ^ M ^ © © —> r-H r-H , - H OH QH 0-I OH OH OH OH

S (N (N (N (N N (S o o o o o o o H **y ""*a

Pgel.

1.5 kb

800 bp

500 bp

co © "* VO © t ^ oo © r~i o <N 0 0

* — » <N o o i — ' i—H i—H <N 1 1 1 i

h—1 i

h—1 t - » 1 i

I—> 1—J 1

1—J i

m PQ PQ PQ PQ


NB: Primer OPI-20 has been confirmed to produce identical profiles with Sebago Line C


67


Cultivar: Sequoia Line C

a gel.

1.5 kb

800 bp

500 bp

ro oo O O • o

r--o

^ "<fr

i n r- O <* l > ON Q Q Q 1 1—1

i i—i

i HH • — H

o o >—i r—* '—• T—> PH PL, PL, PH P H P H P H

£ <N «N CN CM <N <N O O O O O O O ^

Pgel.

800 bp

500 bp

oo o ^ VO O r- OO O r~> ^ H ra oo

T-H CN c_> o r - H * — < 1 — < CN i i | • H H

1 M 1—> t - » H T >

•

» — » 1

• — > i

I—» PQ PQ PQ PQ PQ , PH P H PH PH PH PH PH PH PH PH PH P H PH ,_, ^ o o o o o o o o o o o o O S

qnmiiiniM,.! ppw—mn

1.5 kb ^ |«Msf i^': " l r ':#F4


68


Cultivar: Shepody

a gel.

1.5 kb

800 bp

500 bp

m oo o o

»—i

© t--O

r—1 <*t ir> r O ^J" r-- ON Q Q Q i

l-H H H 1—1 i

I—i

s u <-> > — ' i—( i—i i — i PH PH PH PH PH PH PH s <N (N <N CN <N <N O O O O O O O ;> 1 4

&

Pgel.

1.5 kb

800 bp

500 bp

oo o O o

o oo o CM -0

1 -0

6

T - H i—1 oo

H H • t—t

i—> t — s *—J 1—1 i

» — » • PQ PQ PQ PQ PQ

P H P H P H P H P H P H P H P H P H P H P H P H P H ^ . ,

S o o o o o o o o o o o o o ^


69


Cultivar: Shine

a gel.

CO OO

o o C5 © '—' '-"' >-"H ,— l

QH PH

C N C N C N C N C N C N O O • i f

1.5 kb

^ VO t > T-H 'vf • O O *-H <-<

Q l I I I

I—H HH P-H 1—1 P H P H PH P H PH

O O P O O

* * * •

800 bp

500 bp

Pgel.

oo o ^r vo o f - OO o < — > ^ CN oo

T - H rN o o < — 1 1 - H * — < CN • 1 • i 1

!—1 1

HH 1 •

H-4 H-» t — J H-» t—j PQ PQ PQ PQ PQ

S o o o o o o o o o o o o o S jar

1.5 kb

800 bp

500 bp


70


Cultivar: Simcoe

a gel.

1.5 kb

800 bp

500 bp

fO 00

o o 1 1

I—I *—1

1 v©

o r-o

^ H

m o o ^r r ~ ON Q Q Q 1 t—i

1 h - 1

1 1—A h—1

s < — > i <->

(N

r—1 1

CO (N •

O O O O O O O ^

(3 gel.

1.5 kb

800 bp

500 bp

oo o i i

O i

O O h OO O r-H ^H r-i

o i

i 1 i i ' i ' i i 9 i — 3 i j I j

P H O H P H P L I D - I P H P L I P H

* — I V © *—( C M 00 O O —i ^ H ^H

i i i i i PQ PQ PQ PQ PQ P_| PH P_| PH PH l ^ o o o o o o o o o o o o o S

1 * 1 . . *


71

PT97011 DNA fingerprints and cryopreservation of potato cuitivars for improved quality assurance

Cultivar: Snowchip

a gel.

o o T—i ô i > -—> ^ i i i O O <—i >—1

10 r- o ^ t t~~~ ON Q Q Q >—1 1—I t—1 l-H

5 9 ? T 4 M fN fS

> — ' *—* '—i P k p L i P - i e ^ P - i P H P L i ^ 5 9 ? T 4 M fN fS

<N CN <N O O O O O O O ^

Tm:*. * • * 1 v -sv - ^ ^-"i "*"^*35ESHHHKF

1.5 kb \ r

800 bp -V

500 bp

P gel.

1.5 kb

800 bp

500 bp

00 o o o o r—( T—1

oo o o -06

f—H -12

-18

1 1—1 HH * -s t — s

1

>-> >—> t — > * — > PQ PQ PQ PQ PQ



72


Cultivar: Snowgem

a gel.

1.5 kb

800 bp

500 bp

en oo T—i O O —'

1 1 1 o O i — i ^

>n O O ^T r G\ Q Q Q i i h—1

i

o O i—i *—1 \ — i i — i p i p i p-i CL, n,, n , CL, s i <N <N

1 • i O O O O O O O ^

- - vs\

•was « > rS A v * v- ,-PV «r ^ j r a m

J

Pgel.

1.5 kb

800 bp

500 bp

oo o o o

o oo o <N

O i -0

6 ^ I—1 oo

i i 1—1 HH

H-» >—> i—» 1—> •—J i

• — > PQ PQ PQ PQ PQ


•Z.1\ i';-'*7 • ••:-*.*~KaH'91*i'fife^.^!


73


Cultivar: Spunta

a gel.

1.5 kb

800 bp

500 bp

»o o o ^ i o\ C 5 C D > — ' *—' *—< < - • '

i i i i i i

(S M N (S (S M

m OO 1—1 o

i o 1

^ o O Q Q Q 1

HH M PL, PH PH PH PH

- H ^t-

PH PH O O O O O O O ^

mss

if* 11

PgeL

1.5 kb

800 bp

500 bp

OO o "^1- vo o t ^ 00 o i—i 1—H <N OO

i - H <N o o I - * * - H 1 — * CN i 1 1 1

HH 1

HH t—> i—j H-» H-J 1—1i i -s PQ PQ PQ PQ PQ




74


Cultivar: Toolangi Delight

a gel.

1.5 kb

-03 CO r-<

o —< 1 1

o 1 ^ O

, - H

1—H

ir> r>- O ^J- t-» 0\ Q Q Q h—1 i

M 1

•—1 •—.

s o c_> ""• i — i '—' T — 1 P i PL. PH Pw PL. PL. PH s CN CN c\) CN <N CN o o o o o o o ;>

800 bp s . -.' &•- - . •

Cvn«.W 500 bp iSfiH

v«fe:

Pgel.

1.5 kb

800 bp

500 bp

CO O r-H ( M o o

o t ^ CO O o 1

O —i i i -1

2 CO

• 1 1

• — 1 1 — 1 t -s • — j t-ij 1 i i « PQ PQ CQ pq



75


Cultivar: Trent

a gel.

1.5 kb

800 bp

500 bp

m oo -—•i O O - H *0 £ - —i Tf J . J . i O O ^ - H

i n r - O T f N O \ Q Q Q ^ ^ ^ ^

S <N O - H H ^ H ^ ^ ^ ^ ^ ^ p ^

S <N ( S C S I N N M O O O O O O O ^

Pgel.

1.5 kb

800 bp

500 bp

CO o ^ r \o o r-- 00 o i—1 ( (

^ <N oo i - H <N <_> <—) T — ( T — 1 1 - H <N 1 1 i 1—1 H-1 >—> 1—>

1 *—>

1 >—>

1 i 03 PQ PQ PQ PQ P - l P L i P H P L i f X i P L i p L i P L i P L i P L i P L i P L i p L i



76


Cultivar: White Rose

a gel.

m oo r-i o o ,_, *o r <—i rj-

S ? S ( N ( N ( N ( N ( N O O O O O O O ^

1.5 kb S* &v?"S

fpFff£ 800 bp "IK^ff'f^

K**5f .2*

500 bp

K**5f .2*

PgeL

1.5 kb

800 bp

500 bp

r - H t N O O r - H r - H r - H r ^ V l l l l

P H P H O H P - I P - I P H P - I P - I P - I P H P H P L H P - I ^ . ,

O O O O O O O O O O O O O S

WflHH


77


Cultivar: Whitu

a gel.

1.5 kb

800 bp

500 bp

CO 00 ^H

O O <-H o o 1 1

-si"

i n t ^ O • < * t-« Os Q Q Q 1—1 H H 1 1

h—1 o o •—' '—' *—* 1—1 P-t P* PH p-i OH PH PH

^ c-i <N CN <N CS rsi O O O O O O O ^

Pgel.

1.5 kb

800 bp

500 bp

CO o "* v© o r- oo O r~> , - H (N 00

1—1 CN <_> c_> * — < r—i * - H CN | i 1 1

1—1 t - » 1—J i—> i—> 1—J •—s PQ PQ PQ PQ PQ OH P H PH PH PH PH PH PH PH P H PH PH PH

>! o o o o o o o o o o o o o ^


78


Cultivar: Wilstore

a gel.

1.5 kb

800 bp

500 bp

m co —i O © —H

1 1 1 o o , - H

«r> O O ^ r F- 0\ Q Q Q • • — 1

i • — 1 h-H

s <-> c_> *—* T » H '—i r—1 PH OH PH PH PH PH PH s <N (N fN (N (N CN o o o o o o o ^

Pgel.

1.5 kb

800 bp

500 bp

oo o o o

o T—1

o- oo o o 1 -0

6 i—i 1—H

oo r—I

1

M • l-H H-s 1—> 1—» 1—> »-» » — » m PQ PQ PQ PQ



M = marker (100 base-pair ladder, ex, Amersham Pharmacia Biotech)

79


Cultivar: Wilwash

a gel.

m oo r—i © O r-4

1 1 1 o o ^ ^r

i n f - o ^ r-- Os Q Q Q i t—(

i I—i

i i—i

i

<-> o I—1 i—H i — i I - H P 1 P 1 PH n, n n, C^ s i • 1 i i i O O O O O O O ^ K ^ &

1.5 kb

800 bp

500 bp

Pgel.

1.5 kb

800 bp

500 bp

OO O 1-H CN © o

o I—1

r oo i - H

© «—i

o 1

-06 1 — 4

1—H

1 -12

-18

i i l-H l-H 1—4 H-»

1 >—j

1

>—> i

t—» PQ PQ PQ PQ PQ P H P H P H C M P H P H P H P H P H P H P H P H P H , ^



80


Cultivar: Winlock

a gel.

1.5 kb

800 bp

500 bp

-03

-08

I—1 £5 O o f—I T—1

lO f» o f t-- ON « w ci i

>—< 1 t—i t — 1

o O T—H r-H < — i T—4 PH PH OH PH OH PH PH

2 <N C-l «N (N CS (N o o o o o o o ;>

Pgel.

1.5 kb

800 bp

500 bp

oo o ^ v© O t- oo O (—i

^H <N OO I-H <N o o *-H t — f i — » CN | | 1 1

1—1 t-J t — > 1 1

I — S i

*-i PQ PQ PQ PQ PQ , PH PH PH PH PH PH PH PH PH PH PH PH (^ , ^ o o o o o o o o o o o o o £

KM /:; . ' - £ „ . • , . ^ " T i t ? 'V"'-"-«3

; , * K"i A *

•?^S .f<\.A •n i » • • * ?


81


Cultivar: Winter Gem (90-105-16)

a gel.

1.5 kb

800 bp

500 bp

CO CO —i © © T-H

1 i 1 © © - H "«-

i n 1> O " f r^ Os Q Q Q i H-1 H H H H H H

o O i—i r—t * — i *—I PH P i P i P i P i P i P i >; CN <N CN <N <N «N O O O O O O O ^

*•* ."3 tfW W

Pgel.

1.5 kb

800 bp

500 bp

oo © •sr ô © l > oo © r - i f N OO

1 - H (N © © 1—1 1—1 T - H CN i i 1 1 1

•—I I—» 1 *—» 1

t-1> 1 i

i—» PQ PQ PQ PQ PQ , P i P i PH P i P i P i P i PH P i P i P i PH P i ^ o o o o o o o o o o o o o '£


82


Cultivar: Wontscab

a gel.

1.5 kb

800 bp

500 bp

m oo <—i O O - H

1 1 1 o o i—( ^t-

«n f - O «t O C\ Q Q Q i »—i

i 1 1 — 1

1 • — 1

s i i CN <N

i

O O O O O O O ^

i. . 4 - - - * -- • . . "

Pgel.

1.5 kb

800 bp

500 bp

oo o o o

© 1—1

OO O o 1

* © ,—1 O <-H

1 1 -12 00

i i i

t—1 h-1 1—1 *—i » - j 1 1 1

1—» >—> PQ PQ ffl 0Q m S o o o o o o o o o o o o o S


83


Cultivar: Yanchip (88-59-12)

a gel.

1.5 kb

800 bp

500 bp

m oo -—i o o ^

i i i o o ^ •*t

i n f - O •sf t ^ Q\ Q Q Q 1 i i 1 1—H

o O '—' *—i i—* ' — < PH PH PH PH PH cu PH

2 ( N «N «N <N CN <N O O O O O O O >

K ^

, " • ; ' . • • * * -,1i <*.« " * ) j "

pgel.

1.5 kb

800 bp

500 bp

CO o T f VO o t -- CO O r-> T—t ra CO

I—1

»"—< <N o o * - H i - H 1—1 <N i I i 1

H-1 1 1 1

l - » 1

t — > 1—> • — > H-J PQ PQ PQ PQ PQ , P H PH PH PH PH ft* PH PH PH ft< P H P H PH , £ o o o o o o o o o o o o o £

*

& , I f e f i f c i f e j l r ^ f c ^ j t i L


84


3.0 Cryopreservation of potato cultivars 3.1 Introduction

With clonally propagated crops, cultivars can only be maintained as field or glasshouse grown collection (in situ) or in tissue culture (in vitro). Maintaining

germplasm collections is costly, particularly for cultivars that are less frequently used. Presently potato cultivars at IHD - Knoxfield and Toolangi are maintained in slow-growth tissue culture conditions (incubating 4-15 °C on media with mannitol) and as a field-bank collection. Over the last two decades, methods based on cryopreservation have been investigated for potato germplasm storage (Grout and Henshaw 1980, Renshaw et al. 1985; Bouafia etal. 1996; Schafer-Menuhr et al. 1996; Towill 1996, Sarkar and Naik 1998).

While a number of methods have been successfully used, most have only assessed a small number of cultivars. The droplet freezing technique (Schafer-Menuhr et al. 1996), has been the only method used for the routine cryopreservation of shoot tips from a large number (200+) of potato cultivars.

An important consideration is whether cryopreservation results in genetic changes. Investigations to date, have used flow cytometry and RFLPs (Schafer-Menuhr et al. 1996). PCR-based RAPD analysis offers a fast and simple method to detect possible genetic change that can occur during cryopreservation treatments.

The objective in this study was to evaluate cryopreservation as a long term storage system for potato cultivars and screen regenerated plants using RAPDs.

