ISSN 0095�4527, Cytology and Genetics, 2015, Vol. 49, No. 6, pp. 378–381. © Allerton Press, Inc., 2015.

378

1 INTRODUCTION

Common, bread wheat, Triticum aestivum L. (2n =6x = 42, AABBDD), has many relatives in the Trit�iceae tribe, which are valuable sources for broadeninggenetic diversity and may provide genes for disease andpest resistance and general wheat improvement [1, 2].

Different approaches have been proposed for genetransfer from alien species to wheat depending on theevolutionary relationships of the species involved [3].One important step in alien transfer is the production ofcompensating wheat–alien trans�locations. These canbe readily produced for the targeted chromosomesthrough the mechanism of centric breakage�fusion [4].

Robertsonian translocations (RobTs) arise by cen�tric misdivision of univalents at anaphase I followed bysegregation of the derived telocentric chromosomes tothe same nucleus, and fusion of the broken ends dur�ing the interkinesis of meiosis II. The formation ofcompensating translocations in a double�monosomicplant requires that the derived telocentric chromo�somes from opposite arms of homoeologous chromo�somes in ana�/telophase I migrate to the same spindlepole followed by the rejoining of the broken ends dur�ing the interkinesis [5]. Depending on the chromo�somes involved and environmental conditions, thedesired compensating wheat�alien RobTs can berecovered at fairly high frequencies [5].

1 The article is published in the original.

Amongst the wild wheat relatives, Thinopyrumbessarabicum (Save ex Rayss) A Löve (2n = 2x = 14,EbEb) is an important source of agronomically desir�able genes for wheat breeding. In a previous work weidentified the disomic substitution line DS2Eb(2B) ina BC1F6 population produced from wheat andTritipyrum—an amphiploid between durum wheatand Th. bessarabicum�crosses [6]. The present studydescribes the production of compensating wheat�Th. bessarabicum whole arm Robertsonian transloca�tions involving chromosomes 2B of wheat and 2Eb ofTh. bessarabicum.

MATERIALS AND METHODS

Plant Material

The lines used in this study included T. aestivum cv.“Roushan” (2n = 6x = 42, AABBDD), Tritipyrum(2n = 6x = 42, AABBEbEb), two nulli�tetrasomic(N2B�T2D and N2B�T2A) and a ditelosomic (Dt�2BS)stocks. Wheat disomic chromosome substitution linein “Roushan” background i.e. DS2Eb(2B) in which2B pair of wheat is replaced with 2Eb pair of Th. bessa�rabicum chromosomes was produced at the Universityof Kurdistan, Sanandaj, Iran [6].

In order to produce compensating RobTs involvingchromosomes 2B of wheat and 2Eb of Th. bessarabi�cum, crosses were made between bread wheat “Rous�han” cultivar (as female parent) and DS2Eb(2B) sub�

Production of a Novel Robertsonian Translocation from Thinopyrum bessarabicum into Bread Wheat1

S. Ghazali, G. Mirzaghaderi, and M. MajdiDepartment of Agronomy and Plant Breeding, Faculty of Agriculture, University of Kurdistan, Sanandaj, Iran

e�mail: gh.mirzaghaderi@uok.ac.irReceived November 13, 2013

Abstract—Development of wheat�alien translocation lines will facilitate its practical utilization in wheatimprovement. The objective of the present study was to produce compensating wheat–Thinopyrum bessara�bicum whole arm Robertsonian translocations (RobTs) involving chromosomes 2B of wheat and 2Eb ofTh. bessarabicum through the mechanism of centric breakage�fusion. F2 population from crosses betweenDS2Eb(2B) substitution line and bread wheat “Roushan” (2n = 6x = 42, AABBDD) as female parent weremade. Forty one F2 lines (L1–L41) were screened for their chromosome composition. Three 2Eb specificPCR�based Landmark Unique Gene (PLUG) markers were used for screening F2 progeny derived fromplants double�monosomic for chromosome 2B and 2Eb. Two RobTs (~5%) were observed among F2 plants.Homozygous translocation (T2EbS.2BL) with good plant vigor and full fertility were selected from F3 fami�lies. The T2EbS.2BL stock has longer awn than that of its parents. It is cytogenetically stable, and may be use�ful in wheat improvement.

Keywords: Robertsonian translocation line, EST�based marker, Thinopyrum bessarabicum, wheat

DOI: 10.3103/S0095452715060031

CYTOLOGY AND GENETICS Vol. 49 No. 6 2015

PRODUCTION OF A NOVEL ROBERTSONIAN TRANSLOCATION 379

stitution line. F1 plants with 42 chromosomes thatwere double�monosomic for chromosomes 2B/2Eb

were self�pollinated. The F2 progeny were genotypedby molecular markers for putative RobTs.

