Dissertation Jessica Denning 25300792

BIOL 3034 Lab report Jessica Denning 25300792

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Has parallel domestication occurred

between Aubergine and Tomato in shape

and colour traits?

BIOL 3034 Long Lab Report

Jessica Denning 25300792


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Lay Summary

The process of domestication involves altering the physical attributes of plants and animals

to best suit our provisional human needs. The species of crops that we consume today look

significantly different from those that our ancestors would have initially come across, many

of which would have been inedible. Plants and animals would have originally evolved to have

the highest fitness for the environment that they lived in. They would have only been under

environmental selection pressures i.e. the species best adapted to dry environments under

dry conditions would have thrived and reproduced where others that were not as well

adapted to those conditions could have died out and been removed from the gene pool had

the selection pressure been strong enough.

During the process of domestication humans shaped plants and animals through breeding

them with characteristics to meet their needs as well as being able to thrive under their

environmental conditions. Selecting characteristics to improve crops and animals for our own

consumption is termed artificial selection. It is thought artificial selection began around

10,000 years ago in an area of the Middle East called the Fertile Crescent where the first

evidence of agriculture has been discovered. It has become evident that there are certain

characters that have been selected for in modern cultivars across species such as large fruit

and seed size, loss of natural seed dispersal, pod shattering and reduced branching of plants;

it is this set of attributes that characterise domestication syndrome.

Most fruiting plants (the angiosperms) rely on animals to disperse their fruit and seeds so that

their offspring may grow in an uncrowded environment, because of this requirement they

have developed a mutualistic relationship with animals to carry out their reproductive cycle.

This meant most ancestral crop fruits and seeds were originally small and round as they had

to be easy to carry by animals. Some ancestral plants such as aubergine’s produced spines on

their stems and leaves which although deters predators such as herbivores it makes

harvesting them in large quantities by hand more difficult, so when a chance mutation

occurred where they did not have them that character would have been selected for and bred

into the population, this is the principal of domestication by artificial selection.


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This project has attempted to identify genes involved in domestication syndrome in tomatoes

and compare them to the aubergine genome in order to determine if the same characters

have been selected for in both species, this would infer if parallel domestication has occurred.

Four genes sequenced from tomato were successfully sequenced in aubergine. Identical

colour polymorphisms were found in aubergine in the genes 3GT and DFR, and shape

polymorphisms found in OVATE, suggesting it is possible that parallel domestication has

occurred.

Abstract

In recent years there have been huge advances in genetic sequencing and mapping

techniques which has enabled us to generate a much deeper understanding of evolutionary

processes. Domestication has led to rapid evolutionary changes in both animals and plants

and recent studies have been able to identify and locate the artificially selected genes that

have given rise to the phenotypes that characterise our food crops.

Through these studies it has become evident that in some cases a pattern of parallel

domestication has occurred where the same genes have been conserved in different species.

Solanaceous species seem to have conserved several genes relating to domestication; for

example in aubergine 43% of the QTL for fruit weight, shape and colour have orthologues in

tomato, potato and pepper.

This project has examined genes involved in fruit shape and colour in the aubergine in order

to determine if they share polymorphic correlations between alleles and phenotype with

tomato. This would indicate that at some stage they have both been subjected to parallel

domestication where the same characters have been artificially selected in both species. It

was found that the genes DFR, 3GT, 5GT and OVATE had been conserved in aubergine and

that there were colour and shape polymorphisms present. This indicates these genes may

have similar roles in tomato as aubergine which supports the evidence found in prior studies

suggesting parallel domestication may have occurred.


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Contents Page

1. List of abbreviations 5 2. List of tables and figures 5 3. Introduction - review of the literature 6

3.1 How, when and where did domestication arise 6 3.2 Why was domestication successful 7 3.3 Domestication syndrome 7 3.4. Domestication syndrome in tomato 8 3.5. The genetics of seed shattering 9 3.6. The genetics of reduced branching 9 3.7. Anthocyanins and domestication 10 3.8. Domestication syndrome in animals 11 3.9. Studying domestication using polymorphisms 11 3.10. Evidence of recent domestication 12 3.11. Parallel domestication 13 3.12. Selective Sweeps under strong selection pressures 14 3.13. Aims, hypotheses and Acknowledgements 14

4. Materials and Methods 15

4.1. PCR Primer design 15 4.2. DNA extractions 16 4.3. Calculating DNA concentrations 16 4.5. PCR 17 4.6. Sequence analysis 18

5. Results 19 5.1 Identification of Polymorphisms 20 5.2 Identification of the coding region and amino acid changes 21 6. Discussion 23 6.1. Relation of DFR expression to colour pigment 23 6.2. Relation of OVATE expression to round fruit phenotypes 23 6.3. Relation of 3FT expression to fruit pigmentation 24 6.4. Does my data support colour and shape phenotypes? 25 and does it show evidence of parallel domestication? 6.5 Limitations and further study 27

7. References 28

8. Appendices 32 8.1 All plants used in study 32

8.2 All identified polymorphisms and alleles 33


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1. List of abbreviations

QTL Quantitative trait loci

DFR dihydroflavonol-4reductase

3GT 3-O-glucosyltransferas

LC Locule number

FAS Facilitated

QSh1 Seed Shattering1

Tbx1 teosinte branchx1

RNA Ribonucleic acid

FAB1 Fatty acid biosynthesis1

FAD2-1 fatty acid desaturase

FATB fatty acyl-ACP thioesterases

DNA Deoxyribonucleic

BLAST Basic Local Alignment search tool

PCR polymerase chain reaction

Bp base pairs

CTAB Cetyl trimethylammonium bromide

TE buffer Tris & EDTA buffer

dNTP Nucleoside triphosphate

EDTA Edetic Acid

2. List of tables and figures

Table 1. Master Mix for PCR reaction for one sample 16

Table 2. PCR programme TD_55 16

Table 3. Master Mix for enzyme reaction for one sample 17

Table 4. Enzyme incubation reaction programme 37_80 17

Table 5. Master Mix for sequencing PCR reaction for one sample 17

Table 6. Sequencing PCR programme seq_50 17

Table 7. 18 plant samples and their phenotypes 20

Table 8. Polymorphisms in gene DFR 21

Table 9. Polymorphisms in gene 3GT 21

Table 10. Polymorphisms in OVATE 21

Figure 1. Chromas output from gene DFR individual 1654 18

Figure 2. PCR products 19

Figure 3. DFR coding region identifying changes in amino acid sequence 22

Figure 4. OVATE coding region identifying changes in amino acid sequence 22


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3. Introduction

3.1. How, when, and where did domestication arise?

The development of agriculture has been one of the most significant shapers of modern

civilisation our species has experienced. The crops produced as a result of domestication

which started around 10,000 years ago still feed the world’s population today. (Doganlar et

al., 2002; Doebley & Gaut, 2006). It is thought that farming could not have arisen consciously

(Diamond, 2002), the first farmers would have had no way of learning the functional trait that

enabled agricultural skills from anything or anyone else. It may have been a behavioural trait,

an evolved skill, with no goal orientation of domestication. The first evidence of agriculture

was found at the Fertile Crescent, a highly fertile area in the Middle East surrounding Syria

and Jordan; however this is now being reviewed as evidence suggests that a multi-regional

model of domestication is more likely. The multi-regional model suggests that farming arose

several times independently in different parts of the world leading to convergent evolution.