3.2 Methods and materials 3.2.1 Maintenance of tissue culture plants All cultivars used (Appendix 3) were obtained as certified pathogen tested cultures IHD, Knoxfield. Cultures were maintained by nodal cuttings on MS (Murashige and Skoog, 1962), containing 30 gL"1 sucrose and 3 gL"! phytagel (Sigma, Aust). Cultures were incubated at 21 °C under a 16:8 h light: dark photoperiod. The light source was cool-white fluorescent tubes providing 50 umol mV1 at the culture level.

3.2.2 Cryopreservation Shoot tips were cryopreserved according to Schafer-Menuhr et al. (1996). In brief, shoot tips two to three mm tall were isolated and incubated overnight at 21 °C in MSTo (MS, 30 gL"1 sucrose, 2.5 uM zeatinriboside, 0.6 uM GA3 and 2.5 uM IAA) (Towill 1983). Shoot tips were then transferred to MSTo with 10 % dimethylsulfoxide (MSTo - DMSO) and incubated for two hours at room temperature. After incubation, shoot tips were transferred to three uL droplets of MSTo - DMSO on foil strips (0.7 x 2 cm, six drops per foil). The foil strips were transferred to pre-cooled 1.8 mL Cryotube™ vials (Nalgene Nunc International, Denmark) and plunged directly into liquid nitrogen for storage One-hundred shoot tips were cryopreserved for each cultivar and stored for 12-18 months.

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3.2.3 Recovery Shoot tips were thawed by quickly transferring foil strips into MS liquid medium heated to approximately 50 °C, then transferred to MSTo medium.

Survival and regeneration of shoot tips was monitored weekly over the following 6-8 weeks. Survival was recorded as the mean percentage of total shoot tips with the ability of a treated shoot tip to produce a callus and/or shoot (Towill 1983). Regeneration was recorded as the mean percentage of total shoot tips treated that developed plantlets from at least one shoot. Two replications of 50 shoot tips was used for each cultivar.

3.2.4 Genetic stability Regenerated plants were analysed with RAPDs (Chapter 2), using six primers: 2-07, 2-10, 2-14, 2-17, 2-19 and OPJ-04.

3.3 Results All 37 cultivars survived after cryopreservation (Table 3.1, Fig. 3.1). The survival rate varied considerably with each cultivar, the highest survival rates obtained were with Krantz (58.6% ) and Norgold Russet (56.7%). Low survival rates (<10%) were obtained with cultivars Allegany, Campbell 11, Fontenot 287, Monona (Line A), Oneida, Rosa, Sangre and Yankee Supreme.

Thirty of 37 cultivars have shown regeneration into plantlets (Table 3.1, Fig. 3.1). Regeneration rates were generally lower than survival rates and varied with cultivar, the highest obtained with Gemchip (14%) and Krantz (17%). Shoot tips generally regenerated multiple shoots that can increase the number of explants to obtain if necessary (Fig. 3.2).

It is important to note, that there was endogenous microbial contamination with tissue culture plants used in the cryopreservation experiments which have reduced survival and regeneration. Many shoot tips were discarded with some cultivars and results presented may be underestimated. Contamination issues have been investigated and discussed in more detail in Chapter 6.

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Table 3.1. Mean survival and regeneration rates of 37 potato cultivars cryopreserved. Cultivar Survival (%) Regeneration (%)

Allegany 8.9 0 Atlantic White Flower 16.9 4.3 Brownell 19.0 3.0 Butte 30.0 5.0 Campbell 11 7.2 0 Cariboo (Line B) 25.0 5.0 Chipbelle 10.0 3.0 Fontenot 287 6.0 1.0 Frontier Russet 20.0 3.1 Gemchip 26.0 14.0 Hampton 11.0 3.0 Hudson 24.0 2.0 Itasca 40-3 31.0 8.0 Kanona 23.0 6.0 Katahdin (Line B) 47.2 10.6 Kennebec (Line A) 24.7 7.1 Kenzy 30.0 12.0 Krantz 58.7 17.0 Lenape 12.6 3.1 Merrimack 11.0 1.0 Monona (Line A) 4.6 1.0 Norchip Line A 13.3 1.0 Norgold Russet 56.7 8.4 Norking Russet 19.0 3.0 Oneida 3.0 0.0 Pembina Chipper 13.0 4.0 Pentland Envoy 20.0 12.0 Platte 43.7 1.7 Red Ruby 21.0 12.0 Rosa 4.0 0 Sangre 2.0 0 Snowchip 17.4 0 Somerset 25.0 2.0 Tobique 46.2 3.9 Tolass 10.0 2.0 Whitu 19.0 5.0 Yankee Supreme 5.0 0

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(JO

90

80

60

50 -

40 -

30 -

20 -

10 •

'

Cultivar

Figure 3.1. Percentage of survival (dark/blue bars) and regeneration (light/pink bars) of 37 potato cultivars after cryopreservation using the droplet-freeze technique.

Figure 3.2. An example of shoot regeneration from potato shoot tips 6 weeks after thawing and recovery.

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RAPD analysis detected no genetic change in regenerated plants of cultivars Sebago, Atlantic, Hampton, Gemchip and Norchip. The number of bands produced from the six primers varied from 30 to 40 and ranged in size from 300 bp to 2500 bp. An example is shown with cultivar Norchip (Fig. 3.3).

M N C N C N C N C N C N C M

^ 2.5 kb

— 1.5 kb

•— 800 bp

— 500 bp

Figure 3.3. RAPD profiles of cryopreserved (C) and non cryopreserved (N) potato plants cultivar Norchip with primers 2-07, 2-10, 2-14, 2-17, 2-19 and OPJ-4. Lane M = molecular weight DNA markers.

3.4 Discussion Cryopreservation has shown to be successful and have potential for the long term storage of many potato cultivars. Although survival varied with different cultivars, many have shown the ability to regenerate into normal looking tissue culture plantlets. These results confirmed and expand on those of Schafer-Menuhr (1996), who reported considerable variability in survival and regeneration rates with over 200 potato cultivars. Multiple shoot formation was commonly observed which can increase the number of clones obtained with cultivars that show low survival and regeneration after cryopreservation (Sarkar and Naik 1998).

The two main processes in cryopreservation are freezing and regeneration. As all cultivars survived cryopreservation this indicated that the freezing procedure used was satisfactory. As regeneration of tissue culture plantlets is known to be influenced by cultivar, modifications to media are necessary to improve regeneration (Kariuki et al. 1996). It may not be necessary to obtain high regeneration rates as this can be compensated by multiple shoot formation or a greater number of shoot tips could be frozen to insure the cultivar can be regenerated.

The genetic stability of regenerated plants is of great importance if cryopreservation is to be used for long-term storage of germplasm. RAPD analysis did not detect any genetic changes in cryopreserved plants. This does not necessarily suggest that there was no genetic change and further evaluation is required. Other studies have shown that methylation of DNA occurred in potatoes grown in slow growth mannitol containing medium (Harding 1994), however, it is unknown if this occurs after cryopreservation. RFLP analysis, flow cytometry, cytological and glasshouse studies have found no changes in profiles, ploidy and morphology after cryopreservation (Benson et al. 1996; Schafer-Menuhr et al. 1996).

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3.5 References Benson, E.E., Wilkinson, M., Todd, A., Ekuere, U. and Lyon, J. (1996).

Developmental competence and ploidy stability in plants regenerated from cryopreserved potato shoot tips. Cryo-Letters 17, 119-128.

Bouafia, S., Jelti, N., Lairy, G., Blanc, A., Bonnel, E. and Dereuddre, J. (1996). Cryopreservation of potato shoot tips by encapsulation-dehydration. Potato Research 39, 69-78.

Grout, B.W.W. and Henshaw, G.G. (1980). Structural observations on the growth of potato shoot-tip cultures after thawing from liquid nitrogen. Annals of Botany 46, 243-48.

Harding, K. (1994). The methylation status of DNA derived from potato plants recovered from slow growth. Plant Cell, Tissue and Organ Culture 37, 31-38.

Henshaw, G.G., O'Hara, J.F and Stamp, J. A. (1985). Cryopreservation of potato meristems. In: Cryopreservation of plant cells and organs. (Ed. K.K. Kartha). pp. 159-69. CRC Press.

Kariuki, A., Graham, M.W., Waterhouse, P. and Hutchinson, J.F. (1996). Production of virus resistant potato plants to enable reduced use of insecticides on potatoes. Final report for HRDC project PT338.

Sarkar, D. and Naik, P.S. (1998). Cryopreservation of shoot tips of tetraploid potato (Solanum tuberosum L.) clones by vitrification. Annals of Botany 82,455-61.

Schafer-Menuhr, A., Miiller, E. and Mix-Wagner, G. (1996). Cryopreservation: an alternative for the long term storage of old potato varieties. Potato Research 39, 507-513.

Towill, L. (1983). Improved survival after cryogenic exposure of shoot tips derived from in vitro plantlet cultures of potato. Cryobiology 20, 567-573.

Towill, L. (1996). Vitrification as a method to cryopreserve shoot tips. In: Plant tissue culture concepts and laboratory exercises. (Eds. D.J.Gray and R.N. Trigiano). pp. 297-304. CRC Press, Inc., Boca Raton, FL.

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4.0 Random Amplified Polymorphic DNA analysis 4.1 Introduction T ) / ^ T ) n a s simplified and improved the techniques for generating molecular -L V^/XVmarkers in a diverse range of organisms (Karp et al. 1998). The most commonly used system has been Random Amplified Polymorphic DNA (RAPD) and have been useful in many applications for plant breeding, genetic mapping and DNA fingerprinting (Welsh and McClelland 1990).

RAPD analysis involves the use of a single primer of arbitrary design that amplifies dominant markers that have Mendelian inheritance (Williams et al. 1990). The technique is simple, fast and only requires nanogram amounts of DNA. The DNA fragments are resolved on agarose gels, stained with ethidium bromide and visualised under ultra-violet light. Polymorphisms or genetic differences between individuals are observed by differences in banding patterns (profiles) obtained. These differences allow us to characterise, differentiate and therefore distinguish between individuals making this technique useful for cultivar identification and differentiation (Morell et al. 1995). Molecular markers are also useful for estimating genetic diversity of germplasm collections and examining genetic relationships among potato cultivars (Demeke et al. 1996) (see Chapter 7).

An important requirement for any DNA fingerprinting system is a protocol that is reliable and reproducible (Cobb 1998). A number of factors have shown to effect band profiles such as different thermocyclers, annealing temperature, cycling conditions, polymerase buffer and DNA polymerase type (Innis and Gelfand 1999). This chapter describes experiments used to develop the RAPD-PCR protocol for DNA fingerprinting, and evaluate factors that influence reproducibility.

4.2 Methods and materials 4.2.1 General conditions The plant material used, DNA isolation and gel electrophoresis for DNA fingerprinting with RAPDs are described (Chapter 2).

4.2.2 Development of protocol and reproducibility of RAPD analysis 4.2.2.1 Optimisation of PCR components using a modified Taguchi method A modified Taguchi method (Cobb and Clarkson 1994, Cobb 1998) was used to optimise and evaluate interactions of PCR components. This method is based on using orthogonal arrays that evaluate PCR component interactions using a 34 factorial design with 9 reaction mixtures.

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Table 4.1. Evaluation of four components displayed as an orthogonal array for nine PCR mixtures.

Component (Level) Reaction Mg2+ (mM)(1) dNTPs

(uM)(2) Primer (HM)(3)

DNA (ng)(4)

1 0.75 50 0.2 25 2 0.75 100 0.4 50 3 0.75 200 0.8 100 4 1.5 50 0.4 100 5 1.5 100 0.8 25 6 1.5 200 0.2 50 7 3.0 50 0.8 50 8 3.0 100 0.2 100 9 3.0 200 0.4 25 (1) evaluated at 0.75,1.5 and 3 mM {2) evaluated at 50, 100 and 200 ^M (3) evaluated at 0.2, 0.4, 0.8 |oM (4) evaluated at 25, 50 and 100 ng

Four reaction components Mg2+, dNTP, primer and DNA were evaluated at three concentrations (Table 4.1). Gels were scored in binary format for the presence (1) and absence (0) of bands for each reaction. Signal-to-Noise (SNL) ratios were calculated using the following quadratic loss function:

SNL = -lOlog

•s i

where n is the number of levels (in this case 3) and v is the yield (number of bands). The largest SNL determined was the optimum concentration for each of the PCR components.

4.2.2.2 Thermocycling conditions The effect of annealing temperature (Ta) and alterations to denaturing and annealing

time were evaluated with cultivar Atlantic and primers 2-07 and 2-17.

All reactions were carried out in a final volume of 50 [xL containing 50 ng DNA, 0.4 HM primer, 10 mM Tris-HCl, (pH 8.8), 1.5 mM Mg2+, 150 mM KCl and 0.1 % Triton X-100, 100 \iM dNTPs, and 1 U Dynazyme II polymerase (Finzymes Oy, Finland). Thermocycling was performed in a Corbett Research FTS-960 (Corbett Research, Aust).

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Thermocycling conditions comprised 35 cycles of 94 °C for 10 s, Ta for 30 s, and 72 °C for 1 min. First denaturation cycle for 1 min; last extension was for 5 min. Ta's investigated 30, 35, 40, and 45 °C were tested.

Experiments to test denaturing and annealing time combinations consisted of four treatments (Table 4.2).

Table 4.2. Denaturation and annealing time combinations tested.

Treatment no. Denaturation time (s) Annealing time (s) 1 2 3 4

10 10 30 30

30 60 30 60

4.2.2.3 Effect of DNA polymerase type Five DNA polymerase types were tested (Table 4.3).

Table 4.3. Details of the polymerase types tested. DNA polymerase * Abbreviation Manufacturer Dynazyme II Dynazyme Finzymes Oy, Finland Boehringer Mannheim Taq BM Taq Boehringer Mannheim, Germany Bresatec Taq B Taq Geneworks, Sth. Australia Promega Taq VTaq Promega Corp., USA Perkin Elmer Ampli7a<7 AmpliTaq Perkin Elmer, USA

The biological source of Taq is Thermus aquaticus except with Dynazyme that is T brockianus

DNA from cultivars Coliban and Wilstore were tested with primers 2-13 and 2-17. Reactions were carried out in a final volume 50 uL containing 50 ng DNA, 0.4 \iM primer, x 1 buffer (according to manufactures instructions), 3 mM Mg2+, 100 uM dNTPs, 1 U polymerase.

Thermocycling was performed in a Corbett Research FTS-960 (Corbett Research, Aust.) with 35 cycles of 94°C for 30 s, 45°C for 30 s, and 72°C for 1 min. First denaturation cycle was for 1 min; last extension cycle was for 5 mins.

4.2.2.4 Reproducibility of RAPD fingerprints between two thermocyclers Thermocycling was conducted in a Corbett Research FTS-960 (Corbett Research, Aust.) and a Hybaid PCR Express (Hybaid, UK) with 35 cycles 94°C for 30 s, 45°C for 1 min, and 72°C for 1 min. First denaturation cycle was for 1 min; last extension cycle was for 5 mins. The same operator was used to run the PCR experiment.