Molecular Marker Analysis

DNA was extracted from fresh leaves of F2 progenyat three�leaf stage with CTAB method. The purity andconcentration of DNA was assessed by comparisonwith 1 kb ladder in 1% agarose gel and by spectropho�tometry. The DNA of each genotype was finallydiluted to ~50 ng µL–1 and stored at –20°C. Primersof PLUG markers including LOC_Os04g41910,TNAC1102 and TNAC1140 were made by Bioneer Co.(South Korea) and used in 20 mL PCR reactions in orderto identify 2Eb chromosome arms in the lines (table). Ourprevious study revealed that LOC_Os04g41910 andTNAC1102 produce specific bands for the short armof 2Eb in wheat background [6]. The PCR mixturecontained 25 ng template DNA, 5 pmol of eachprimer, 2.5 mM of each dNTP, 2.5 mM MgCl2 and 0.5 UTaq polymerase. Amplification was done for 5 min at94°C, followed by 30 cycles of 45 s at 94°C, 45 s at 61–63°C according to the different primers, 90 s at 72°Cand a final step of 7 min at 72°C. A 10�µL aliquot ofthe mixture was digested overnight with 2.0 U ofHaeIII or TaqI (table) in incubators set at 37 or 65°C,respectively. Three percent agarose gel electrophoresiswas used for separating digested PCR products.

Cytogenetic Analysis

Actively growing roots of about 1–1.5 cm in lengthwere cut and pretreated in ice cold water for 24 h forchromosome staining. Root tips were then fixed inethanol : acetic acid (3 : 1) for 48 h at 4°C. Chromo�some preparations and staining were done usingsquash method [7]. Slides were analyzed with anOlympus BX51 microscope and images were cap�tured, using a DP72 digital camera. For genomic insitu hybridization (GISH), the Th. bessarabicumgenomic DNA was labeled with biotin�16�dUTP

using a nick translation kit (Roche), according to themanufacturer’s instructions. Probe was purified withethanol precipitation. In situ hybridization and detec�tion were carried out based on published protocols [8].

RESULTS AND DISCUSSION

A total of 41 F2 plants derived from F1 plants dou�ble�monosomic for chromosomes 2B/2Eb werescreened with PLUG markers for homoeologousgroup�2. To ensure about the correct assignment ofspecific alleles to homoeologous chromosomes andarms of wheat group�2, the three polymorphic mark�ers were amplified in group�2 nullisomic/tetrasomicand ditelosomic stocks. LOC_Os04g41910 andTNAC1102 were specific markers for short arm andTNAC1140 was specific marker of long arm ofhomoeologous group 2 (Fig. 1). The 2Eb�specificpolymorphic markers were observed in four plants outof which two plants had positive markers for both 2EbSand 2EbL indicating that these two plants had com�plete 2Eb chromosome. The other two plants showedpositive bands specific for the short arm of 2Eb usingLOC_ Os04g41910 and TNAC1102 markers. Theselines showed specific band for long arm of 2B (i.e.2BL) using the long arm specific marker TNAC1140indicating the presence of 2BL arms (Fig. 1b) so theywere putative Robertsonian translocations and werefurther characterized by chromosome counting andGISH analysis. Chromosome counting of one of theselines (i.e. L20) revealed that it has 42 chromosomes(Fig. 2a) and genomic in situ hybridization confirmedthe presence of 2EbS in this line (Fig. 2b).

The PLUG markers used in the present study areco�dominant and can be used to distinguish betweenhomozygous and heterozygous wheat�alien transloca�tion lines as previously described [9] so they allowed usto select homozygous translocations (Fig. 1).

The frequency of compensating wheat�alien Rob�ertsonian translocations in progenies of double mono�somic plants depends on the targeted chromosomes,

PLUG [17] and LOC_Os [9] primer sequences on the wheat group�2 chromosomes and primer/enzyme combinations pro�ducing polymorphic PCR products

Marker

Arm

loca

tion

An

nea

lin

gte

mpe

ratu

re,

°C

Primer sequence (5' → 3')

En

zym

epr

oduc

ing

poly

mor

phic

PC

R p

rodu

ct

forward reverse

LOC_Os04g41910 2S 61 AGA CCA TCT TTC CGGTCT TTG

GAGAGGAATGCGTGAAGTTTG

HaeIII

TNAC1102 2S 63 GGA GAG GTG AAGGAC CAA CTC

CCT TGC AGC GTA GTGAGA TTT

TaqI

TNAC1140 2L 63 TCC CAG AAA TTA CAAGGC TCA

AGGAACCCTATGCATTGGAAA

HaeIII

380

CYTOLOGY AND GENETICS Vol. 49 No. 6 2015

GHAZALI et al.

genetic background and environmental conditionsand ranges from 4% to almost 20% [5, 10–12].