(Brown et al., 2009). It is likely farming arose in areas only where there were high abundances

of progenitor crops such as teosinte the ancestor of maize. It has been suggested only in these

areas of high density progenitor species that farming was able to out- compete the traditional

hunter-gathering lifestyle that up until that point had been the dominant life-history strategy

for human beings (Doganlar et al., 2002).

The Fertile Crescent has produced several progenitors of modern crops and animals including

wheat, barley, peas, sheep and goats and emerged as the first real centre of agriculture

around 8,500 years ago. There are however a further eight proposed regions that arose

independently; China, Mesoamerica, Andes/Amazonia, Eastern United States, Sahel tropical

West Africa, Ethiopia and New Guinea which gives supporting evidence to the multi-regional

theory. The Fertile Crescent community spread their genetics and knowledge, subsequently

migrating to areas like Italy and Greece which may not have otherwise developed agriculture

and the early great civilisations would not have been as successful as they were (Doganlar et

al., 2002).

In order for a living and evolving system to have remained viable for so long, continued and

successful domestication would have involved generating genetic diversity by selection both

natural and artificial (Brush, 2000). Artificial selection has been one of the main drivers of


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domestication through the ages as it involves selecting the plants we wish to breed into the

future based on the genetic traits they possess best meeting our needs and wants as a

consumer. We could infer that early domestication probably focussed mostly on fruit or grain

size and stem strength to support the enlarged fruit as most wild species relying on animal

dispersal had very small fruit that would have not been adequate for human consumption

other than in large quantities.

3.2 Why was domestication successful?

Farming led to the formation of settlements; despite these causing higher levels of infectious

diseases due to population explosion and overcrowding agriculture remained the dominant

way of life. These farming settlements increased the available food to early populations

leading to an increase in carrying capacity. This life history strategy also gave rise to

technology; sedentary living allowed the pooling of ideas and the creation of technology that

could not be carried by nomadic people e.g. forges that allowed for the further development

of agriculture by creating advanced tools. Farming enabled food surplus that could feed full

time inventors, but also lead to the formation of social stratification creating social politics

(Doganlar et al., 2002). It was these advantages that gave the agriculture and domestication

of wild species the evolutionary advantage over the hunter gatherer strategy.

3.3 Domestication syndrome

Modern cultivars differ greatly from their wild progenitors, they typically have more robust

stems, larger fruit and do not shed their seeds (Doebley et al., 2006). The production of fruit

and flowers is the fundamental way in which plants reproduce and which has led to the great

success of the angiosperms (Midgley et al., 1991). The floral meristem produces both male

(the stamen) and female plant organs (petals, sepals, and pistil), fertilisation of the ovules by

pollen stimulates fruit development. (Monforte et al., 2013). The fruit produced by the plant

contains the seeds, these require dispersing and that is where the mutualistic relationship

between plants and animals arose (Schupp, 1993); therefore originally wild progenitor fruits

would have needed to be small enough to be carried potentially long distances by animals but

be large and colourful enough to attract them (Frary et al., 2000). Dispersal of seeds is

important for all plants, it reduces crowding of the natal environment and also helps prevents

inbreeding. Large fruit in the wild progenitor populations would not have been advantageous


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for dispersal, however it was maximised during the domestication process for human

consumption (Monforte, et al., 2013).

Domestication is defined as the selective breeding of species by humans in order to

accommodate human needs (Allaby, 2009). Early domestication involved increased seed or

fruit size, more flowering, reduced branching, suppression of natural seed dispersal and the

loss of seed dormancy (Burke et al., 2007). Reducing the energy used to grow surplus

branches increases the amount available for fruit and seed production which could have

facilitated the growth of larger fruit. Increasing the fruit and seed size from small wild varieties

meant one plant could provide more nutrition to more people, these traits would have

enabled humans to increase their own fitness and so would have been selected for.

Supressing natural seed dispersal would have also been important, having the seed remain

on the plant would have increased ease of harvest as well as reducing spoilage and increasing

yield. These archetypal changes have been characterised as ‘domestication syndrome’

(Poncet, et al., 2004).

Current consensus suggests that there have been two stages of domestication, one initial

stage followed by a current and ongoing stage of improvement. For example plants selected

with disease and pest resistance, increased yield and nutritional value like the biofortification

of rice with vitamin A (golden rice) (Paine et al., 2005) and also for drought intolerance would

be going through the second phase of domestication.

3.4. Domestication syndrome in tomato

The tomato is one of the most well studied crops, through domestication it has reached a size

that is over several times larger than its progenitor Solanum pimpinellifolium which measures

less than 1cm across and weighed only a few grams (Monforte et al., 2013). Many studies

have been conducted in attempts to identify and clone the genes that underlie the phenotypic

changes in domesticated crops. Changes in tomato fruit shape have been accounted for by

mutational changes to four genes: SUN and OVATE regulate fruit elongation and LC and FAS

determine the locule number and flat fruit shape respectively (Rodriguez et al., 2011; Liu &

Van Eck, 2002).


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Fw2.2 was found responsible for changes in fruit weight by up to 30%, it is thought that the

effect is caused by ORFX which is expressed early on in floral development as this gene

controls the number of cells present in the carpals (Frary et al., 2000). This suggests that it is

not just fw2.2 that is responsible for their gigantic size Cong et al. (2008) imply two QTLs, LC

and FAS both affect carpel number rather than size and this in turn could increases fruit size

by up to 50%

3.5. The genetics of seed shattering

The prevention of seed shattering would have been fundamental to early domestication, it

would have made harvesting more efficient and also prevented the seeds from damage as

well as increasing yield (Zhongwei et al., 2012). Taking away cereals natural seed shedding

ability would have made them very dependent on humans for seed dispersal which is another

trait associated with domestication syndrome. The genes responsible for seed shattering have

been identified in a select few cereals: Qsh1 and Sh4 have been cloned from rice and recently

the Sh1 gene has been cloned from several cereals including wheat.

Studies show that even though many of the early progenitors of cereal cultivars were

domesticated in different regions globally they share the same conserved genes (Patterson et

al., 2005). The recently identified Sh1 gene possibly has the most orthologues, initially

sequenced from sorghum, rice has ortholog OsSh1 and there are structural variations in Maize

(ZmSh1-1 and ZmSh1-5.1+ZmSh1-5.2) (Zhongwei et al., 2012). The evidence from these

studies does suggest that parallel evolution did occur during domestication due to selection.

It would depend of course on each species having the same genes to be conserved in the first

place, or being in similar conditions that would encourage similar mutations and the

development of similar traits.

3.6. The genetics of reduced branching.

The cultivar maize and its progenitor teosinte are so vastly different and it its thought that

this drastic alteration in plant architecture is controlled by five loci with major effect genes.