All reactions were carried out in a final volume of 50 uL containing 50 ng DNA, 0.4 uM primer (Geneworks, S.Aust), xl buffer (10 mM Tris-HCl, 3mM MgCl2, 50 mM KC1, pH 8.3), 100 \iM dNTPs, 1 U Taq DNA polymerase (Boehringer Mannheim, Germany).

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There were two separate PCR runs using cultivars Coliban and Wilstore with primers 2-07 and 2-17 and cultivars Cadima, Wilstore, and Norchip with primers 2-11 and 2-17.

4.2.2.5 Other indicators of reproducibility Seven separate DNA extractions of the cultivar Wontscab was amplified with 12 primers to check reproducibility across the individual extractions.

The cultivar source comparison from the two collections described in Section 2.3.1 was also an useful indicator of reproducibility between two different tissue sources.

Taguchi optimisation determined that 0.6 uM primer concentration was optimum however, 0.4 iM primer was used routinely. To investigate if the 0.2 |uM primer difference would affect the RAPD profile, these two concentrations were evaluated with cultivars Atlantic and Kennebec with primers 2-07 and 2-17.

4.2.3 Sensitivity of RAPD analysis for detection of different cultivars in the field The sensitivity of RAPD analysis to potentially detect different cultivars in the field was tested by DNA titration experiments with primers that revealed cultivar distinguishing markers.

DNA titration with admixtures of DNA from two different cultivars were made with a stepwise decreasing ratio of 5:0, 4:1, 3:2, 1:1, 2:3, 1:4, 0:5, with cultivars Atlantic and Patrones with primer 2-17 and cultivars Atlantic and Trent with primers 2-17 and 2-20. The primers selected reveal markers that can readily distinguish the cultivars tested.

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4.3 Results 4.3.1. Development of protocol and factors influencing reproducibility 4.3.1.1 RAPD-PCR optimisation using a modified Taguchi method Based on the SNL ratio, the concentration of Mg2+, dNTPs, primer and DNA influenced the number and size of amplification products (Fig. 4.1).

With 0.75 mM Mg2+ the SNL ratio was zero and increased up to 18 at 3 mM (Fig. 4.1a). At 50 and 100 fxM, dNTP concentration had little influence on the SNL ratio (about 4.5), whereas at 200 uM it was reduced to two (Fig. 4.1b). With 0.2 \xM primer the SNL was 1.7 and increased to 4.5 at 0.4 and 0.8 yM (Fig. 4.1c). Altering DNA concentration produced an ambiguous result where SNL was 4.5 at 25 ng and reduced to about 1.5 at 50 ng and increased to about 4.5 at lOOng (Fig. 4.1d).

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(a)

SN,

20

18

16

14

12

10

8

' ' ! i ! I I I 1 1 1 1 I 1 I 1

0.6 0.8 1.0 1.2 1.4 16 1R " 1 " 2.4 2.6 2.8 3.0 ,2+

Mgz+ (mM)

(b

SNL

70 90 110 130 150 170 190 210

dNTPs (uM)

Figure 4.1. Effect of reaction components on the SNL ratios for the RAPD amplification as determined by polynomial regression.

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(C)

SNL

(d)

SNL

-J I L_

0.2 0.3 n a na n R 0.7 0.8

Primer (\iM)

20 30 40 50 60 70 80 90 100

DNA (ng)

Figure 4.1. (cont'd.) Effect of reaction components on the SNL ratios for the RAPD amplification as determined by polynomial regression.

97


Optimum PCR conditions as determined by SNL ratio were based on the total number of products scored in the profile (Table 4.4; Fig. 4.2).

Table 4.4. PCR conditions determined by using the modified Taguchi method. Reaction component Concentration Mg dNTP Primer DNA

3mM 100 uM 0.4-0.6 ^M 25-50 ng

M 1 2 3 4 5 6 8 9 M

>¥*>?

2.5 kb

1.0 kb

500 bp

Figure 4.2. Amplification of nine PCR mixtures using the modified Taguchi method with cultivar Atlantic and primer 2-07. Details of lanes 1 -9 can be found in Table 4.1. Lanes M = molecular weight DNA markers (Amersham Pharmacia Biotech).

4.3.1.2 Thermocycling conditions The evaluation of Ta and denaturation and annealing times were based on the intensity and the number of products generated.

With primer 2-17 the band intensity increased with Ta, 45 °C was subsequently was chosen (Fig. 4.3).

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30 35 40 45 X M

1.5 kb

800 bp

Figure 4.3. Effect of annealing temperature on cultivar Atlantic and primer 2-17. Lane X is the cycling regime of Collins and Symons (1993). Lane M = 100 bp ladder molecular weight marker (Amersham Pharmacia Biotech).

There was no major difference in the banding patterns between denaturation and annealing times, except that product intensity was slightly stronger with denaturation of 30 s and annealing of 60 s with both primers tested (Fig. 4.4, lane 4). This denaturing and annealing time combination was used for further experiments.

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2-07

1 2 3 4 M

V.

. ^ „ „ _ . , 100 bp

Figure 4.4. Effect of denaturation and annealing time combinations with cultivar Atlantic and primers 2-07 and 2-17. Lanes 1 = denaturation 10 s/annealing 30 s (10/30), 2 = 10/60, 3 = 30/30 and 4 = 30/60. Lane M = 100 bp ladder molecular weight marker (Amersham Pharmacia Biotech).

4.3.1.3 Effect of polymerase type The intensity and number of products varied with the type of polymerase used. With BM Taq and AmphTag, profiles were similar when compared to P Taq, Dynazyme and B Taq. BM and AmphTag were more efficient at amplifying greater than 1.2 Kb bands than other polymerases (Fig. 4.5, Lanes 2 and 3). Dynazyme II polymerase was more efficient at amplifying bands below 500 bp and with greater intensity compared to other polymerases (Fig. 4.5, Lane 1).

1 2 3 4 5 M

Art!*

800 bp

Figure 4.5. Effect of different types of polymerases with cultivar Coliban and primer 2-13. Lane 1 = Dynazyme II, Lane 2 = ArcvpliTaq, Lane 3 = Boehringer Mannheim Taq, Lane 4 = Promega Taq, Lane 5 = Bresatec Taq, Lane M = 100 bp ladder molecular weight marker (Amersham Pharmacia Biotech).

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4.3.1.4 Reproducibility of DNA fingerprints between thermocyclers The thermocyclers tested generated identical DNA fingerprint profiles with all DNA and primer combinations tested (Fig. 4.6).

2-07 2-17 1 2 3 4 M 5 6 7 8

Figure 4.6. Reproducibility of RAPD profiles between two thermocyclers Corbett Research FTS-960 (lanes 1,3,5, 7) and Hybaid PCR Express (lanes 2, 4, 6, 8) with primers 2-07 and 2-17. M = 100 bp ladder molecular weight marker (Amersham Pharmacia Biotech).

4.3.1.5 Other indicators of reproducibility From seven individual DNA extractions of cultivar Wontscab, reproducible DNA fingerprints were generated with all 12 primers tested (Fig. 4.7).

1 2 3 4 5 6 7 M 8 9 10 11 12 13 14

Figure 4.7. Reproducibility of RAPD profiles from 7 individual DNA extractions of cultivar Wontscab with primers OPD-3 (lanes 1-7) and OPD-8 (lanes 8-14). Lane M = 100 bp ladder molecular weight marker (Amersham Pharmacia Biotech).

An additional useful indicator of the reproducibility of the system developed, has been the generation of indistinguishable fingerprints between 72 accessions (64 cultivars and 8 clonal variants) from the cultivar collections maintained in tissue culture and the field (Section 2.3.1).

Evaluation of RAPD amplification with 0.4 and 0.6 pM primer concentration showed that no differences in RAPD profiles were observed with the two DNA and two primers tested (Fig. 4.8).

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2-07 2-17

A K A K

0.4 0.6 0.4 0.6 M 0.4 0.6 0.4 0.6

Figure 4.8. Evaluation of 0.4 and 0.6 uM primer concentration with RAPD profiles produced with cultivars Atlantic (A) and Kennebec (K) with primers 2-07 and 2-17. Lane M = Kilobase DNA ladder molecular weight marker (Amersham Pharmacia Biotech).

4.3.3 Sensitivity of RAPDs for detection of different cultivars in the field With primer 2-17 cultivar distinguishing markers were produced with cultivar Atlantic at 750 bp (2-1775o) and with cultivar Patrones at 1000 bp (2-17i00o)- The Atlantic marker 2-1775o could be detected in admixture ratios from 4:1, 3:2, 1:1, 2:3 and 1:4 with cultivar Patrones (Fig. 4.9). The amplification signal weakened as the dilution increased. The cultivar Patrones marker 2-17iooo was found to amplify at the same ratios tested with cultivar Atlantic. The amplification signal or intensity was stronger than Atlantic marker 2-17750 at the highest dilution 4:1.

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Atlantic : Patrones M 5:0 4:1 3:2 1:1 2:3 1:4 0:5 M

2-17 1000

2-17 750

1.0 kb

800 bp

500 bp

200 bp Figure 4.9. DNA fingerprints from admixture ratios of cultivars Atlantic and Patrones with primer 2-17. Markers that distinguish these cultivars are arrowed. Lane M = 100 bp ladder molecular weight marker (Amersham Pharmacia Biotech).

The validity of this approach was confirmed with cultivar distinguishing markers for Atlantic (2-17750) and Trent (2-17i000) (Fig. 4.10).

103


Atlantic: Trent M 5:0 4:1 3:2 :1 2:3 1:4 0:5

>v.

2-17iooo

2-17750

1.0 kb

800 bp

500 bp

300 bp Figure 4.10. DNA fingerprints from admixture ratios of cultivars Atlantic and. Trent with primer 2-17. Markers that distinguish these cultivars are arrowed. Lane M = 100 bp ladder molecular weight marker (Amersham Pharmacia Biotech).

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4.4 Discussion The modified Taguchi method has been demonstrated to be a cost-efficient approach to optimise and evaluate the interactions of chemical conditions required for PCR. With conventional experimentation, evaluating 4 components (Mg2+, dNTP, DNA, primer) at 3 concentrations would involve a 34 factorial experiment of 81 reaction mixtures. Setting up this number of reactions is time consuming, costly and can easily result in mistakes. This method is based on using orthogonal arrays and can evaluate 34

factorial design with 9 reactions (Cobb and Clarkson 1994, Cobb 1998). The Taguchi method has also been useful for the optimisation of DNA amplification fingerprinting (Caetano-Anolles 1998) which is similar to RAPDs but uses smaller primers and profiles are visualised by silver staining.

Changes in Ta, also affect the RAPD profile. Although amplification was achieved at Ta of 30, 35 and 40 °C, bands with of stronger intensity were obtained at 45 °C and this was used for all subsequent work. Selecting Ta is usually a compromise because more efficient amplification can be achieved at lower temperatures (eg. 37 °C) however, mispriming can significantly increase the number of spurious products that are likely to be less reproducible. At higher temperatures (45-50 °C), less amplification products were generated but reproducibility and the detection of Mendelian inherited bands is improved due to the reduction in spurious amplification (Rychlik et al. 1990; Atienzar et al. 2000).

Evaluation of denaturation and annealing times can be useful to improve the efficiency of thermocycling time without reducing product yield and reproducibility (Yu and Pauls 1992). In this case, the original thermocycling regime used has been shown to work efficiently at 35 cycles. Evaluation of altered denaturation and annealing time resulted in no major changes to amplification profiles. Slightly stronger signals were observed with 60 second annealing time and this could be because more time was allowed for primers to bind and therefore allow for more sites to be amplified that will increase product yield and therefore intensity. By having short thermocycling regimes, at least two PCR experiments can be performed per day generating results quickly. In comparison to non-PCR based systems, for example RFLPs, results can take up to four days to obtain and larger amounts of DNA are required for analysis. PCR-based methods can be more efficiently used for DNA fingerprinting applications (Morell et al. 1995).

Different polymerases were shown to generate different RAPD profiles. It is not certain why different profiles are produced, but it may be due to PCR conditions not being favourable or optimised for each polymerase (Schierwater and Ender 1993). This suggests that the reproducibility of DNA fingerprints depends on the type of polymerase used and therefore it is important to not only use the same PCR conditions but also the same type of polymerase specified.

Reproducible DNA profiles were obtained with different thermocyclers using identical PCR conditions. This suggests that the routine application of DNA fingerprinting of potato cultivars could be performed using other thermocyclers and laboratories located at IHD. There was no testing conducted in laboratories external to IHD and therefore it is not certain if reproducible RAPD profiles could be obtained, as it has been shown

105


that RAPDs were unreliable for achieving reproducible band profiles amongst eight European laboratories (Jones et al. 1997).

Reproducible DNA fingerprints has been achieved with individual DNA extractions and different leaf tissue sources, which suggests that these factors should not influence comparisons of DNA fingerprints to an established database. The 0.2 LXM difference in primer concentration showed no affect on RAPD profiles generated, and this indicates that 0.4 to 0.6 \xM primer can be used to reproduce DNA fingerprints.

Cultivar distinguishing markers were shown to be detectable in admixtures of DNA from two different cultivars. The sensitivity was limited shown by the reduced band intensity as DNA dilution increased. In both experiments the effect of dilution was greater on the 750 bp band compared to the 1000 bp. These effects is due to competition, which is quantitative and directly influences the amount hence the intensity of the amplified band (Hallden et al. 1996). The data shows that mixed cultivars can be detected in the field by screening bulked samples of up to five plants at a time.

Optimising the PCR components was necessary and useful as it revealed that different conditions could result in different DNA fingerprint profiles, for example with Mg2+

and polymerase type. As alterations to the conditions affect reproducibility, it is essential that the PCR conditions defined in this report are used for generating DNA fingerprints of potato cultivars. In addition, an experimental design with the potential to detect cultivar mixes in the field has been demonstrated.

4.5 References Atienzar, F., Evenden, A., Jha, A., Sawa, S. and Depledge, M. (2000). Optimized

RAPD analysis generates high quality genomic DNA profiles at high annealing temperature. Biotechniques 28, 52-4.

Blackmore, K. (1998). Operations Report. In: Victorian Seed Potato Authority annual general meeting report, September 1998. ViCSPA Inc.

Caetano-Anolles, G. (1998). DAF optimization using Taguchi methods and effect of thermal cycling parameters on DNA amplification. Biotechniques 25,472-480.

Cobb, B. (1998). Optimization of RAPD fingerprinting. In: Fingerprinting methods based on arbitrarily primed PCR. (Eds. M.R. Micheli and R.Bova). Springer.

Cobb, B.D and Clarkson, J.M (1994). A simple procedure for optimising the polymerase chain reaction (PCR) using modified Taguchi methods. Nucleic Acids Research 22, 3801-5.