Surprisingly no 2BS.2EbL Robertsonian transloca�tion was recovered. The total recovery rate of homolo�gous RobTs in the present study was 2 out of 41 (~5%).This recovery rate of RobTs indicates that the produc�tion of compensating wheat�alien RobTs is fairly com�mon [5].

Molecular markers indicated that two lines areRobTs and they involved the target wheat chromo�some 2B so that the 2BS fragment was absent but the2BL fragment was present (Fig 1). GISH observationsusing Th. bessarabicum genomic DNA as probe wereconsistent with the marker data. GISH revealed thattwo progenies had wheat�2Eb RobTs (Fig. 2b). Theselines had longer awn than that of their parents (Fig. 3).

Classical genetic and molecular data show thatgenes conferring stress tolerance and disease resis�tance in plants are frequently clustered in the genome,so RobTs often confer multiple desirable resistancetraits. The most successful RobTs used in wheatimprovement are the T1BL.1RS and T1AL.1RS trans�locations [13]. RobTs T6AS.6V#3L derived from

Dasypyrum villosum conferred resistance to race Ug99of stem rust [12]. Translocation T2EbS�2BS2BLderived from Th. bessarabicum had higher yield perplant, which suggests that the alien segment carriesyield�related genes [14]. Our newly developed translo�cation T2EbS.2BL may prove to be a valuable source ofresistance to biotic or abiotic stresses.

By producing F1 plants double�monosomic forchromosomes 2B of wheat and 2Eb of Th. bessarabi�

2BL

2D

2A

2BS

2Eb

100

bp la

dder

T. a

esti

vum

cv.

Rou

shan

N2B

�T2D

N2B

�T2A

Dt2

BS

DS

2Eb (2

B)

L20

Tri

tipy

rum

100

bp la

dder

Tri

tipy

rum

T. a

esti

vum

cv.

Rou

shan

N2B

�T2D

N2B

�T2A

Dt2

BS

DS

2Eb (2

B)

L20

(a) (b)

Fig. 1. PCR pattern of nullisomic–tetrasomic (NT) and ditelosomic (Dt) lines of wheat group�2 chromosomes, wheat–Thinopy�rum bessarabicum disomic substitution line DS2Eb(2B) and Tritipyrum amplified by TNAC1102 (a) and TNAC1140 (b) primers.The TNAC1102 were previously mapped to the short arm and TNAC1140 to the long arm of wheat group�2 chromosomes. PCRproducts were digested with the enzyme HaeIII or TaqI. Arrows indicate specific fragments of different group�2 chromosome.

(a) (b)

Fig. 2. Acetoorcein staining of mitotic chromosomes in aroot tip cell of the RobT line L20 (2n = 42) (a) and agenomic in situ hybridization (GISH) pattern of one of theT2EbS.2BL translocation line (L20) (b). Scale bar repre�sents 5 µm.

(a) (b)

Fig. 3. Spikes of the bread wheat parent “Roushan” (a) andL20 which is homozygous translocation T2EbS.2BL (b).

CYTOLOGY AND GENETICS Vol. 49 No. 6 2015

PRODUCTION OF A NOVEL ROBERTSONIAN TRANSLOCATION 381

cum we targeted the formation of RobTs between thesetwo chromosomes. In the present study two RobTswere identified among the 41 progenies screened, cor�responding to a frequency of 5%.

Cereal plant centromeres consist of highly con�served retrotransposon�like elements [15, 16].Sequence homology between the centromeres ofhomoeologous chromosomes can provide the struc�tural basis for chromosome pairing and recombina�tion, which may also result in formation of RobTsalbeight this hypothesis was tested previously but noevidence of recombination events in the centromeresof plants double�monosomic for chromosomes 1A ofwheat and 1Ht of E. trachycaulus was obtained [5].However, the centromere structure of some chromo�some may be unique and may allow the occurrence ofcrossover between homoeologous centromeres. Moststudies suggest that the random event of breakage�fusion�bridge leads to arising RobTs producing bothcompensating and non�compensating type.

We are pursuing further characterization of theidentified translocations and directed chromosomeengineering aimed at shortening the Th. bessarabicumsegment in the T2EbS.2BL translocation.

ACKNOWLEDGMENTS

The wheat nulli�tetrasomic and ditelosomic lines werekindly provided by Dr. W.J. Raupp and Dr. B.S. Gill,Wheat Genetic and Genomic Resources Center, Kan�sas State University, USA. This research was funded bythe University of Kurdistan.

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