(tbx1) corresponds to one of these loci and has been found to effect the apical dominance;

this causes the lower buds on the stem to fail to form resulting in less branching. Although

the gene is expressed in both the cultivar and the progenitor it is shown that there is a greater

amount of tbx1 RNA in maize and higher expression in certain parts of the plant which may


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cause such major variation. The gene teosinte glume architecture has also been described

and interacts with tbx1 and is involved in the growth of the stalk and the orientation of the

floral bract (Kellogg, 1997). These genes would most likely have been selected for because a

plant that produces fewer branches and has more energy for producing fruit, less branching

also reduces shading and allows plants to be planted closer together all of which are

advantageous for producing higher yielding plants.

3.7. Anthocyanins and domestication

Anthocyanins are flavonoid compounds that control pigment and have been shown to be vital

to plant growth and development. Colour creates a visual target attracting pollinating insects

and animals to aid in seed dispersal (Vogt et al., 1994). The evolution of this mutualistic

relationship with insects and animals has led to the angiosperms becoming the dominant

plant species on Earth (Crepet & Niklas 2008) and anthocyanins have played a pivotal role in

the development of this important relationship. Anthocyanins protect plants from the

damage of ultra violet light irradiation, insect attack and pathogen infection (Li et al., 2014).

This role would have been vital during domestication along with a robust immune response

as there were no pesticides which we rely heavily on today due to the high pest prevalence

within monocultures.

Around 35% of crop plants globally rely on insect and animal pollination and throughout

domestication ancestral varieties would have also (Winifree et al., 2011). Maintaining species

genetic diversity so that they maintained or enhanced the level of pigmentation in their

flowers and fruit would have been very advantageous to ensure that crops continued to have

a high fertilisation rate and produced high yields.

Anthocyanins clearly have a role in biotic environmental plant interactions however a recent

study identified the role of DFR in abiotic stress tolerance. It was found that anthocyanin

biosynthesis increased under stress conditions and that miR156-SPL9 may directly influence

anthocyanin biosynthesis through DFR. Blocking anthocyanin biosynthesis increased the

sensitivity of the plants to salt and drought stress. DFR then is an important part of the

anthocyanin pathway for surviving abiotic stress (Cui, et al., 2014). It is likely that abiotic stress

such as drought would put strong selection pressure on plants and so it is likely that DFR may

have been conserved across domesticated species because of this.


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Interesting work with poplar under elevated CO2 conditions has discovered an up regulation

in anthocyanin production with DFR upregulating by 39%. This work was in response to

studies looking at senescence in late autumn of trees. It was found that increasing levels of

anthocyanin production due to elevated CO2 conditions was the likely cause of a prolonged

growing season; in domesticated crops this could increase yields and the shelf life of the

commercial product which would be very beneficial. Up-regulation of anthocyanins also

increased the levels of antioxidants produced by the plant reducing oxidative damage and

prolonging the life of the plant (Tallis et al., 2010). Raised antioxidant activity has been found

present in fruiting plants such as Vaccinium uliginosum L. that have high anthocyanin content

(Wang, et al., 2014). Not only do anthocyanins potentially have a role in prolonging growing

seasons and the shelf life of the fruit but many studies have shown that high levels of

flavonoids in grapes, cranberries and blackcurrants can have benefits to human health

including having anti-inflammatory and anti-cancer properties so selecting for anthocyanin

content would have been very advantageous (Pietta, 2000).

3.8 Domestication syndrome in animals

The domestication syndrome characterised by domesticated animals tended to be the

opposite in most respects to plants. Herders would have wanted animals smaller than their

progenitors that were easy to handle and less aggressive. The domestication of animals was

thought to have also occurred in the Fertile Crescent with goats and sheep around 1000 years

after crop domestication began. With emphasis on breeding herds archaeological evidence

suggests that it was mainly females that were culled beyond their fertile years and much like

today there were few males as surpluses were not needed for breeding (Zeder, 2008). It

seems although we now have access to technology that enables greater speed and

understanding of the principals underlying domestication, the traits that we have been

selecting for over generations has changed very little.

3.9. Studying domestication using polymorphisms

There are ways of testing for selection that do not require complex genetic mapping but which

instead involve polymorphism (Chapman, et al., 2013). Strong selection can reduce patterns

of diversity among populations of crop plants for example molecular markers exhibit a

reduced state of polymorphism compared to wild gene pools. Once identified this data can


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be applied to sequence data and loci under selection can be discovered. It becomes evident

that genes that have undergone positive or directional selection show a greater than

expected loss in diversity between progenitor and cultivar specimens. This can be measured

using a ML-HKA program as used by (Chapman et al., 2013). This analysis uses maximum

likelihood analysis on multi-locus polymorphism and divergence data and tests for natural

selection in genes you are interested in.

3.10. Evidence of recent domestication

Work on sunflower genetics has shown evidence of recent secondary domestication, the

second phase of improvement and refinement. Using phylogenetic studies it is possible to

test for selection and whether it occurred before/during domestication or in the subsequent

improvement stage. In a study by Chapman et al. (2012) fatty Acid biosynthesis genes were

used for comparison and it was found that four of the five analysed (FAB1, FAD2-1, FAD6, and

FATB) only showed selection in the improved–outgroup comparison only; this would suggest

that these genes were fixed more recently as part of the improvement process of

domestication due to the increased demand for sunflower oil. As our needs have changed

over the previous 10,000 years of agriculture, plants have been selected to meet that change

which is why we see both ancient and more recent traces of domestication.

Domestication much like evolution is a continuing process we are always looking for ways of

breeding a better plant and now with greater scientific understanding of genetics we are able

to do this at an accelerated rate which means more useful traits are being recognised and

fixed quickly. Improving cultivars requires selection of mutations that may have arisen

naturally or by genetic manipulation (Paran & Van der Knaap, 2007). A mutation that has

arisen through natural means recently is the delayed fruit deterioration (DFD) in the tomato.

The delayed deterioration is due to the reduction of water loss in the ripe fruit. This trait is

thought to be displayed phenotypically because of a change in the chemical composition of

the cuticle (Saladie et al., 2007). This trait will be valuable in increasing the longevity of the

harvested fruit which is important in terms of its commerciality as some fruits travel long

distances which takes time before they are eventually consumed.


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3.11. Parallel Domestication

One of the major questions that science is unravelling at present is if the same genes are

responsible across species for causing the domestication syndrome, i.e. do the same genes

underlie the same phenotypic changes? QTL is a statistical analysis that enables the

comparison of genes with phenotypic traits (Miles, & Wayne, 2008). Its aim is to discover the

cause of variation in specific traits and to also discover if a few major QTL are responsible for

the majority of genetic change, or whether several QTL are responsible for only a few changes.

As comparative QTL studies and fine mapping techniques have improved light has been

gradually shed on these theories. In some species it is thought that only a few major genes

can alter phenotypes (Doganlar et al., 2002) enabling plants which were inedible to us

previously to be made very useful. Being able to identify these genetic traits could conceivably

allow us help people thrive in areas where agriculture is extremely difficult or unprofitable.