Collins, G.G. and Symons, R.H. (1993). Polymorphisms in grapevine DNA detected by RAPD-PCR technique. Plant Molecular Biology Reporter 11,105-12.

Demeke, T., Lynch, D.R., Kawchuck, L.M., Kozub, G.C. and Armstrong, J.D. (1996). Genetic diversity of potato determined by random amplified polymorphic DNA analysis. Plant Cell Reports 15, 662-7.

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Innis, M. and Gelfand, D. (1999). Optimization of PCR: Conversations between Micheal and David. In: PCR Applications: Protocols for functional genomics. (Eds. Micheal A. Innis, David H.Gelfand and John. J. Sninsky). Academic Press.

Jones, C.J., Edwards, K.J., Castaglione, S., Winfield, M.O., Sala, F., Wiel van de, C , Bredemeijer, G., Vosman, B., Matthes, M., Daly, M., Brettschneider, R., Bettini, P., Buiatti, M., Maestri, E., Malcevschi, A., Marmiroli, N., Aert, R., Volckaert, G., Rueda, J., Linacero, R., Vazquez, A. and Karp, A. (1997). Reproducibility testing of RAPD, AFLP and SSR markers in plants by a network of European laboratories. Molecular Breeding 3,381 -90.

Karp, A., Isacc, P.G. and Ingram, D.S. (1998). Molecular tools for screening biodiversity: Plants and Animals. Chapman and Hall. UK.

Morell, M.K., Peakall, R., Appels, R., Preston, L.R. and Lloyd, H.L. (1995). DNA profiling techniques for plant variety identification. Australian Journal of Experimental Agriculture 35, 807-19.

Rychlik, W., Spencer, W.J. and Rhoads, R.E. (1990). Optimization of the annealing temperature for DNA amplification in vitro. Nucleic Acids Research 18, 6409-12.

Schierwater, B. and Ender, A. (1993). Different thermostable DNA polymerases may amplify different RAPD products. Nucleic Acids Research 21s 4647-8.

Welsh, J. and McClelland, M. (1990). Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Research 18, 7213-18.

Williams, J.G.K., Kubelik, A.R, Livak, K.J., Rafalski, J.A. and Tingey, V. (1990). DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Research 18,6531-35.

Yu, K. and Pauls, K.P. (1992). Optimization of the PCR program for RAPD analysis. Nucleic Acids Research 20, 2606.

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5.0 Simple Sequence Repeat analysis 5.1 Introduction

Although RAPD analysis has been useful for DNA fingerprinting of potato cultivars, there are potential limitations such as the ability to detect small genetic

differences between clones and endophytic microbial contamination can sometimes alter profiles. During this project, additional research was carried out to investigate the feasibility and application of Simple Sequence Repeat (SSR) or microsatellites markers. These markers are different to RAPDs in a number of ways and research has shown that they are well suited for DNA fingerprinting and genetic analysis of potato (Provmetal. 1996; Kawchuck£tfa/. 1996).

SSRs are a highly informative, robust and reproducible DNA markers, that are co-dominant with Mendelian characteristics that are amplified from hypervariable repeated sequences located in regions of the genome. In comparison to RAPDs, SSRs have been shown to generate a higher degree of polymorphisms (Milbourne et al. 1996), and have been more reproducible and robust in different laboratories (Jones et al. 1997). However, the major drawbacks of developing SSRs are the labour input and expertise required to generate DNA libraries and screen for appropriate sequences for the species of interest. Much of this sequence information is available from DNA databases in the public domain and encompasses a wide range of species including potato (http://www.ncbi.nlm.nih.gov/).

This chapter describes preliminary work to evaluate Ta and the resolving system on PCR-based SSRs for potato. In addition, SSRs were also applied to differentiate 12 important cultivars to the Australian potato industry.

5.2 Methods and materials 5.2.1 General conditions DNA from the cultivars Coliban and Wontscab was used with primer sets SSR 1, SSR 2 and SSR 3 (Table 5.1).

Table 5.1. SSR primer sets used ( i )

Primer set Gene Repeat Sequence 5'-3' SSR1 Potato wound- (TGAAA)2

induced genes WINlandWIN2

(ATA)6

SSR 2 Potato inhibitor UK (T)12(A)9

ATTCTTGTT (TA)2CA(TA)7

SSR 3 Potato proteinase inhibitor pseudogene

(TA)23

SSR 24 Starch synthase (TCAC)m

SSR 25 Starch synthase (TCAC)m

(CTT)n ~TT\ : : v ' Primer sequences obtained from Kawchuck et al.

TGT TGA TTG TGG TGA TAA TGT TGG ACG TGA CTT GTA

TTC GTT GCT TAC CTA CTA CCC AAG ATT ACC ACA TTC

TGT ACT GGG GAG CCT CAA AG AAT TTT AAC CTC GTG ACA TGG G

TCT CTT GAG ACG TGT CAC TGA AAC TCA CCG ATT ACA GTA GGC AAG AGA TCT CTT GAC ACG TGT CAC TGA AAC TTG CCA TGT GAT GTG TGG TCT ACA A

(1996) and Provan et al. (1996).

All reactions were carried out in a final volume of 50 uL containing: 50 ng DNA, 0.4 uM primer, xl buffer (10 mM Tris-HCl pH 8.3, 3mM Mg2+, 50 mM KC1), 100 uM dNTPs, 1 U Tag DNA polymerase (Boehringer Mannheim, Germany).

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http://www.ncbi.nlm.nih.gov/


5.2.2 Effect of Ta The following Ta were evaluated 40, 45, 50, 55, 60 and 65 °C. Thermocycling was conducted in a Hybaid PCR Express (Hybaid, UK) with 35 cycles 94°C for 30 s, Ta °C for 1 min, and 72°C for 1 min. First denaturation cycle was for 1 min; last extension cycle was for 5 mins.

PCR product (20 \xL) was separated in a 5 mm thick 3.0 % agarose (Sigma) gel in xl TAE buffer with constant voltage at 80V for 3 hrs. containing 0.5 jag mL"1 ethidium bromide and visualised under ultra violet light (305 nm).

5.2.3 Evaluation of agarose type The aim of this experiment was to evaluate different agarose types for resolving SSR-PCR products with three primer sets and Ta (Table 5.2 and 5.3).

Table 5.2. Agarose types used. Agarose type Source / supplier Abbreviated term used in text Agarose Type I-A GIBCO Agarose-1000 Metaphor® agarose

Sigma Life Technologies FMC Bioproducts

Sigma Gibco Metaphor®

Table 5.3. Agarose type, SSR primer sets and Ta evaluated.

Agarose(1) Primer set(2)

SSR1 SSR 2 SSR 3 Sigma 45,50,55 45,50,55 50,55,60 Gibco 45,50,55 45,50,55 50,55,60 Metaphor® 45,50,55 45,50,55 50,55,60 Sigma: Gibco (1:1) 45,50,55 45,50,55 50,55,60 Sigma: Metaphor®(1:1) 45,50,55 45,50,55 50,55,60 1 ' Individual agaroses were tested at 3 % (w/v) and combinations are 1.5 % [(w/w)/v] (2) The Ta selected on the basis of the Tm of the primer design

5.2.4 Evaluation of agarose and polyacrylamide To test the resolution and sensitivity of gel electrophoresis, Metaphor® agarose was compared to polyacrylamide.

DNA samples from cultivars Redsen, Wontscab, Exton and Red Rascal were used with primer sets SSR 1 and SSR 2 (Table 5.1). PCR and thermocycling conditions were described above. The PCR product (15 ^L ) were run on both a 3 mm thick 3% Metaphor® gel in TAE buffer and 10% acrylamide gel (Biorad, USA) in TBE buffer, and stained with ethidium bromide.

5.2.5 Cultivar differentiation Primer sets of SSR 2, SSR 3, SSR 24 and SSR 25 were tested (Table 5.1) with 12 cultivars (Atlantic, Coliban, Sebago, Russet Burbank, Sequioa, Desiree, Exton, Crystal, Red La Soda, Denali, Pontiac and Kennebec line 7). PCR was conducted as described above and resolved on 3-4 % Metaphor® agarose gel.

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5.2.6 Case study: Effect of contaminated tissue cultures on SSR profiles While generating DNA fingerprints with RAPDs, the cultivar Simcoe produced aberrant bands (see Chapter 2 for full details). In this case study, three SSR primer sets SSR 1, 2 and 3 were used to fingerprint the samples that revealed aberrant RAPD markers and to confirm that these bands were not generated from the potato genome.

5.3 Results 5.3.1 Effect of Ta Generally, the number of PCR products decreased as Ta increased with all DNA and primer combinations tested (Fig. 5.1a, b, c). Generally, amplification with SSR 1 and 2 resulted in more intense bands at 100 to 300 bp as Ta increased with both cultivars Coliban and Wontscab.

With SSR 1, bands at 400 bp and greater disappeared as Ta increased to 50 and 55 °C but amplified a faint band of about 700 bp at 60 and 65 °C.. Amplification of identical bands at about 170 bp were detected with 50 and 55 °C. At 65 °C bands at about 170 bp were not detected.

With SSR 2 at Ta 40 to 60 °C, identical bands at about 200 bp were detected. Whereas only faint bands greater than 400 bp were detected at Ta 40 and 45 °C (Fig. 5.1a, b). At Ta 65 °C no amplification occurred.

SSR 3 amplified bands that ranged from about 100 to 800 bp with cultivars Coliban and Wontscab. As for SSR 1 and SSR 2, the number of PCR products decreased as Ta increased. The number of products had been reduced as Ta increased. A faint blurry band at 150 bp became visible at Ta 55 to 60 °C. At 65 °C with cultivar Coliban, a 500 bp band was produced where with cultivar Wontscab an additional 150 bp band was produced (Fig. 5.1c).

110


(a)

cv. Coliban

(b)

cv. Wontscab

SSR1 M40 45 50 55 60 65

SSR 2 40 45 50 55 60 65 M

SSR1 M 40 45 50 55 60 65

SSR 2 40 45 50 55 60 65 M

800 bp

500 bp

300 bp

200 bp

100 bp

(c) cv. Coliban cv. Wontscab

M 40 45 50 55 60 65 40 45 50 55 60 65 M

800 bp

500 bp

300 bp

200 bp

100 bp

SSR 3

1 Al- *S Mi^HL

800 bp

500 bp

300 bp

200 bp

100 bp

Figure 5.1. Evaluation of Ta with primer sets SSR 1, 2 and 3 and cultivars Coliban and Wontscab. Lane M = 100 bp ladder molecular weight marker (Amersham Pharmacia Biotech).

i l l


5.3.2 Evaluation of agarose type Better resolution, such as sharper bands and improved separation of 10 to 20 bp bands, was obtained with Gibco and Metaphor® agarose when compared to sigma agarose gels. This is clearly shown with SSR 1 and SSR 2 using Metaphor® and Sigma agaroses (Fig. 5.2).

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SSR 1 SSR 2 SSR 3 M 45 50 55 45 50 55 50 55 60 M

Metaphor - 800 bp

- 400 bp

- 200 bp

- 100 bp

SSR1 SSR 2 SSR 3 Sigma M 45 50 55 45 50 55 50 55 60 M

— 800 bp

— 400 bp

— 200 bp

— 100 bp Figure 5.2. Comparison of Metaphor® and Sigma agarose at three Ta and three primer sets. Lanes M = 100 bp ladder molecular weight DNA marker (Amersham Pharmacia Biotech).

5.3.3 Evaluation of agarose and polyacrylamide Both agarose and polyacrylamide were able to clearly resolve bands that differed by 10 bp apart (Fig. 5.3).

With SSR 1, a monomorphic band at approximately 175 bp was observed in all four cultivars screened. A polymorphic band at 160 bp was observed with cultivar Red Rascal. When resolved on polyacrylamide, the 175 bp monomorphic band was only observed in cultivars Redsen, Wontscab and Exton, and a 160 bp band was observed in cultivar Red Rascal. At 300 bp and above faint bands were observed in cultivars Exton and Red Rascal in the polyacrylamide gel but not in the agarose gel (Fig. 5.3 a, b).

With SSR 2 and agarose, all 4 cultivars could be distinguished by at least 1 to 3 distinct PCR products ranging from approximately 190 to 250 bp. On polyacrylamide, with SSR 2 the same major bands in the 190 to 250 bp range resolved on agarose were also detected. Faint bands that ranged at about 400 bp and higher were also resolved on polyacrylamide but were not on agarose (Fig. 5.3 a, b).

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(a)

Metaphor agarose SSR1 SSR 2

R W E R r M l M 2 R W E Rr

Polyacrylamide SSR 1 SSR 2

R W E R r M l M 2 R W E R r • 4 ' _ " . ,*' r<

, - r . rt i * . . - 1

< * • , " • , * ** -• * r • «. .

• • « ' • . 1

. - • '

II

JEWS*;:,. '- 5"* *

jBfffetihîTiM 1L liÊiffiMM&tfSM

^ ^ ^H l "D iWIWIM »III! iHI 1 IIJB |ffTWri»Tff3WWHWP^V*

(b)

Figure 5.3. Comparison of Metaphor and polyacrylamide with primer sets SSR 1 and SSR 2 and four cultivars Redsen (R), Wontscab (W), Exton (E) and Red Rascal (Rr). Lane Ml = 10 bp ladder (Gibco BRL) and M2 = 100 bp ladder molecular weight markers (Amersham Pharmacia Biotech).

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5.3.4 Cultivar differentiation All 12 cultivars could be distinguished by profiles generated collectively from SSRs 2, 3, 24, and 25, an example of results is only represented with SSR 2 (Fig. 5.4).

Primer set SSR 2 generated banding patterns from two to five bands ranging from about 165 to 260 bp. SSR profiles could clearly distinguish 10 of the 12 cultivars. Profiles for cultivars Russet Burbank (lane 4) and Sequoia (lane 5) were ambiguous and could not be distinguished with certainty (Fig. 5.4).

SSR 3 generated 3 to 9 bands ranging from about 150 to 650 bp and could distinguish nine of the 12 cultivars with the exception of cultivars Sebago, Sequioa and Kennebec (data not presented).

SSR 24, generated one or two bands that ranged from 200 to 260 bp, and could only distinguish three of the 12 cultivars (Fig. 5.5). Cultivars that produced ambiguous profiles were considered indistinguishable from each other: Atlantic/Russet Burbank/Desiree; Coliban/Sebago/Sequoia/Red La Soda/Denali; Exton/Crystal/Pontiac; and it was uncertain if Kennebec could have a unique profile or be indistinguishable from Atlantic/Russet Burbank/Desiree.

SSR 25 generated one or two bands that ranged from 350 to 390 bp and could distinguish three of the 12 cultivars. A unique profile was generated for cultivar Crystal. Cultivars that produced indistinguishable profiles from each other are: Atlantic/Coliban/Sebago/Russet Burbank/Sequoia/Desiree/Exton and Red La Soda/Denali/Pontiac/Kennebec (data not presented).

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Ml 1 2 3 4 5 6 7 8 9 10 11 12 M2 Ml

*•» " . " ' S p * * ^ 100 bp

Figure 5.4. Negative of DNA fingerprints generated for 12 cultivars with SSR 2: Atlantic (1), Coliban (2), Sebago (3), Russet Burbank (4), Sequoia (5), Desiree (6), Exton (7), Crystal (8), Red La Soda (9), Denali (10), Pontiac (11) and Kennebec (12). Lane Ml = 100 bp Ladder (Amersham Pharmacia Biotech) and lane M2 = 10 bp ladder molecular weight markers (Gibco BRL).

Ml 1 2 3 4 5 6 7 8 9 10 11 12 M2 Ml

_ •*" — 300 bp ^: ^ î ^r» m m m <m ^ m ^ •

— 200 bp

Figure 5.5. Negative of DNA fingerprints generated for 12 cultivars with SSR 24: Atlantic (1), Coliban (2), Sebago (3), Russet Burbank (4), Sequoia (5), Desiree (6), Exton (7), Crystal (8), Red La Soda (9), Denali (10), Pontiac (11) and Kennebec (12). Lane Ml = 100 bp Ladder (Amersham Pharmacia Biotech) and lane M2 = 10 bp ladder molecular weight markers (Gibco BRL).

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5.3.5 Case study: Effect of contaminated tissue cultures on SSR profiles Six primers (OPI-6, OPI-7, 2-10, OPI-14, 2-17, OPJ-18) revealed banding patterns that distinguished the two samples. Additional RAPD analysis with two tissue cultured samples showed that sample A (originally screened) was distinguished from the other three samples (Fig. 5.6). It was likely that either sample A was a mislabelled culture or the aberrant bands were the result from an endogenous bacterial contaminant which was later observed in the tissue culture media weeks after the DNA was isolated. SSR analysis was used and showed that all four tissue culture samples tested were indistinguishable (Fig. 5.7). This indicates that sample A was not mislabelled and the aberrant bands were likely to have been amplified from the tissue culture contaminant.

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OPI-14 2-17 OPJ-18 A B C D M A B C D M A B C D

1.5 kb

800 bp

500 bp

200 bp

Figure 5.6. RAPD profiles of four samples of cultivar Simcoe (A, B, C, D) amplified with RAPD primers OPI-14, 2-17 and OPJ-18. Sample A shows amplification of aberrant markers (arrowed). Lanes M = 100 bp ladder molecular weight marker (Amersham Pharmacia Biotech).

SSR1 SSR 2 SSR 3 A B C D M A B C D M A B C D

800 bp

500 bp

300 bp

200 bp

100 bp

Figure 5.7. SSR profiles of four samples of cultivar Simcoe amplified with primer sets SSR 1, 2 and 3. All four samples were indistinguishable. Lane Ml = 100 bp ladder (Amersham Pharmacia Biotech) and lane M2 = 10 bp ladder molecular weight markers (Gibco-BRL).

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5.4 Discussion Different Ta affects the product range and size of SSR markers. Provan et al. (1996) who used Ta for SSR 1, SSR 2 and SSR 3 of 48, 50 and 60 °C respectively. Our results confirm this observation and demonstrate that at 55 °C amplification of expected product size is found allowing a single Ta to be used for the three primer sets.

Testing different agarose types have shown to influence the resolution of SSR banding patterns. Although standard grade Sigma (Sigma, USA) agarose is adequate for resolving RAPDs, it could not resolve bands as clearly and sharply as Metaphor® agarose. Metaphor agarose was designed for fine resolution of DNA bands especially fragments ranging from 20 to 800 bp, such as SSRs (Cregan and Quigley 1997). Technical information provided by the manufacturer (www.bioproducts.com), claims that the resolution of a 4 % gel equates to a 8% polyacrylamide gel and up to 4 bp differences can be separated. We have found that bands less than 10 bp can sometimes be difficult to resolve depending on the complexity of the band patterns.

When Metaphor® agarose was compared to polyacrylamide, some differences in resolution and detection were observed. For example, with agarose and an extra band were detected with cultivar Red Rascal that was not detected on polyacrylamide. The reason for this is unknown but has been reported to be a limitation of using high resolution agarose (Cregan and Quigley 1997). As higher molecular weight bands were detected with polyacrylamide and not on with agarose, this indicates that the sensitivity of acrylamide was greater than agarose for bands in this range, however, these bands may not be true loci. Sequencing these products will confirm the whether they are really SSRs and therefore true loci. Nevertheless, the technical simplicity of using agarose was advantageous and satisfactory for resolving some SSR fragments.

SSRs have been demonstrated to be useful for differentiating the 12 potato cultivars tested. This is an extremely valuable result as the 12 cultivars tested represent the majority of cultivars presently grown in Australia (Blackmore 1998). Only primer set SSR 2 are required to differentiate 10 of the 12 cultivars and any other of the SSR primer sets tested can be used to distinguish the remaining cultivars. This compares favourably with Provan et al. (1996), where 18 cultivars could be differentiated with SSR 2. It is highly possible that SSR 2 can distinguish more than 12 Australian grown cultivars but only further work can confirm this.

Our SSR system shows promise, but is not ready for routine commercial use as more testing is required on polyacrylamide gels and silver staining. This could improve the system significantly and result in a procedure that is reliable and reproducible between laboratories (Jones et al. 1996).

In the case study, it was demonstrated that RAPD analysis can be affected by endogenous tissue culture contaminants because of the arbitrary design of primers. The use genome specific SSR primers has been demonstrated to overcome this problem.

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http://www.bioproducts.com


5.5 References Blackmore, K. (1998). Operations Report. In: Victorian Seed Potato Authority annual

general meeting report, September 1998. ViCSPA Inc.

Cregan, P.B. and Quigley, C.V. (1997). Simple sequence repeat DNA marker analysis. In: DNA Markers (Eds. G. Caetano-Anolles and P.M Gresshoff). pp. 173-185. Wiley-Liss, Inc.

Jones, C.J., Edwards, K.J., Castaglione, S., Winfield, M.O., Sala, F., Wiel van de, C , Bredemeijer, G., Vosman, B., Matthes, M., Daly, M., Brettschneider, R., Bettini, P., Buiatti, M., Maestri, E., Malcevschi, A., Marmiroli, N., Aert, R., Volckaert, G., Rueda, J., Linacero, R., Vazquez, A. and Karp, A. (1997). Reproducibility testing of RAPD, AFLP and SSR markers in plants by a network of European laboratories. Molecular Breeding 3,381 -90.

Kawchuck, L.M., Lynch, D.R., Thomas, J., Penner, B., Sillito, D. and Kulcsar, F. (1996). Characterization of Solanum tuberosum simple sequence repeats and application to potato cultivar identification. American Potato Journal 73, 325-35.

Provan, J., Powell, W. and Waugh, R. (1996). Microsatellite analysis of relationships within cultivated potato {Solanum tuberosum). Theoretical and Applied Genetics 92,1078-84.

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6.0 Investigation of genetic variability in clonal variants 6.1 Introduction

The potato tissue culture collections at IHD have a number of lines that are maintained as "clonal variants". The history of these introductions is vague and

there is little understanding on how they were established. There is a common belief that some of these introductions have different genetic attributes and phenotypic differences in the field. It is possible that some of the clonal variants have resulted from somatic mutations, genetic drift or DNA methylation. It can also be possible that these introductions are genetically identical and just have been duplicated in the collection.

In this study, RAPD and SSR analysis were used to investigate if clonal variants of five cultivars can be distinguished.

6.2 Methods and materials 6.2.1 Plant material Cultivars (and clonal variants) Atlantic (Line A and B), Russet Burbank (ex.Tas 95., Tooheys, British Columbia, Line A Ruen, and Netted Gem), Kennebec (Line 2, 7 and B), Sebago (Line C, D, and New Brunswick), Delaware (Line A, B, D, E, and G) were obtained from the cultivar collection at IHD - Knoxfield.

6.2.2 RAPD and SSR analysis DNA isolation, PCR conditions and primer sequences have been described in Chapter 2 (RAPD) and Chapter 5 (SSR).

6.3 Results 6.3.1 RAPD analysis DNA fingerprints generated from 20 to 26 primers were used (Table 6.1). Up to a total of 150 bands showed that clonal variants of cultivars Russet Burbank, Sebago, Atlantic, Delaware and Kennebec, were indistinguishable. An example of DNA fingerprints obtained with cultivar Kennebec is presented (Fig.6.1).

121


Table 6.1 . Results from comparing RAPD profiles in cultivar clones. Cultivar - clone No. RAPD primers used Result Atlantic -Atlantic -

Line A LineB

26 Indistinguishable

Kennebec - Line 2 Kennebec - Line 7 Kennebec - Line B

26

Russet Burbank Russet Burbank Russet Burbank Russet Burbank Russet Burbank

Tas. '95 Line A Ruen British Columbia Netted Gem Tooheys

26

Indistinguishable

Indistinguishable

Sebago - Line C Sebago - Line D Sebago - New Brunswick

20-26

Delaware - Line A Delaware - Line B Delaware - Line D Delaware - Line E Delaware - Line G

20

Indistinguishable

Indistinguishable

OPD-03 2 7 B

OPD-08 OPI-06 2 7 B M 2 7 B

OPI-18 2 7 B

!*fS AJiflftfcfi'&Si.-S *diMi)liBte&tti

Figure 6.1. DNA fingerprints of clonal variants of Kennebec Line 2 (2), Kennebec Line 7 (7) and Kennebec Line B (B) with primers OPD-03, OPD-08, OPI-06 and OPI-18 (Operon Technologies). Lane M = Kilobase DNA molecular weight marker (Amersham, Pharmacia Biotech).

122


6.3.2 SSR analysis DNA fingerprints generated from 12 SSR primer sets amplified about a total of 40 to 50 alleles. Clonal variants of cultivars Russet Burbank, Sebago, Atlantic, Delaware and Kennebec, were found to indistinguishable with major bands observed (Table 6.2). Some faint bands were amplified with SSR 6 in clonal variants of Russet Burbank, Sebago and Delaware. An example of DNA fingerprints obtained with cultivar Russet Burbank is presented (Fig. 6.2).

Table 6.2. Results from comparing SSR profiles in cultivar clones. Cultivar - clone No. SSR primers used Result Atlantic - Line A Atlantic - Line B


Kennebec - Line 2 Kennebec - Line 7 Kennebec - Line B


Russet Burbank - Tas. '95 12 Indistinguishable Russet Burbank - Line A Ruen Russet Burbank - British Columbia Russet Burbank - Netted Gem Russet Burbank - Tooheys Sebago - Line C 12 Indistinguishable Sebago - Line D Sebago - New Brunswick Delaware - Line A 12 Indistinguishable Delaware - Line B Delaware - Line D Delaware - Line E Delaware - Line G

123


M 1 2 3 4 5

800 bp ft "-- »& ;,,- &§* "-- »& ;,,- &§*

100 bp

Figure 6.2. SSR profiles generated with SSR 6 for clones of cultivar Russet Burbank Ex. Tas. 95 (1), Russet Burbank Line A Ruen (2), Russet Burbank British Columbia (3), Russet Burbank Netted Gem (4), Russet Burbank Tooheys (5). Potential polymorphisms indicated with arrows. Lane M = 100 bp ladder molecular weight marker (Amersham, Pharmacia Biotech).

6.4 Discussion RAPD analysis was not able to distinguish between the clonal variants of the five cultivars used in this study. Sosinki and Douches (1996), were able to distinguish Burbank and its sport Russet Burbank by one band, with one primer from a total of 29 primers tested. In another study with Russet Burbank clonal variants (Demeke et al. 1993), Russet Burbank Idaho D and Russet Burbank White Skin were able to distinguish the two lines with one of 20 primers tested. Clonal variants of the cultivar Viking have been distinguished with one of 20 primers tested however, when tested with clonal variants Sebago, Superior, Norgold and Norland they could not distinguish the lines (Demeke et al. 1993).

While RAPD analysis have been useful to distinguish between clonal variants of Russet Burbank and Viking it is likely that this is more fortuitous than by good experimental design, as RAPD primers are 10-mers and only bind to a very small part of the genomic DNA. To increase the chance of detecting polymorphisms more primers need to be screened.

Milbourne et al. (1997) have reported that SSRs produce more polymorphisms than RAPDs and Amplified Fragment Length Polymorphism (AFLP) in potato and could be useful in detect differences in clonal variants. In the only published work screening clonal variants with SSRs, Kawchuck et al. (1996), found that 7 clonal variants of Russet Burbank could not distinguished.

The faint bands that were amplified using SSR 6 with clonal variants of Russet Burbank, Sebago and Delaware are unlikely to be true polymorphisms as they are not in the expected size range and are most likely due to non-specific annealing. This can

124


only be determined by sequencing these products. SSR primers may not be suitable for distinguishing clonal variants as the polymorphism may not exist in the gene.

Another possible limitation with microsatellites for detecting polymorphisms in clonal variants could be the resolution of agarose gels. The use of high resolution Metaphor® agarose has demonstrated to be useful in assessing SSRs in potato, however, the resolution and accuracy in determining molecular size of bands and the sensitivity for detection of polymorphisms is greater with polyacrylamide sequencing gels.

Inter Simple Sequence Repeats (ISSRs) have detected variation in potato plants regenerated from tissue culture (Albani and Wilkinson 1998), but have not been used to study clonal variants and this approach is warranted.

The detection of clonal variants has proven to be difficult using marker systems such as RAPDs and SSRs, and detailed field studies to assess the agronomic performance with clonal variants may be required to determine if the differences are real or perceived.

6.5 References Albani, M.C. and Wilkinson, M.J. (1998). Inter simple sequence polymerase chain

reaction for the detection of somaclonal variation. Plant Breeding 111, 573-575.

Demeke, T., Kawchuck, L.M. and Lynch, D.R. (1993). Identification of potato cultivars and clonal variants by random amplified polymorphic DNA analysis. American Potato Journal 70, 561-9.

Kawchuck, L.M., Lynch, D.R., Thomas, J., Penner, B., Sillito, D. and Kulscar, F. (1996). Characterization of Solanum tuberosum simple sequence repeats and application to potato cultivar identification. American Potato Journal 73, 325-335.

Milbourne, D., Meyer, R., Bradshaw, J.E., Baird, E., Bonar, N., Provan, J., Powell, W., Waugh, R. (1997). Comparison of PCR-based marker systems for the analysis of genetic relationships in cultivated potato. Molecular Breeding 3, 127-136.

Sosinski, B. and Douches, D.S. (1996). Using polymerase chain reaction-based DNA amplification to fingerprint North American potato cultivars. HortScience 31, 130-3.

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7.0 Genetic relationship between potato cultivars 7.1 Introduction

Comprehensive knowledge of the genetic relationships of potato cultivars grown in Australia is lacking. This knowledge can assist in breeding programs to

acknowledge diverse parental lines and also estimate the level of diversity that exists in the germplasm collection. The RAPD markers used for DNA fingerprinting (Chapter 2) were also useful for examining the genetic relationships between these cultivars.