FThis will be increasingly important as the effects of climate change are realised and areas

like Southern Europe and Africa struggle to maintain yields under high drought conditions.

Comparative QTL studies have revealed some interesting results when investigating parallel

domestication. The Solanacaea family which includes the eggplant, pepper, tomato and

potato contain several orthologues relating to domestication syndrome. These genes have

been conserved despite the potato being domesticated on a completely different continent

(Doganlar, et al., 2002). The three QTL that control fruit weight in the eggplant (fw2.1, fw9.1

and fw11.1) are found as orthologues in all the aforementioned plants and fruit shape

orthologues from the eggplant in the tomato and pepper (fs7.1 and ovs4.1) have also been

identified (Grandillo et al., 1999). There seems to be substantial number of QTL that have

been conserved between Solanaceous species including plant height and flowering time and

leaf lobbing which isn’t agronomical. The eggplant shares forty four different QTL with the

other species and 27% of these have putative orthologues in the tomato and potato (Frary,

et al., 2003); and also 43% of the QTL for fruit weight, shape and colour in eggplant have

orthologues in tomato, potato and pepper (Doganlar et al., 2002). These studies have shown

that there is conservation in related species of the same genes that are related to

domestication and also those that are not. This could indicate that some genes are linked- the

non-agronomical i.e. leaf lobbing to an agronomical trait that is selected for intentionally or

it may persist due to epistasis.


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3.12. Selective Sweeps under strong selection

During intense selection, a selective sweep may occur whereby advantageous traits are

selected for along with genes that ‘hitchhike’ or are associated closely with the advantageous

gene on the chromosome and are so inherited together. These genes are fixed quickly by

increased prevalence in the population whereas less beneficial alleles are lost

(Schaffner & Sabeti, 2008). Given that domestication can put such strong selection pressure

of progenitor crops it is possible that selective sweeps may have occurred in cultivars. Rice

has been cultivated from O. rufipogon, in Asia different starch qualities have been selected

for during domestication depending on human preference (Whitt et al., 2002). A study of the

Waxy gene responsible for the absence of amylose found that it was present in temperate

japonica rice varieties, but rare or absent in tropical japonica, indica, aus, and aromatic

varieties (Olson et al., 2006). Analysis found that the gene had undergone strong selection in

temperate japonica and is likely to have undergone a selective sweep as it is not found in the

other domesticated varieties even though they are closely related.

A selective sweep was also identified in a study by Wang et al. (2005) in the gene responsible

for the loss of the hard casing around the maize kernel, this has been beneficial for farmers

allowing less processing of the crop. The gene tga1 is a transcriptional regulator and the trait

is controlled by just this one gene, being such a beneficial trait it would have been under

strong selection and does show evidence of a selective sweep at the promoter region (Wang

et al., 2005). Selective sweeps although reducing genetic diversity in domesticated crops does

lead to the most beneficial trait being fixed in the population quickly and eliminates the

chance of less advantageous traits being displayed in the phenotype which could decrease

yields.

3.13. Aims, hypotheses and acknowledgements

This study aims to sequence domestication genes related to size such as fw2.2, yield, colour

and shape such as LC and FAS in the aubergine based on the domestication of the tomato to

determine if parallel selection has occurred. The study aims to take sequences from the

tomato and identify them in the aubergine by designing PCR primers to amplify aubergine

genes and relate them to phenotypic measurements. The hypotheses for this study are H0:


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Genes under selection in one species will not be under selection in another species. HA: Genes

in one species will be under selection in another species.

I would like to thank Dr Mark Chapman for all his help, support and guidance throughout the

entirety of my project.

4. Materials and Methods

4.1 PCR primer Design

Firstly it was necessary to locate the gene of interest from the eggplant genome database

(Hirakawa et al., 2014) by searching for a key word e.g. ovate or its gene name (e.g.

Sme2.5_00099.1_g00011.1).On the resulting webpage the top hit was checked against the

sequence number and the nucleotide sequence copied into the query box in the BLAST tab

with an expected setting of 0.01. From the output the genomic hit was used to calculate the

size and position of the introns. Using the Primer 3 website, (Untergrasser et al., 2013) the

intron positions were copied into the excluded regions box and an appropriate product range

was selected for the size of the DNA fragment and pick primers was selected. From the

output the Left primer which is closest to the start of the DNA fragment and any Right primer

closest to the end of the DNA fragment were selected. Some pieces of DNA are large and may

need two sets of primers for effective PCR up to about 1500 bp.

4.2 DNA Extractions

All fruiting plants were sampled in the greenhouse (see Appendix 1 for plants used). 200mg

of meristem tissue taken from fruiting plants was submerged in liquid nitrogen then ground

to a fine dust before adding 700µl of CTAB. Samples were incubated at 60⁰ C for one hour

inverting once. **700µl 24:1 Chloroform: Iso Amyl Alcohol was added once samples had

cooled and were mixed for 15 minutes and centrifuged for 10 minutes at 13, 000rpm. 650µl

of the aqueous phase was pipetted into a new Eppendorf. Repeated from ** once, pipetting

only 600µl of aqueous phase. 0.08 volumes of 7.5M ammonium acetate (48 µl) and 0.5

volumes freezer cold isopropanol (324 µl) were added the samples frozen for 2 hours.

Centrifuged for 10mins at 13,000rpm. The liquid was poured off leaving the pellet each time

the. 500µl of freezer cold 70% ethanol was added to the pellet and centrifuged for 3 minutes

at 13rpm and the liquid poured off. 500 µl of freezer cold 95% ethanol was added to the pellet


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and centrifuged for 3 minutes at 13rpm. The liquid was poured off and the eppendorf left to

dry on a tissue for 15 minutes. The samples were re-suspended samples in 100 µl of TE buffer

and frozen until needed.

4.3 Calculating DNA concentration (ng/µl)

Using the Nano drop (ND-1000 Spectrophotometer 6070/1584) it was first calibrated using

sterile H2O. 1 µ𝑙 was placed on the receptacle and a measurement taken; then a blank

measurement using the buffer my sample was suspended in e.g. TE. Each of the DNA samples

(1µ𝑙) was pipetted onto the Nanodrop to calculate the concentration. Measurements were

taken for each of the DNA samples ensuring the receptacle was wiped clean each time to

prevent contamination.

4.4 PCR

Six DNA samples were selected to test each of the primers with. Primers were diluted with

TE buffer at 1:100µl. DNA should be diluted to 10ng/ µl. Master mixes for each of the primer

pairs were made using Table 1. enough for as many DNA samples as being tested. All mixtures

and dilutions were shaken and spun down. All DNA samples were plated out (1.5µl) into wells

and (13.5 µl) of the Master Mix was added into the relevant wells. The plate was firmly sealed

with clear film and centrifuged for 10s up to 1000rpm. The plate was placed in the PCR

machine program TD55_1 was run (See table 2). Use electrophoresis to run the PCR products

on Agarose gel with 5µl of UV marker. Using Gensnap, set the programme to 500ms and take

an image of the gel to view bands of DNA and determine if PCR was successful.

for 1 sample

10x buffer 1.5µl

dNTP 1.5µl

F primer 0.3µl

R primer 0.3µl

Taq 0.2µl

H2O 9.7µl

DNA 1.5µl

Table 1. Master Mix for PCR reaction

for one sample. TD_55

Incubate/cylce temperature ⁰C time mins/sec

Incubate 94 3

Incubate 94 /30s

incubate 65 /30s

decrease 1 each cycle

incubate 72 1

cycle steps 2 n/a 9 more times

incubate 94 /30s

incubate 55 /30s

incubate 72 1

cycle steps 6 n/a 29

incubate 72 7

incubate 10 5

Table 2. PCR programme TD_55


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To prepare for the sequencing PCR first the dNTP’s and Taq must be denatured. The reaction

required a master mix (see table 3) again enough was made for just more than the samples

to sequence. The incubation was run using the programme 37_80 (see table 4).

The final reaction is the Sequencing PCR, another master mix is made (see table 5) this PCR is

carried out separately for the forward and reverse reactions, and requires making master

mixes for the forward primer and for the reverse. To run the PCR programme Seq_50 was

used (see table 6). The sequencing PCR products were then cleaned up before being sent off

to be sequenced. 2.5 µl of 125mm EDTA and 30µl of 100% Ethanol were added to each of the

wells before covering and inverting. Samples were then left in the dark for 10 minutes. The

sequencing plate was then centrifuged at 2500g for 30 minutes, removed and the ethanol

gently tipped out of the wells. Following this all samples were re-suspended in 70% ethanol

and centrifuged on the same programme. The ethanol was tipped out and the plate left to

dry upside down for 30 minutes. The samples were sent to Oxford University’s department of

Zoology for sequencing.

Enzyme reaction master mix for 1 sample

thermosensitive alkaline phosphate 0.5µl

Exonuclease 1 0.25µl

dH2O 0.75µl

PCR product 7µl

Table 3. Enzyme reaction master mix for

one sample

37_80

incubate/cycle temperature ⁰C time mins/ sec

incubate 37 45

incubate 80 15

incubate 7 5

Table 4. Enzyme reaction programme

sequencing PCR master mix for 1 sample

dH2O 5.9µl

Sequencing Buffer 2µl

Big Dye 0.5µl

Primer 0.6µl

PCR Product 1µl

Table 5. Sequencing PCR reaction master

mix for one sample

Seq_50

incubate/cycle temperature ⁰C time mins/sec

incubate 95 5

incubate 95 /30s

incubate 50 /10s

incubate 60 2

cycle step 2 n/a 34 times

incubate 7 forever

Table 6. Sequencing PCR programme

Seq_50


18

4.5. Sequence analysis

The forward and reverse sequences needed to then be joined together and cleaned up. Using

the programme Chromas (Chromas 2015 Informer Technologies, Inc.). The ends of the

sequences that weren’t useful were cut off (see fig 1) and the reverse sequences reverse

complimented using the tools tab.

To join the forward and reverse sequences together the sequences were pasted into a text

file with the line breaks deleted. The beginning of the forward sequence was searched for in

the reverse sequence to find where they overlapped. The forward was then lined up with the

reverse where they overlapped and the end of the forward deleted. The end of the reverse is

then added to the end of the forward joining where they overlapped.

Ensuring all the sequences are in FASTA format in the text file, the joined sequences were

opened and viewed using the software (Proseq 2013). The sequences were aligned, the gaps

deleted, and the ambiguous bases were corrected where possible. The sequences could then

be viewed in the dotted view which highlighted where the polymorphisms were.

Polymorphisms were recorded in a spreadsheet along with the phenotype for comparison to

identify patterns (for tables of all polymorphisms see appendix 2)

Further analysis was then carried out to determine if the region containing the polymorphism

is in the coding region of the gene and therefore not an intron and then if the sequence

differences underlie differences in amino acids.

Figure 1. Chromas output showing the DFR gene from individual 1654. Yellow area shows

the cut end sequence.


19

To translate the coding region the website (ExPACy 2011) on the compact setting was used

which highlighted any introns making it easy to identify the coding region. This was performed

for each of the alleles with polymorphisms once the coding region had been translated and

identified the two could be compared to identify whether there were amino acid changes,

this was achieved by aligning them in a text file.

5. Results

A variety of 40 aubergine plants grown under glasshouse conditions were initially sampled;

their DNA was extracted and several attempts at PCR were completed as described previously

in the methods. Primers for the genes OVATE, Fw2.2, SUN, SUN2, FAS,

Anthocyanidinsynthase, CHS, S1_anthocyanin1, S1_anthocyanin2, Sm_5GT, St_3GT were

designed in order to result in a range of colour and shape genes to be sequenced. There was

great difficulty however in getting the genes to amplify in the majority of the DNA samples

and only four were ultimately successful. For the 18 DNA samples that were the most

consistent in amplifying the genes during PCR four genes: DFR, 3GT, 5GT and OVATE were

amplified successfully though still, not all resulting sequences were usable Figure 2. Shows

the PRC products from the most successful PCRs.

Figure 2. PCR products from 18 individulas in (Table 7.) with genes top left – bottom right

DFR. 3GT, 5GT, OVATE.


20

Table 7. The 18 Individual plants whose DNA samples had the most consistent gene

amplifications that were used in the analysis; including their phenotypic characters, variety and

amount of DNA extracted from each during the first stage of the experiment.

5.1 identification of Polymorphisms

The aligned sequences showed some evidence of polymorphisms between the individuals of

different phenotypes and some patterns were visible. For tables of all polymorphisms and

phenotypes see (appendix 2.) For the gene DFR (Table 8.), it is evident that there are two

individuals that share the same allele (b) and have the colour phenotype of green compared

to the common allele (n). This may suggests that the presence of alleles (a) & (b) and correlate

with green colour and it may be these alleles are causing that phenotype to be displayed.

Interestingly the same pattern is visible in polymorphisms of the gene 3GT (see Table 9). This

time with two individuals sharing the same allele (a) correlating to the colour yellow, this

allele may then be responsible for the yellow colour phenotype being expressed in this case.

Polymorphisms in alleles of the gene OVATE also gave some interesting preliminary findings,

OVATE regulates fruit elongation in tomato (Rodriguez et al., 2011; Liu & Van Eck, 2002) and

Table 10 shows a correlation with the (a) allele and round fruit shape which is what would be

expected if the gene was being expressed at the same locus in aubergine.