7.2 Methods and materials Cultivars and RAPD-PCR conditions are described (Chapter 2). RAPD markers were scored for the presence and absence of bands and transcribed into binary format (1,0 respectively). The primers that amplified faint, non-consistent or complicated band patterns were not used for cluster analysis. A similarity matrix using the simple matching coefficient was calculated and cluster analysis was done using Genstat v.5.0.

7.3 Results From 17 useful primers, a total of 210 bands were scored from the potato cultivars, clonal variants and one tobacco outgroup (Table 7.1). An average of 12.3 bands per primer were obtained, with molecular sizes ranging from 0.3 to 2.5 kb. The highest number of bands (19) was obtained with primer OPI-06 while the lowest number (6) was obtained with OPB-12. Sixty bands were found to be unique to tobacco which contributed to a higher number of bands assessed.

126


Table 7.1. RAPD primer sequences and number of bands scored for potato. Primer1 Sequence (5'-3') Total number 1 Polymorp

of bands scored bands 2-05 CAA TCG CCG T 8 8 2-07 GAA CGG ACT C 15 15 2-10 GGT GAT CAG G 18 18 2-14 GTC CCG TGG T 13 13 2-17 CAC AGG CGG A 9 9 2-19 GGA CGG CGT T 17 17 OPD-03 GTC GCC GTC A 12 12 OPD-08 GTG TGC CCC A 17 16 OPI-06 AAG GCG GCA G 19 19 OPI-07 CAG CGA CAA G 11 11 OPI-18 TGC CCA GCC T 12 11 OPJ-04 CCG AAC ACG G 15 15 OPJ-06 TCG TTC CGC A 11 11 OP J-10 AAG CCC GAG G 9 9 OPB-01 GTT TCG CTC C 10 9 OPB-06 TGCTCTGCCC 8 8 OPB-12 CCT TGA CGC A 6 6

Primers obtained as either custom synthesised oligonucleotides from former Bresatec FAPD 10-mer kit (Geneworks P/L, SA, Aust.) or from Operon Technologies Inc. (USA).

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Overall the genetic similarity ranged from about 0.29 to 0.91, with tobacco clustering independently and showing the least similarity at 0.29. The genetic similarity of the potato cultivars ranged from 0.69 to 0.91, indicating low genetic variation and very close relationships between the Australian, European and North American cultivars. No distinct separation between cultivars based on the country of origin was found.

Cluster analysis, illustrated by a dendrogram revealed very close genetic relationships with many cultivars that share part of the same parentage or pedigree (Fig. 7.1). For example, White Rose and Russet Burbank have the same female parent Early Rose; Riverina Russet and My Fry have Maris Piper as a male parent; Pontiac and Red La Soda have the same parents Triumph and Katahdin; and Sequoia and Sebago have Katahdin as a male parent. Some very close cultivar relationships were revealed with a sibling and a parent. For example, Crispa and Knox where Knox is the female parent of Crispa; Coliban and Kennebec where Kennebec is the female parent of Coliban; Dynamite and 85-30-12 where 85-30-12 is the female parent of Dynamite; Wilwash, 92-19-10 and Wintergem where Wilwash is the male parent to both 92-19-10 and Wintergem; and Dalmore and Atlantic where Atlantic is the female parent of Dalmore. Not all relationships shown on the dendrogram could be correlated to known pedigree, partly because breeding history was either unknown or not available.

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Tobocco PCongo

Nooksock Nicolo-

Pinkeye RCReyol

Potrontt Rubylou Desire* Simcoe

NorpchA Evont

Shepody Kipfler Crispo

Knox Bntje

Codimo Crystal Oenoli Whitu

Snowchip RonRus Riv Rut

WRose RBRuenA

R8TH RBNG

R8 Tos95 RBBC Oslo A KingEd

Donnelly Foxton

Bsmork Wonlscob

Col B Ken 2 Ken 7 Ken B Cotoni

Trent-88-59-12-

ToolDel-Shine-

Mondial MacRus Winlpek Uortifry-Nodine-

85-30-12-Dynomite Dolmore

AUA All 8

Wilslore Exton

Snowgem Spunto

WinCem Wilwash

92-19-10 SeqC-

SebB SebNB BIsonB Bremer Redsen

Onto Rideou

PontioeB RedSodo

u

J

tf

- I 1 I 1.0 0.9 0.8 0.7 0.6 0.5

Figure 7.1. Dendrogram showing genetic relationships of 64 potato cultivars, clonal variants of Russet Burbank, Kennebec, Sebago, Atlantic and a tobacco outgroup.

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7.5 Discussion The dendrogram has revealed many cultivar relationships to correlate with known breeding history. This result can be indicative of the heritability and stability of the RAPD markers used. Quiros et al. (1993), reported that RAPD markers segregated with expected ratios in potato and are therefore of use in potato genetics, breeding and evolutionary studies. The genetic relationships shown between Russet Burbank and White Rose and also between Pontiac and Red LaSoda, were found to be consistent with previous work reported (Sosinki and Douches 1996). Other studies have also successfully used RAPD analysis to examine genetic relationships with potato cultivars using 84 loci (Hosaka et al. 1994), 43 loci (Sosinki and Douches 1996), and 158 loci (Demeke ef a/. 1996).

The close genetic relatedness found between the potato cultivars in this study indicated a narrow genetic base. The origins of many modern North American potato cultivars have been restricted to a small number of early introductions which have resulted in a lack of genetic variability (Mendoza and Haynes 1974; Demeke et al. 1996). Australian cultivars were found to be closely related to North American and European cultivars which implies the probable source of Australian grown germplasm.

RAPD analysis has shown to be useful in examining genetic relationships and indicated the level of genetic diversity among the potato cultivars used in this study. Molecular markers are useful tools for breeding and genetic applications in potato.

References Demeke, T., Lynch, D.R., Kawchuck, L.M., Kozub, G.C. and Armstrong, J.D. (1996).

Genetic diversity of potato determined by random amplified polymorphic DNA analysis. Plant Cell Reports 15, 662-7.

Genstat 5.0 Committee (1987). Genstat 5.0 reference manual. Clarendon Press, Oxford.

Hosaka, K., Mori., M., and Ogawa, K. (1994). Genetic relationships of Japanese potato cultivars assessed by RAPD analysis. American Potato Journal 71, 535-46.

Mendoza, H.A. and Haynes, F.L. (1974). Genetic relationship among potato cultivars grown in the United States. HortScience 9, 328-30.

Quiros, C.F., Ceada, A., Georgescu, A, and Hu, J. (1993). Use of RAPD markers in potato genetics: segregations in diploid and tetraploid families. American Potato Journal 70, 35-42.

Sosinski, B. and Douches, D.S. (1996). Using polymerase chain reaction-based DNA amplification to fingerprint North American potato cultivars. HortScience 31, 130-3.


8.0 Detection of bacterial contaminant Baccillus circulans ('White Ghost') using PCR 8.1 Introduction T ^ V T A fingerprinting of potato cultivars sourced from tissue culture and field \-J J .N -/x.bank collections (see Chapter 2), revealed an aberrant 500 bp fragment in 12 cultivars but only with tissue culture material and RAPD primer 2-10. The tissue cultured plants from which DNA was extracted were later found with a bacterial contaminant, Bacillus circulans (aka. White Ghost), which was symptomless at the time of DNA isolation. The hypothesis was that the aberrant band originated from this endogenous contaminant and therefore was not a true polymorphism from the potato cultivars.

The origin of this band was of interest as it could have ramifications to the potato cultivar germplasm collection. A molecular approach was used to resolve the origin of the aberrant band and information generated was used to develop a diagnostic tool.

8.2 Methods and materials 8.2.1 Isolation of bacterial contaminant Leaves and stems from tissue culture plants were crushed in sterile distilled water, spread either onto nutrient agar and incubated at room temperature for several days until colonies grew. Single colonies were streaked and subcultured at least three times before being used for DNA isolation and PCR experiments.

The isolate was assessed for morphology, gram-staining and tested for growth on a range of bacteriological media at 30 and 46 °C. It was also tested for anaerobic growth at 30 °C.

8.2.2 Cloning and sequencing of RAPD products The aberrant 500 bp fragment generated with primer 2-10 (2-IO500) from was excised from an agarose gel and purified using Bresa-clean DNA purification kit (Geneworks, Sth. Aust.). The 500 bp fragment from cultivars Onka and Trent. A 750 bp fragment generated with primer 2-17 (2-17750) from cultivar Riverina Russet was also isolated for a control.

PCR products were cloned using TA overhang method into pGEM-T Easy vector (Promega, USA), and transformed into E.coli JM109 competent. Selected colonies were grown on LB media containing 100 u.g mL"1 ampicillin.

The 500 and 750 bp inserts were sequenced using the ABI-Prism system (Perkin Elmer Applied Biosystems, USA).

8.2.3 Database searches for sequence similarity The BLAST and FAST search programs were used to search for homologous DNA sequences to 2-lOsoo and 2-17750, and LALIGN was used for sequence alignment (ANGIS).

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8.2.4 SCAR primer design SCAR primers (Paran and Michelmore 1993) and internal primers for the 2-105oo fragment were designed using PRIME (ANGIS) (Table 8.1).

Table 8.1. SCAR and internal primer sequences. Primer name Sequence Expected

product size (bp) S2-10 F GGT GAT CAG GGT AC A TAA CAT T 500 S2-10 R GGT GAT CAG GCA GCA CGC SE2-10 F GGT GAT CAG GGT ACA TAA CAT TCG 500 SE2-10 R GGT GAT CAG GCA GCA CGC GTG AAA IS2-10 F GAT GCT TCA AAA AAC AGA AGC C 300 IS2-10R CAC GAT TCT CAA CCA AGC C 2-101 GGT GAT CAG G n/a (1) Original random 10-mer primer (Geneworks, Sth. Aust.)

8.2.5 Effect of Ta Primer set S2-10 and IS2-10 were tested at 55, 60, 65, 70 °C Ta. In addition, primer sets S2-10 and SE2-10 were tested at 55, 56, 58, 61, 66, 67 °C Ta. All experiments were performed using positive (ex. tissue culture) and negative (ex. field) potato DNA samples from cultivar Onka. PCR conditions have been described (Chapter 2).

8.2.6 PCR primer specificity To screen the specificity of S2-10, SE2-10 and IS2-10, a range of DNA types were tested including mammalian, avian, fungal and bacterial.

8.2.7 Dot Blot hybridisation Dot Blot hybridisation was used to confirm sequence homology of genomic DNA types and compared to results from PCR. Fragment 2-lOsoo (BC500 probe) was labelled and hybridisation and X-ray development of nylon membrane was done with using the DIG system (Boehringer Mannheim, Germany).

8.2.8 PCR-RFLP analysis PCR products amplified with S2-10, SE2-10 and IS2-10 were subject to restriction endonuclease digests. Restriction enzymes were selected by using WAG-Mapplot (ANGIS) to scan for unique cutting sites and Bse Gl and Mse 1 were selected.

8.2.9 Sensitivity of detection The threshold for the detection by PCR of B. circulans was determined with a suspension culture. The concentration of cells was estimated by the relationship between ODeoo and the number of cfu mL"1. Bacterial suspensions from one day old cultures were adjusted to 108 cfu mL"1 and 10-fold dilutions prepared before and after DNA preparation. For PCR reactions, 1 mL of each dilution was boiled for 5 min, cooled on ice and a 1 uL aliquot used as template DNA with primers SE2-10 and IS2-10.

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8.3 Results 8.3.1 Bacterial isolate identification The isolate was found to grow on brain heart infusion agar at 30 °C and 46 °C and on orange serum agar (OSA) at 30 °C but with scanty growth at 46 °C. There was no anaerobic growth at 30 °C. The isolate was tested using an API 50 CHB test kit (bioMerieux, France). The isolate was identified as Bacillus circulans with 97.2 % probability.

8.3.2 Sequence alignment and database searches The cloned 2-lOsoo fragments from cultivar Onka and Trent were found to have 100 % sequence homology, and therefore were identical.

The 2-IO500 fragment was found to have no sequence homology to potato or plants sequences in the databases searched. There was partial homology of the 2-IO500 fragment to a number of different microrganisms (Table 8.2).

The 2-IO750 fragment was found to have partial sequence homology to genes in tomato and potato (Table 8.2).

Table 8.2. Examples of sequence database similarity searches for 2-IO500 and 2-10 750 RAP D fragments 1 using BLASTN and FASTA Database Search

type Number positive nucleotides (Identity %) Search

type 2-IO500 2-10750

E. coli BLASTN E. coli 27/36 (75) none none Bacillus subtilus

BLASTN B. subtilus 28/37 (75) none none

Haemophil us influenza

BLASTN Flu virus 32/48(66) none none

Bacterial Genebank

BLASTN Rickettsia 60/105 (57) prowazeki

Synechocystis 39/55 (70) sp.

Bacterial Nucleic

BLASTN Rickettsia 60/105 (57) prowazeki

Synechocystis 39/55 (70) sp.

Genebank BLASTN None found Human 37/50 (74) EMBL FASTA None found Potato 476 (58) EMBL FASTA None found Tomato 126 (78.6)

8.3.3 Evaluation of Ta With S2-10 and IS2-10 a Ta of 65 °C produced the least number of unspecific amplification products in positive control and B. circulans (Fig. 8.1a). With SE2-10 a Ta of 67 °C produced a 500 bp fragment with little unspecific amplification (Fig. 8.1b). With S2-10 and IS2-10, non-specific amplification was observed with the negative control sample (non-White Ghost contaminated potato DNA) but was reduced as Ta increased. No 500 bp fragment was amplified with the negative control sample with S2-10 and SE2-10 at all Ta tested.

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+ + Primer IS2-10

+ + + Primer S2-10

Figure 8.1a. Evaluation of Ta on amplification of S2-10 and IS2-10 fragments with positive (+) and negative (-) DNA samples. Lane M = 100 bp ladder molecular weight marker (Amersham Pharmacia Biotech).

+ + + + + +

M 55 56 58 61 66 67 55 56 58 61 66 67

t —!«, Jflg r-g" t~-r - , T - , -*. fc— -» - *-C -An- £ f c

I - _ - •* • 1

•4

500 bp

Figure 8.1b. Evaluation of Ta on amplification of SE2-10 fragment with positive (+) and negative (-) DNA samples. Lane M = 100 bp ladder molecular weight marker (Amersham Pharmacia Biotech).

8.3.4 PCR primer specificity Screening S2-10 with different DNA types specifically amplified the expected 500 bp fragment with a number of samples, for example contaminated tissue cultures of potato and tobacco and B. circulans (Fig. 8.2a. see samples 3, 4 and 6). Amplification of expected 500 bp fragment and some non-specific products occurred with a number of samples, such as, Botrytis, Lichenostomus melanops (Yellow Tufted Honey Eater), Erwinia and Agrobacterium (Fig. 8.2a. see samples 8, 9, 17 and 19). IS2-10 primers amplified the expected 300 bp fragment with a number of DNA samples, for example contaminated tissue cultures of potato and tobacco and B. circulans (Fig. 8.2a. see samples 3, 4 and 6). Amplification of a non-specific product occurred with Erwinia and Agrobacterium (Fig. 8.2a; see samples 17 and 19). While the amplification product for Agrobacterium appears to be 300 bp, gel image analysis indicates it is approximately 280 bp.