Plant Number Cultivated date planted Spines? Fruit colour Fruit Shape fruit size L/W (mm) flower colour where from variety DNA extracted ng/ul1654 yes 09/06/2014 no Purple elongated 74/41 n/a INDONESIE TERUNG UNGU 58

1659 yes 09/06/2014 no green round 37/32 purpleINDONESIE(JAVA

)TERONG BULAT HIJAU 246

1792 yes 02/06/2014 no yellow round 47/40 purple VIETNAM BIG & ROUND FRUIT 190

710 no 09/06/2014 yes greee round 22/21 n/a THAILANDE BIRM/S.2440 39

195 no 09/06/2014 yes green round 15/17 n/a TUNISIA Solanum linneanum 59

1597 yes n/a no yellow elongated 165/37 n/a INDE MYSORE GREEN LONG 268

693 no 02/04/2014 yes purple round 87/48 n/a SRI-LANKA BIRM/S.1961 71

1571 no 09/06/2014 no yellow round 27/22 n/a THAILANDE TS76/X468 171

1581 no 09/06/2014 no purple/white round 22/27 n/a INDONESIE FARMER LONG/TS99/X492 793

1637 yes n/a no purple elongated 153/50 n/aCHINE

(LIAONING)DONGLING FENGCHENG 842

706 no 09/06/2014 yes green round 27/25 n/a MADAGASCAR BIRM/S.2310 306

1567 yes 09/06/2014 no green round 67/57 n/a INDONESIE MARUKAN/TS66/X461 525

1298 yes 09/06/2014 no yellow elongated 123/55 n/a BHUTAN PARO LOCAL 63

1290 yes n/a no green elongated 60/36 n/a PHILIPPINES BATANGAS 163

356 yes 09/06/2014 yes purple round 57/97 n/a CHINE JIO YE CHIE (A 9 FEUILLES) 110

415 yes n/a no yellow elongated 96/51 n/a PHILIPPINES MAYON 302

197 yes 09/06/2014 no yellow round 62/68 n/a CHINE LIU YE QIE (A 6 FEUILLES) 446

1838 no n/a no yellow round 21/26 n/a VIETNAM VIR-1034 190


21

Table 8. The sequenced individuals for the gene DFR that shared the same phenotype (green)

and the same allele (b) compared to the common allele (n)

Table 9. The sequenced individuals for the gene 3GT that shared the same phenotype (yellow)

and the same allele (a) compared to the common allele (n)

Table 10. The sequenced individuals for the gene OV that shared the same phenotype (green)

round and (Purple) round and elongated with the same allele (a) and (f) respectively compared

to the common allele (n)

5.2 Identification of the coding region and amino acid alterations

The second part of the sequence analysis as mentioned in the materials and methods was to

determine whether a change in the amino acid sequence could have been responsible for the

phenotypic polymorphisms. The alignment of the translated coding sequence for the gene

DFR was the most interesting, individual 1654 had the common allele (n) – (CTA) whereas

individuals 415 and 1290 shared the same allele (b) - (CCA) resulting in a green phenotype

and the common allele (n) – (CTA) resulting in a purple phenotype. Individual 1955 (see

appendix 1) also had the green round phenotype but had the allele (a) (ATA). When the coding

regions of these alleles were aligned in see figure 3. there is a difference in the amino acid

Individual 3GT Colour Long? Spines? Sample name Allele

96

1654 Purple Long None D1 T n

1792 yellow round None D3 C a

1597 yellow long None D6 C a

Individual OV Colour Long? Spines? Sample name Position Position Position Position Position allele

98 100 103 413 472

1654 Purple Long None G1 T G T C G n

1659 green round None G2 T C A C G a

706 green round Spines G11 T C A C G a


1290 green round None H2 T C A C G a


1581 purple round None G9 T A A C G f

1298 Purple Long None H1 T A A C G f

Individual DFR Colour Long? Spines? Sample name Allele

236 310 368

1654 Purple Long None A1 C A T n

1290 green round None B2 C G T b

415 green round None B4 C G T b


22

sequence circled in red with 1654 the common allele amino acid changing in that position

from an (L) to a (P) in 415 and an (I) in 195. This may suggest that the phenotype expressed

by the fruit may be down to a change in amino acid sequence in the gene DFR.

The translated coding region of OVATE (see figure 4.) is also quite interesting; as

aforementioned OVATE regulates shape and there was a correlation between round shape

and the (a) allele. In the individual 1659 along with the others that share the same allele there

is a change in amino acid sequence from the (n) haplotype seen in individual 1654 –(D) (GAT)

which is elongated to an (E)- (ATA) in 1659 which is round. This could suggest that the change

in amino acid sequence is responsible for the expression of the round phenotype. However

the same change in sequence is found for the haplotype (f) and one of the individuals: 1298

is elongated which may mean that the change does not cause the round phenotype but a

different allele may be responsible at another point in the sequence which we are not aware

of as we were only able to sequence a portion of the gene, consequently there may be more

than one gene that causes the shape change i.e. there may be a mutation in ovate but there

may be a mutation in another locus also.

Figure 3. Shows the coding region of the gene DFR in individuals (Top 1654 with the (n)

allele, middle individual 195 with the (a) allele and bottom individual 1290 with the allele

(b). The changes in amino acids are highlighted in red.

Figure 4. Shows the coding region of the gene OVATE in individuals (Top 1654 with the

(n) allele, middle individual 1659 with the (a) allele and bottom individual 1298 with the

allele (f). The changes in amino acids are highlighted in red.


23

6. Discussion

6.1 Relation of DFR expression to colour pigment

Studies have shown that the gene DFR does relate to colour pigment in aubergine. Stommel

& Dunn (2015) undertook PCR analysis to determine the role of DFR in anthocyanin

accumulation and how that related to colour phenotype. They compared the accumulation

levels of DFR as amongst three transcriptomes and found the purple phenotype to have

significantly higher anthocyanin concentration, increasing 9 times compared to the other

variety of white fruit. It was shown only two of the transcriptome levels MybC and Myc

increased in developing the purple fruit coincident with increasing anthocyanin content. In

my study I found a correlation with polymorphisms at DFR and green colour (see table 8) and

managed to identify the coding region of the gene (see figure 3) which indicated there had

been a change in amino acid sequence from one of the common alleles which resulted in the

purple fruit phenotype compared to the other two which had the green phenotype. This may

suggest that the change in amino acid sequence was underlying the green colour caused by

reduced expressed of DFR and therefore had lower levels of anthocyanin concentrations

rather than purple phenotype expressed by the different allele which has a higher level of

anthocyanin concentration.

Another study grew aubergine seedlings under red light which were irradiated with

ultraviolet-containing white light. It was found anthocyanin synthesis was induced in the

hypocotyl tissues, especially when a UV light was added. The accumulation of pigments was

found to be closely associated with the expression of genes involved in flavonoid synthesis

genes including chalcone synthase (CHS) and DFR. This again highlights the link between DFR

and anthocyanin biosynthesis in aubergine. They also interestinly identify the gene CYP75

which may be responsible for anthocyanin biosynthesis requiring the presence ultra violet

light (Toguri et al., 1993).