134


J _ _2_ 3 4 5 6 7 M I S I S I S I S I S I S I S

500 bp

300 bp

~-^^lkîlA:^mMm 10 11 12 13 14

500 bp

300 bp

15_ 16. 17. 18_ 19_ 20_ M I S I S I S I S I S I S

500 bn 300 bp tasr- • # ? ^-U-jg^SS^ M !

i i

Figure 8.2a. Representative gels of the specificity of amplification with IS2-10 (I) and S2-10 (S) primers with a range of DNA types. DNA samples are: 1 = +ve control cv. Onka; 2 = -ve control; 3 = 5 . circulans; 4 = Tobacco tissue culture (TC); 5 = Tobacco glasshouse (GH); 6 = potato TC; 7 = potato GH; 8 = Botrytis sp.; 9 = Yellow Tufted Honey eater (avian); 10 = Hydrangea TC; 11 = Hydrangea GH; 12 = Hosta TC; 13 = Macropeadia TC; 14 = Macropeadia gh; 15 = Santalum lanceolatum; 16 = Blue Mondo Grass TC; 17 = Erwinia sp.; 18 = Clavibacter sp.; 19 = Agrobacterium sp.; 20 = Daphne TC; M = 100 bp ladder molecular weight marker (Amersham Pharmacia Biotech).

135


With SE2-10 primers, amplification of the expected 500 bp fragment occurred with the positive control, B .circulans and Hosta and potato tissue culture material (Fig. 8.2b. see lanes 1, 11 and 13). In addition, the 500 bp fragment has been amplified from a number of bacteria isolated from plant tissue cultures (tentatively identified as B. circulans) (lanes 2 to 8). Weak amplification occurred with E. coli, Botrytis and Hydrangea tissue culture (lanes 8, 10 and 12). No amplification occurred with yellow tufted honey eater.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 M

Figure 8.2b. Specificity of amplification with SE2-10 primers. DNA samples are: 1 = +ve control cultivar Onka; 2 - 8 = unidentified bacterial tissue culture contaminants; 9 = E. coli; 10 = Botrytis sp.; 11 = potato tissue culture (TC); 12 = Hydrangea TC; 13 = Hosta TC; 14 = Yellow Tufted Honey Eater (avian); M = 100 bp ladder molecular weight marker (Amersham Pharmacia Biotech).

8.3.5 Dot blot Dot blot hybridisation produced strong signals with the positive control plasmid and B. circulans . Weak ambiguous signals were detected with unidentified bacterial contaminants, E. coli, Agrobacterium sp. and Botrytis sp. (Table 8.3; Fig. 8.3). No signal occurred with the remaining samples.

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A B C D E

# * * " •

6

* # • •

Figure 8.3. Dot blot hybridisation using BC500 probe with a range of DNA types. DNA samples are: Al = B. circulans; Bl , CI, Dl = unidentified bacterial contaminants; El = E. coli; A2 = Agrobacterium; B2 = Clavibacter, C2 = Bacterial wilt; D2 = Erwinia; E2 = Tomato Big Bud phytoplasma; A3 = Apple scab; B3 = Pear scab; C3 = Botrytis; D3 = potato tissue culture (TC); E3 = potato glasshouse (GH); A4 = tobacco TC; B4 = tobacco GH; C4 = Hydrangea TC; D4 = Hydrangea GH; E4 = Hebe TC; A5 = Hosta TC; B5 = Daphne TC; C5 = Macropeadia TC; D5 = Blue Mondo Grass TC; E5 = Yellow Tufted Honey Eater (avian); A6, B6, C6, D6 = +ve control plasmid DNA.

8.3.6 PCR-RFLP analysis PCR-RFLP analysis of products amplified with S2-10 was used to further test samples that produced the 500 bp fragment. Sequence analysis of the 2-IO500 fragment showed it contained unique restriction sites for Bsr Gl and Mse 1. Fragment sizes of 180 and 320 bp with Bsr Gl and 100 and 400 bp with Mse 1 are predicted after digesting DNA amplified with S2-10 and SE2-10. This is the diagnostic cut to distinguish PCR products of B. circulans from other 500 bp products.

Predicted PCR-RFLP profiles were produced for positive control, and tissue cultures of tobacco, potato, Hydrangea, Hosta, and B. circulans (Fig. 8.4., lanes 1, 2, 3, 4, 5 and 12). With unidentified bacteria isolated from potato tissue cultures the predicted result was obtained with all samples, although some were only partial digests (lanes 13 to 18). The sample in lane 15 contains excessive RNA and no DNA and in lane 18 there Was non-specific amplification. PCR-RFLP profiles for Botrytis, E. coli, Clavibacter, Santalum and Yellow Tufted honey Eater could be distinguished from the positive control sample, B. circulans, and unidentified bacteria isolated from potato tissue cultures. With Blue Mondo Grass the result was inconclusive.

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Table 8.3. Summary of results from screening different DNA types. DNA type IS2-10 S2-10 SE2-10 Dot RE

primers primers primers blot digest B. circulans + + + + + E. coli - ? - +? -

Agrobacterium - - - ? nt Clavibacter - - nt - nt Erwinia - - nt - nt Ralstonia solanacearum - - nt - nt Phytoplasma (Tomato Big Bud) - - nt - nt Apple scab - - nt - nt Pear scab - - nt - nt Botrytis + ? +? + -

Tobacco TC + + nt - ? Tobacco GH - - nt - nt Potato TC + + + + + Potato GH + ? nt - nt Hosta TC + + + - + Hydrangea TC + +? + - +? Hydrangea GH + + nt - nt Begonia TC - - nt nt nt Begonia GH - - nt nt nt Daphne TC - - nt nt nt Daphne GH - - nt nt nt Macropidia TC +? - nt - nt Macropidia GH - - nt nt nt Hebe - - nt - nt Raspberry - - nt nt nt Blue Mondo Grass - - nt - -

Santalum lanceolatum - - Nt nt nt YTHE - - ? - -

Honeybee - - nt nt nt Ratus arsus - - nt ? = ambiguous result nt = not tested

138


M 1 2 3 4 5 6 7

Bsr Gl

8 9 M 10 11 12 13 14 15 16 17 18

SdN^W*?'"? #*iKr 320bp

M 1 2 3 4 6 7

'..¥?.'.-

Mse 1

9 M 10 11 12 13 14 15 16 17 18

'Z3S7.**

•^fltHt ^ j j & s M ^ * # b f i i k ^ ^ a . - .. i«*.".,".-aarfr_ . « -Vs _ „ _ _ _ _ _ _ _ ^ _

Figure 8.4. PCR-RFLP analysis with restriction enzymes 5 j r Gl and Mse 1, of SCAR-PCR products from primer set S2-10. DNA samples are: 1 = +ve control cv. Onka; 2 = tobacco TC; 3 = potato TC; 4 = Hydrangea TC; 5 = Hosta TC; 6 = Santalum lanceolatum; 7 = Blue Mondo Grass TC; 8 = YTHE (avian); 9 = Botrytis; 10 = Clavibacter, \\=E. coli; 12 = B. circulans; 13-18 = unidentified bacterial tissue culture contaminants; M = 100 bp ladder molecular weight marker (Amersham Pharmacia Biotech).

8.3.7 Sensitivity of detection For DNA preparations diluted after boiling, the sensitivity threshold occurred at 103

cfu mL"1 with IS2-10 and SE2-10 (Fig. 8.5). With DNA preparations diluted before boiling, the sensitivity threshold was one order of magnitude less than for samples diluted before boiling (Fig. 8.6).

175 bp

400 bp

100 bp

139


IS2-10 SE2-10 M 108 107 106 105 104 103 102 101 M 108107 106 105 104 103102 101

300

bp

Figure 8.5. Sensitivity threshold of IS2-10 and S2-10 primers for detecting B. circulans with suspension diluted after boiling DNA preparation. Lane M = 100 bp ladder molecular weight marker (Amersham Pharmacia Biotech).

IS2-10 SE2-10 M 108107 106 105 104 103 102 101 M 108107 106105 104 103 102 101

300 - _ r I00

Figure 8.6. Sensitivity threshold of IS2-10 and S2-10 primers for detecting B. circulans with suspension diluted before boiling DNA preparation. Lane M = 100 bp ladder molecular weight marker (Amersham Pharmacia Biotech).

8.4 Discussion A range of endophytic microorganisms have been identified from plant tissue cultures for example, species of; Agrobacterium, Bacillus, Corynebacterium and Pseudomonas (Cassels 1991). The bacterium isolated from the plant tissue cultures in the present study has been identified as B. circulans using a range of microbiological tests. This is one of the first examples where this species has been identified from plant tissue cultures (ref ex. Cassels 1997). This is suprising considering that B. circulans is a commonly occurring soil-borne organism (Jensen per.com.).

The detection of the 500 bp aberrant DNA fragment with primer 2-10 could have had ramifications if the data was misinterpreted and not investigated. Interpreting the DNA fragment as a genetic polymorphism in the potato cultivars could have caused discrepancies in the cultivar collections and unnecessary loss may have occurred. Comparing two different sources of tissue and investigating the origin of the aberrant band demonstrated to be a valuable in this project as it not only resolved the correct interpretation of the data, but also provided information that could develop a rapid detection method for White ghost contamination. A limitation associated with using arbitrary primers for DNA fingerprinting was also indicated.

The 100 % homology between the two cloned 2-1050o fragments from cultivars Onka and Trent strongly suggests that the DNA fragment was from the same origin. The BLASTN and FASTA database searches indicate that the 2-1050o fragment is not of plant origin and could not be a polymorphism from the potato cultivars. However, some sequence homology was found to other microorganisms such as B. subtilus, E.

140

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coli, H. influenza, and Rickettsia prowazeki. It is not suprising that the 2-IO750 fragment found homology to potato and tomato DNA as it was generated from plant tissue. The fact that 2-IO750 found some homology to human and Synechocytis DNA indicates there are parts of the genomes that are similar.

A range of assays including PCR, dot blot hybridisation and PCR-RFLPs have been tested to detect the presence of B. circulans in tissue cultures. In addition, the specificity of the RAPD marker 2-IO500 has been improved by developing a number of SCAR primers and evaluation of Ta.

The specificity of the PCR assay with S2-10 and IS2-10 was improved by increasing Ta as it reduced a number of non specific products (Rychlik et al. 1990). A further improvement to the assay was increasing the primer length as indicated with the SE2-10 primers. Most improvements to the specificity of RAPD markers have been made by increasing the primer length and converting them into SCAR markers (Paran and Michelmore 1993; Zijlstra 2000) or extended random primer amplified region marker (Wang et al. 2000).

Hybridisation of the BC500 probe to a range of DNA types used for PCR testing produced inconsistent results which caused confusion about its usefulness as a diagnostic test. Such a result could be due to an inappropriate stringency being used for the assay (Sambrook et al. 1989). Other possible reasons the probe did not hybridise to other DNA types that tested positive to PCR, was due an insufficient amount of target DNA from B. circulans in the sample, that DNA quality was poor and contained inhibitors that could have hindered hybridisation. Due to the inconsistent results and the poor sensitivity of the dot blot hybridisation, it is not a suitable method to detect the presence of B. circulans in plant tissue cultures.

A problem with the PCR assay was with SE2-10, a 500 bp fragment was amplified with Botrytis, Clavibacter, E. coli and Yellow Tufted Honey Eater DNA. This indicates that priming sites for 2-IO500 fragment is not specific to B. circulans and is found in other species. The B. circulans DNA can be differentiated from the other species by the use PCR-RFLPs with the restriction enzymes Bsr Gl and Mse 1 as the restriction sites are only found in the 2-lOsoo fragment isolated from B. circulans.

A PCR diagnostic test to detect the presence of B. circulans in plant tissue cultures has been developed. The assay involves the amplification of DNA with primer set SE2-10 to generate a 500 bp fragment. DNA from B circulans can be differentiated from other 500 bp fragments generated with this primer set by restriction enzyme digests with Bsr Gl and or Mse 1.

The method developed can detect 103 and 104 cfu mL"1 which is the level of sensitivity commonly achieved with PCR. Some additional studies with Ralstonia solonacearum (Bacterial Wilt) have increased the level of sensitivity to a range oflOôlO^fumL"1

by using nested-PCR (Poussier and Luisetti 2000) or selective media (Granada and Sequeira 1983). Neither of these approaches have been tested for the detection of B. circulans in plant tissue cultures.

141


The management of microbial contamination is a serious problem in tissue culture laboratories (Cassels 1991; Reed and Tanprasert 1995; Benjama and Charkaoui 1997; Leifert and Woodward 1997). The B. circulans problem manifests itself due to the recent use of clear gelling agents such as Phytagel™ where microbial contamination is simpler to visualise, compared to opaque gelling agents such as agar where observation of the contaminant is masked. While B. circulans is probably ubiquitous in potato and many other plant tissue cultures, it is a problem that can be readily managed by routine and frequent subcultures of material to maintain vigorous growth and multiplication of plantlets. The detection of microbial contaminants using PCR has considerable advantages to standard microbiological methods as it is rapid and sensitive.

142


8.5 References Benjama, Ah. and Charkaoui, B. (1997). Control of Bacillus contaminating date palm

tissue in micropropagation using antibiotics. In: Pathogen and Microbial Management in Micropropagation, 237-244. (Ed. A.C. Cassels). Kluwer Academic Publishers. NL.

Cassells, A.C. (1991). Problems in tissue culture: Culture contamination. In: Micropropagation Technology and Application, 31-44. (Eds. P.C. Debergh and R.H. Zimmerman). Kluwer Academic Publishers. NL.

Granada, G.A. and Sequeira, L. (1983). A new selective medium for Pseudomonas solanacearum. Plant Disease 67', 1084-88.

Leifert, C. and Woodward, S. (1997). Laboratory contamination management; the requirement for microbiological quality assurance. In: Pathogen and Microbial Management in Micropropagation, 237-244. (ed. A.C. Cassels). Kluwer Academic Publishers. NL.

Paran, I. and Michelmore, R.W. (1993). Development of reliable PCR-based markers linked to downy mildew resistance genes in lettuce. Theoretical and Applied Genetics 85, 985-93.

Poussier, S. and Luisetti, J. (2000). Specific detection of biovars of Ralstonia solanacearum in plant tissues by Nested-PCR-RFLP. European Journal of Plant Pathology 106, 255-65.

Reed, B.M. and Tanprasert, P. (1995). Detection and control of bacterial contaminants of plant tissue cultures. Plant Tissue Culture and Biotechnology 1,137-142.

Rychlik, W., Spencer, WJ. and Rhoads, R.E. (1990). Optimization of the annealing temperature for DNA amplification in vitro. Nucleic Acids Research 18, 6409-12.

Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning: A Laboratoty Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, USA.