6.2 Relation of OVATE expression to round fruit phenotypes

Although my data suggests a correlation between round shape and the allele (a) see (table

10) there has been difficulty mapping the QTL of OVATE and it has given unexpected results

although ones that can be explained. A study by Doganlar et al. (2002) found that only one of

the five OVATE genes in aubergine mapped to the region of the genome that contains genes


24

relating to fruit shape; this suggests that the genes responsible for determining the final size

and shape of the eggplant ovaries are controlled by loci that are not directly involved in the

final size of the mature fruit.

QTL studies of OVATE also showed that the genes mapped by Doganlar et al. (2002) explained

fairly little of the variation in ovary shape and size. Three QTL fitted simultaneously for ovary

diameter only explained 24% of the variation in aubergine, the QTL for ovary shape ovs4.1

explained 36% of phenotypic variation and was the most significant. In terms of my study this

could mean that sequencing only a small portion of the gene alone would not give indicative

results of a correlation as I also found that one of the (f) allele individuals had an elongated

shape. Although we are aware that ovate does control fruit shape in aubergine it only appears

to explain a portion of the variation in shape. It may have been more useful if it were possible

to also successfully amplify the genes involved in fruit length fl2.1 and fl9.1 as well as fl11.1.

as they were shown to explain 47% of the phenotypic variation but they are yet to be

sequenced (Doganlar et al., 2002).

6.3 Relation of 3GT expression to yellow fruit pigmentation

Althought there is little work in the literature relating to 3-O-glucosyltransferase in aubergine

most of the research has been on colour pigments in flowers and it is one of the most studied

glycosylation enzymes. 3GT has been identified and cloned from numerous plant species

which suggests parallel domestication or selection. 3GT is responsible for the modification of

the anthocyanin by the addition of a glucose, this stage is the last in the anthocyanin pathway

and appears to have been conserved across species (Grotewold 2006).

A study by Ono et al. (2006) examined the anthocyanin pathway in flower petals and describes

that the Aurones class of flavonoids exhibit a bright yellow colour in ornamental flowers, such

as the snapdragon (Scrophulariaceae, Antirrhinum majus). However the pathway involving

3GT (see fig four) was found to give a pink/purple pigment so it is unlikely that the correlation

I found between the polymorphisms and 3GT (see table 9.) and the yellow phenotype is

unlikely to actually be causal gene in that case. The gene was sequenced in purple individuals

indicating that the polymorphism identified could be interfering with the anthocyanin

pathway by knock out which may cause an alteration in phenotype; however when the coding

region was sequenced it was found that there was no change in amino acid sequence so it is


25

more likely that a portion of the gene we did not sequence may be responsible for yellow

phenotype.

A study of 3GT in tomato by De Jong et al. (2004) found that it had been conserved in based

on partially sequenced tomato cDNA clone that shares approximately 81% sequence identity

with Petunia 3GT which was used as a mapping probe. Tomato 3GT mapped to the lower arm

of chromosome 10 however because it was found that the two contigs do not overlap in

sequence it is not yet possible to conclude that they are derived from the same gene,

however the gene may have undergone parallel selection.

6.4 Does my data support correllations to colour and shape phenotypes and does it show

evidence of parallel domestication?

Although my data has managed to identify some patterns in polymorphisms as discussed

previously, it is possible but unlikely that the changes in amino acid sequences are the causal

factors giving the resultant phenotypes. The result from which we can infer the most likely

correlation is in the gene DFR and the green fruit phenotype. The change in amino acid

sequence identified in figure 3 could be responsible for the green colour as the (n) phenotype

was purple in most cases (see appendix 1) which would indicate that DFR was expressed in

Figure 5. The anthocyanin pathway

showing the role of 3GT resulting in a

purple phenotype compared to 4GT &

6GT resulting in the yellow aurone

pigment. (Ono et al., 2006)


26

higher levels with the (n) haplotype than in the (a) and (b) haplotypes which resulted in a

green phenotype which express lower levels of anthocyanin (Stommel & Dumm 2015).

3GT although showing a polymorphic correlation to colour and haplotype had no change in

amino acid sequence in the coding region so it is unlikely that the resultant phenotypes are

caused by the polymorphism identified in Table 9. Also studies have identified 3GT normally

results in a purple rather than yellow phenotype however different alleles may not code for

purple pigment although there were no changes identified in the amino acid sequence there

may be genetic changes for example stop codons or insertions which could knock out the

gene expression of 3GT.

OVATE did show a strong correlation in haplotype and phenotype with a number of

individuals showing the round phenotype (a) (see table 10) and also there was a change in

the amino acid sequence of the coding region (see figure 4) from (D) – (GAT) in the (n) allele

to (E) – (GAA) which is suggestive that the alteration in amino acids could be the causal factor

in the altered phenotype as the (n) phenotype has an elongated phenotype. There is a

discrepancy however as the other polymorphism identified (f) shows the same alteration in

coding region (D) to (E), and one of the individuals with the (f) allele has an elongated

phenotype so this may suggest that the polymorphism in this case is not underlying the

change in phenotype.

Although genes from tomato were successfully sequenced in the aubergine it is not possible

to define that they are serving the same purpose from the data I have collected so it is not

definitive to say that parallel domestication has occurred in this case, although other studies

mentioned previously including one by Doganlar et al. (2002) have shown significant

similarities between the two genomes.

6.5 Limitations and Further Study

The main limiation to this study was the difficulty in getting the genes to amplify sucessfully

in the individual plant samples. This factor was also limiting in the variety of genes and

phenotypes that it was possible to sequnce, in the end most of the genes that were sucessful

were anthocyanins and it would have been better if some of the fruit size and shape genes

were also successful as they play an equal role in domestication syndrome. Having a limited

number of phenotypes to measure against also was not ideal, with a more numerous and


27

varied sample size it is likely that stronger less ambiguous correlations would have been found

giving more difinitive results. The study also only sequenced a portion of each gene so it is

possible that the correlations we found may be the result of a genetic change in a portion of

the gene that we did not sequence so being able to sequence the whole of each gene would

have been beneficial.

The fact that we were able to sequence the genes from tomato in aubergine suggests that

they have been conserved in the two species which does suggest that there has been parallel

domestication although we were unable to explicitly identify that the genes served the same

function this study does support the comprehensive analysis by (Doganlar et al., 2002) who

found highly significant evidence of the conservation of domestication genes relating to

shape, size and colour QTL in tomato and aubergine. The study also highlighted that the major

phenotypic changes that occurred during domestication are controlled by only a few loci for

instance size and shape in aubergine are controlled by only 2 major loci.

The discoveries made in identifying the genetics underlying domestication and parallel

domestication could have significant applications in the future especially as gentic techniques

and analyses improve. If we are able to detect which genes control certain traits important to

commercialisation and improved growth, it may be possible to genetically engineer wild

species that grow well insitu under climate change conditions, to be cultivated and feed more

people more efficiently rather than trying to import crop varieties that are ill suited to their

alien conditions. If other groups of species also show evidence of having few major loci

controlling large aspects of the phenotype then it may be posisble to fast track domestication

and plant breeding. By selecting specifically for these gentic changes we could effect an

artificial selective sweep that could maintain yeilds in the future when the effects of climate

change become apparent and growing crops in certain areas becomes near impossible.