Wang, X., Fang, Z., Huang, S., Sun, P., Liu, Y., Yang, L., Zhuang, M. and Qu, D. (2000). An extended random primer amplified region (ERPAR) marker linked to a dominant male sterility gene in cabbage (Brassica oleracea var. capitata). Euphytica 112,267-73.

Zijlstra, C. (2000). Identification of Meloidogyne chitwoodi, M.fallax andM hapla based on SCAR-PCR: a powerful way of enabling reliable identification of populations or individuals that share common traits. European Journal of Plant Pathology 106, 283-90.

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9.0 Technology transfer

Results from the project have been presented in conferences, IHD laboratory visits, and published or referred in press articles.

9.1 Publications/Conferences Isenegger, D. and Hutchinson, J.F. (1997/8). DNA fingerprinting and cryopreservation

of potato cultivars. IHD Annual Report, No. 5.

Anonymous (1998). Checking the fingerprints on your varieties. Eyes on Potatoes 4, 10.

Carrington, D., Isenegger, D., Tregenza, J., Horstra, C , Bates, J., Rodoni, B. and Hutchinson, J. (1998). Identified by their fingerprints. Potato Australia 9, 62.

Isenegger, D. and Hutchinson, J.F. (1998). DNA fingerprinting of plant cultivars. Seed Potato Industry Workshop, Colac.

Isenegger, D. and Hutchinson, J.F. (1998). DNA fingerprinting helps identify the best spuds. HortReport 98 (p. 31). HRDC.

Isenegger, D., Taylor, P.W.J., McGregor, G. and Hutchinson, J.F. (1998). DNA fingerprinting and cryopreservation of potato (Solanum tuberosum L.). Proceedings of the Fifth Annual Symposium for the Joint Centre for Crop Improvement, The University of Melbourne, Parkville.

Isenegger, D. and Hutchinson, J.F. (1999). Potato DNA fingerprints. National Potato Growers Field Day, Toolangi.

Isenegger, D. and Hutchinson, J. (1999). DNA fingerprints and cryopreservation of potato cultivars for improved quality assurance. Potato Australia 10,15. (Project update).

Isenegger, D. and Hutchinson, J. (1999). DNA fingerprints. Fresh News, October issue. Published under auspices of the Victorian Farmers Federation, Victorian Potato Growers Council.

Isenegger, D., Mulliris, K., Barlass, M. and Hutchinson, J.F. (1999). Detection of bacterial contamination in tissue culture plants using PCR. Proceedings of the International Association for Plant Tissue Culture and Biotechnology, VI National Meeting Sydney.

Isenegger, D., Taylor, P.W.J., McGregor, G. and Hutchinson, J.F. (1999). DNA fingerprinting of potato {Solanum tuberosum L.). Proceedings of the Sixth Annual Scientific Symposium of the Joint Centre for Crop Improvement, Horsham.

Nadesan, S., Isenegger, D., Waterhouse, P. and Hutchinson, J. F. (1999). Potato Biotechnology - Achievements and Opportunities. Proceedings of the International Association for Plant Tissue Culture and Biotechnology, VI National Meeting Sydney.

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Hutchinson, J.F., Isenegger, D., Nedasan, S., Smith, N. and Waterhouse, P. (2000). Potato Biotechnology - Achievements and Opportunities. Potatoes 2000 -"Linking Research to Practice". Adelaide.

Isenegger, D., Taylor, P.W.J., McGregor, G., Barlass, M. and Hutchinson, J.F. (2000). Detetcion of a bacterial contaminant in tissue culture plants by PCR. Proceedings of the Seventh Annual Symposium for the Joint Centre for Crop Improvement, Rutherglen.

9.2 Miscellaneous This research project has been referred to in: • Good Fruit and Vegetables (8 [7], 1997). • Good Fruit and Vegetables (9[11], 1999). • Eyes on Potatoes (7, 1999).

9.3 Laboratory visits A number of grower and industry groups have visited IHD, including: • APIC R & D committee. • Victorian Potato Industry Council. • The University of Melbourne (Institute of Land and Food Resources). • Delegation from Ministry of Food and Agriculture, Indonesia. • IHD Open Day.

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10.0 Recommendations 10.1 DNA fingerprinting

The DNA fingerprint database developed from this research can be used by industry to identify and differentiate potato cultivars.

A level of technical expertise and appropriate laboratory facilities are required. For reproducibility it is important that the specified PCR conditions are used.

Additional DNA fingerprinting systems based on Simple Sequence Repeats (SSRs) can be extremely useful for cultivar identification, differentiation and genetic studies (Chapter 5). Additional research to develop these systems should be considered.

There are a number of options to consider with clonal variants of the cultivars Atlantic, Kennebec, Sebago, Delaware and Russet Burbank:

1. Combine agronomic studies in the field with marker studies in the laboratory (most recommended).

2. Discard the clones that are not distinguishable.

3. Keep clones regardless of results.

4. Screen more RAPD primers and investigate other DNA fingerprinting systems based on, for example, SSRs, Inter Simple Sequence Repeats and Amplified Fragment Length Polymorphisms.

Bacillus circulans (aka. White Ghost) has been identified as an endogenous bacterial contaminant in tissue cultured plants. It is recommended that tissue culture laboratories use appropriate management by routine and frequent subculturing of material to maintain vigorous growth and multiplication of plantlets (Chapter 8).

The detection of White Ghost by PCR can be used as a rapid diagnostic for quality assurance associated with the seed schemes and germplasm maintained in tissue culture. The routine adoption of this PCR diagnostic tool be considered.

10.2 Cryopreservation Cryopreservation has demonstrated potential use as a long term storage system of potato cultivars (Chapter 3).

It is recommended that additional research to improve the regeneration efficiency of cultivars be conducted.

The routine adoption of cryopreservation be considered for the long term storage of potato germplasm.

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Appendix 1. Public good potato cultivars used in this project. Cultivar Cultivar 85-30-12 88-59-12 92-19-10 Allegany Atlantic Line A Atlantic Line B Atlantic White Flower Bintje Bismark Bison Line B Bremer Brownell Butte Cadima Campbell 11 Cariboo Line B Catani Chipbelle Coliban Line B Crispa Crystal Dalmore Delaware Line A Delaware Line B Delaware Line D Delaware Line E Delaware Line G Denali Desiree Donnelly Dynamite Evans Exton Fontenot 287 Foxton Frontier Russet Gemchip Hampton Hudson Itasca 40-3 Kanona Katahdin Line B Kennebec Line 2 Kennebec Line 7 Kennebec Line A Kennebec Line B Kenzy

Mortifry My Fry Nadine Nicola Nooksack Norchip Line A Norgold Russet Norking Russet Norpchip Line A Oneida Onka Patrones Pembina Chipper Pentland Envoy Pinkeye Platte Pontiac Line B Purple Congo Ranger Russet Red Craigs Royal Red La Soda Red Ruby Redsen Rideau Riverina Russet Rosa Ruby Lou Russet Burbank British Columbia Russet Burbank Line A Ruen Russet Burbank Line Tas95 Russet Burbank Netted Gem Russet Burbank Toohey's Sangre Sebago Line B Sebago Line C Sebago New Brunswick Sequoia C Shepody Shine Simcoe Snowchip Snowchip Snowgem Somerset Spunta Tobique Tolass

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King Edward Kipfler Knox Krantz Lenape MacRusset Merrimack Mondial Monona Line A

Trent White Rose Whitu Whitu Wilstore Wilwash Winlock Winter Gem Wontscab Yankee Supreme


Appendix 2. Potato cultivars and clonal variants^ used for DNA fingerprinting. Cultivar Dendrogram code Country of origin 85-30-12 85-30-12 Australia 88-59-12 88-59-12 Australia 92-19-10 92-19-10 Australia Atlantic Line A (*} AtlA USA Atlantic Line B n AtlB USA Bintje Bismark Bison Line B

Bintje Bismark BisonB

Europe Germany USA

Bremer Bremer Australia Cadima Cadima Australia Catani Catani Australia Coliban Line B ColB Australia Crispa Crystal Dalmore

Crispa Crystal Dalmore

Australia USA Australia

Delaware Line A(** DelaA USA Delaware Line B (** n/a USA Delaware Line D ( t) n/a USA Delaware Line E ( i) n/a USA Delaware Line G (*J n/a USA Denali Denali USA Desiree Desiree Netherlands Donnelly Dynamite Evans

Donnelly Dynamite Evans

Australia Australia Australia

Exton Exton Australia Foxton Foxton Great Britain Kennebec Line 2 (** Ken 2 USA Kennebec Line 7 (** Ken 7 USA Kennebec Line B (<) KenB USA King Edward KingEd Great Britain Kipfler Knox

Kipfler Knox

Austria Australia

MacRusset MacRus Australia Mondial Mondial ? Mortifiy My Fry Nadine

Mortifiy My Fry Nadine

Australia Australia Great Britain

Nicola Nooksack

Nicola Nooksack

Europe USA

Norpchip Line A Onka

NorpchA Onka

USA Australia

Patrones Patrones Netherlands Pinkeye Pontiac Line B

Pinkeye PontiacB

?

USA Purple Congo PCongo ?

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Ranger Russet Red Craigs Royal Red La Soda

RanRus RCRoyal RedSoda

USA Great Britain USA

Redsen Redsen USA Rideau Rideau USA Riverina Russet RivRus Australia Ruby Lou Russet Burbank British Columbia (*}

RubyLou RBBC

Australia USA

Russet Burbank Line A Ruen (*} RBRuenA USA Russet Burbank Line Tas95 n RB Tas95 USA Russet Burbank Netted Gem (*J RBNG USA Russet Burbank Toohey's (* RBT USA Sebago Line B (*} SebB USA Sebago Line C (4) n/a USA Sebago New Brunswick(4) SebNB USA Sequoia C Shepody Shine

SeqC Shepody Shine

USA USA Australia

Simcoe Simcoe Canada Snowchip Snowgem

Snowchip Snowgem

USA Australia

Spunta Toolangi Delight

Spunta ToolDel

Netherlands Australia

Trent Trent USA White Rose White Rose USA Whitu Whitu New Zealand Wilstore Wilstore Australia Wilwash Wilwash Australia Winlock Winlock Australia Winter Gem WinGem Australia Wontscab Wontscab Australia


Appendix 3. Potato cultivars used for cryopreservation. Cultivar Allegany Atlantic White Flower Brownell Butte Campbell 11 Cariboo Line B Chipbelle Fontenot 287 Frontier Russet Gemchip Hampton Hudson Itasca 40-3 Kanona Katahdin Line B Kennebec Line A Kenzy Krantz Lenape Merrimack Monona Line A Norchip Line A Norgold Russet Norking Russet Oneida Pembina Chipper Pentland Envoy Platte Red Ruby Rosa Sangre Snowchip Somerset Tobique Tolass Whitu Yankee Supreme


Appendix 4. Glossary of technical terms.

AFLP

allele

CAPS

cryopreservation

chromosome

DNA

DNA fingerprint

(Amplified Fragment Length Polymorphism) a technique that involves restriction enzyme digestion and PCR for generating molecular markers and DNA fingerprints

an alternative form of a genetic locus. A single allele for each locus is inherited separately from each parent

(Cleaved Amplified Polymorphic Sequences) PCR products that are digested with restriction enzymes and produce polymorphisms (also known as PCR-RFLP)

a technique for storing living biological material at very low temperatures in liquid nitrogen (-196 C).

the self replicating genetic structures of cells containing the cellular DNA, that bears in its nucleotide sequence, the linear array of genes

(deoxyribonucleic acid) the molecule that encodes genetic information. It is a double stranded molecule held together by weak bonds between the base pairs of nucleotides. The four nucleotides in DNA contain the bases: adenine (A), guanine (G), thymine (T) and cytosine (C). Base pairs only form between A and T and between C and G, thus the base pair sequence from each strand can be deduced from its partner

a DNA profile that is characteristic to distinguish or identify a cultivar

DNA polymerase an enzyme which copies a DNA (or an RNA) molecule to produce a DNA copy

electrophoresis a method for separating DNA or RNA molecules according to size by 'sieving' them through a gel.

ethidium bromide a dye used to stain DNA or RNA that fluoresces when viewed with UV light

gene

genetic drift

an ordered sequence of nucleotides located in a particular position (locus) on a chromosome that encodes a specific protein

naturally occurring mutations of DNA

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ISSR

locus

microsatellite

(Inter Simple Sequence Repeat) are SSR regions that are generated by PCR and differ to STMS by the use of universal primer sets

(plural loci) the position on a chromosome of a gene or other chromosome marker

(also known as SSR) repetitive DNA sequences that are highly polymorphic and usually flanked by conserved DNA regions (also see STMS and ISSR l (also see STMS and ISSR)

molecular markers informative pieces of usually DNA that can be used for applications in genetic studies (detecting genetic variation or polymorphisms) and DNA fingerprinting. There are a number of methods based on DNA hybridisation and PCR.

PCR

PCR-RFLP

(Polymerase Chain Reaction) a method of amplifying DNA using a heat stable polymerase and (usually) two primers. Because the newly synthesised strands can subsequently serve as additional templates for the same primer sequences, successive rounds of primer annealing, strand elongation and dissociation produce rapid and highly specific amplification of the desired sequence in a DNA template

see CAPS

primer

polymorphism

restriction enzyme

RAPD

RFLP

a short single strand DNA sequence that recognises complementary sites which 'primes' the addition of nucleotides to a DNA strand by DNA polymerase during PCR

difference in DNA sequence among individuals

a protein that recognises short specific nucleotides and cuts DNA at those sites. Bacteria contain over 400 such enzymes that recognise and cut over 100 different DNA sequences

(Random Amplified Polymorphic DNA) a PCR-based method that uses a single primer of arbitrary design for generating molecular markers and DNA fingerprints. An individual primer will produce an identical DNA fingerprint (or profile) for the same cultivar (in potato). If the DNA is different (eg. a different cultivar) a primer will produce a different DNA fingerprint, providing the primer detects a polymorphism

(Restriction Fragment Length Polymorphism) variation between individuals in DNA fragment sizes cut by specific restriction enzymes and detected by Southern blots. Polymorphic sequences that result in RFLPs are used as markers on both

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physical maps and genetic linkage maps. RFLPs are usually caused by a mutation at a restriction enzyme cutting site

RNA (ribonucleic acid) a nucleic acid found in the nucleus and cytoplasm of cells and plays a role in protein synthesis and other chemical activities. The structure of RNA is similar to DNA but uracil (U) replaces T

SCAR (Sequence Characterised Amplified Region) a RAPD marker that has been cloned, sequenced and has primers designed to specifically amplify a DNA fragment

SSR (Simple Sequence Repeat) synonymous term to microsatellite

STMS (Sequence Tagged Microsatellite Site) microsatellite loci that are cloned and sequenced and have designed primers flanking the microsatellite region

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Documents

PT97011 DNA fingerprints and cryopreservation of potato ... · for improved quality assurance Final report for project PT97011 Principal Investigator James F. Hutchinson Agriculture