28

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32

8. Appendices

8.1 All plants used in study including their phenotypes

Plan

t N

um

ber

C

ult

ivat

edd

ate

pla

nte

dSp

ines

?Fr

uit

co

lou

rFr

uit

Sh

ape

fru

it s

ize

L/W

(m

m)

flo

wer

co

lou

rw

her

e fr

om

vari

ety

DN

A e

xtra

cted

ng/

ul

1654

CV

09/0

6/20

14n

oPu

rple

elo

nga

ted

74

/41

IND

ON

ESIE

TER

UN

G U

NG

U58

1493

CV

02/0

6/20

14n

ogr

een

elo

nga

ted

10

1/47

CH

INE

(SI

CH

UA

N)

NO

IRE

MEI

ZH

OU

74

1659

yes

09/0

6/20

14n

ogr

een

rou

nd

37/3

2p

urp

leIN

DO

NES

IE(J

AV

A)

TER

ON

G B

ULA

T H

IJA

U24

6

1792

yes

02/0

6/20

14n

oye

llow

rou

nd

47/4

0p

urp

leV

IETN

AM

BIG

& R

OU

ND

FR

UIT

190

710

no

09/0

6/20

14ye

sgr

eee

rou

nd

22/2

1TH

AIL

AN

DE

BIR

M/S

.244

039

195

no

09/0

6/20

14ye

sgr

een

rou

nd

15/1

7TU

NIS

IASo

lan

um

lin

nea

nu

m59

290

ov

no

yello

w/w

hit

ero

un

d40

/35

INC

ON

NU

EPL

AN

TE A

UX

OEU

FS36

3

1597

yes

no

yello

wel

on

gate

d16

5/37

IND

EM

YSO

RE

GR

EEN

LO

NG

268

1661

yes

09/0

6/20

14n

op

urp

le/w

hit

eel

on

gate

d15

5/57

IND

ON

ESIE

(JA

VA

)D

AD

ALI

181

1808

yes

no

yello

wro

un

d95

/75

IND

E

(KA

RN

ATA

KA)

MA

TTU

GU

LLA

43

5

1826

yes

09/0

6/20

14n

oye

llow

rou

nd

112/

53C

HIN

E (

YUN

NA

N)

ZI C

HA

NG

QIE

(II6

BO

139)

130

693

no

02/0

4/20

14ye

sp

urp

lero

un

d87

/48

SRI-

LAN

KAB

IRM

/S.1

961

71

1571

ov

09/0

6/20

14n

oye

llow

rou

nd

27/2

2TH

AIL

AN

DE

TS76

/X46

817

1

1581

ov

09/0

6/20

14n

op

urp

le/w

hit

ero

un

d22

/27

IND

ON

ESIE

FAR

MER

LON

G/T

S99/

X492

793

1584

yes

06/0

6/20

14n

oye

llow

rou

nd

54/5

4PH

ILIP

PIN

ES

TS10

8/X5

0232

9

1637

yes

no

pu

rple

elo

nga

ted

153/

50C

HIN

E (

LIA

ON

ING

)D

ON

GLI

NG

FEN

GC

HEN

G84

2

1712

yes

06/0

6/20

14n

op

urp

le/y

ello

wel

on

gate

d10

3/50

CH

INE

(LI

AO

NIN

G)

LIA

ON

ING

N°4

980

706

no

09/0

6/20

14ye

sgr

een

rou

nd

27/2

5M

AD

AG

ASC

AR

BIR

M/S

.231

030

6

1565

ov

no

yello

wro

un

d26

/36

IND

ON

ESIE

TS62

/X45

735

0

1567

yes

09/0

6/20

14n

ogr

een

rou

nd

67/5

7IN

DO

NES

IEM

AR

UKA

N/T

S66/

X461

525

1789

yes

06/0

6/20

14n

ow

hit

ero

un

d15

/17

VIE

TNA

MSM

ALL

& G

REE

N F

RU

IT17

8

1663

yes

no

wh

ite/

pu

rple

elo

nga

ted

154/

26p

urp

leIN

DO

NES

IE(J

AV

A)

TER

ON

G N

AG

A U

NG

U17

0

1300

yes

06/0

6/20

14n

ow

hit

e/p

urp

leel

on

gate

d15

1/47

SRI-

LAN

KATI

NN

EVEL

Y33

3

1298

yes

09/0

6/20

14n

oye

llow

elo

nga

ted

123/

55B

HU

TAN

PAR

O L

OC

AL

63

1294

yes

09/0

6/20

14n

oye

llow

elo

nga

ted

101/

38IN

DO

NES

IETE

RO

NG

PA

N J

AN

G 2

7578

1290

yes

no

gree

nel

on

gate

d60

/36

PHIL

IPPI

NE

SB

ATA

NG

AS

163

620

yes

no

yello

wro

un

d50

/47

IND

EM

AN

JAR

I GO

TA25

6

1543

yes

09/0

6/20

14n

oye

llow

rou

nd

65/5

2M

ALA

ISIE

TS3/

X412

177

414

yes

06/0

6/20

14n

ogr

een

elo

nga

ted

81/3

5PH

ILIP

PIN

ES

MA

KILI

NG

309

356

yes

09/0

6/20

14ye

sp

urp

lero

un

d57

/97

CH

INE

JIO

YE

CH

IE (

A 9

FEU

ILLE

S)11

0

152

yes

no

pu

rple

/wh

ite

elo

nga

ted

111/

46SR

I-LA

NKA

CEY

LAN

SM

164

319

96ye

s09

/06/

2014

no

gree

nel

on

gate

d96

/42

IND

EB

RIN

JAL

LON

G 2

198

1276

yes

06/0

6/20

14n

oye

llow

rou

nd

93/7

8IN

DE

IND

ES 1

115

656

yes

no

pu

rple

/wh

ite

elo

nga

ted

160/

35TA

IWA

NM

AC

HIH

LO

NG

217

415

yes

no

yello

wel

on

gate

d96

/51

PHIL

IPPI

NE

SM

AYO

N30

2

398

yes

09/0

6/20

14n

ow

hit

ero

un

dTH

AIL

AN

DE

THA

ILA

D42

2

197

yes

09/0

6/20

14n

oye

llow

rou

nd

62/6

8C

HIN

ELI

U Y

E Q

IE (

A 6

FEU

ILLE

S)44

6

97ye

s06

/06/

2014

no

pu

rple

/wh

ite

rou

nd

73/6

8p

urp

leIN

DE

BR

INJA

L R

OU

ND

372

4

1838

ov

no

yello

wro

un

d21

/26

VIE

TNA

MV

IR-1

034

190

1790

yello

w/w

hit

ero

un

d26

/30

VIE

TNA

MSM

ALL

& W

HIT

E FR

UIT

59


33

8.2 All identified polymorphisms

Individual DFR Colour Long? Spines? Sample name position position position Allele Individual 3GT Colour Long? Spines? Sample name position Allele

236 310 368 96

1654 Purple Long None A1 C A T n 1654 Purple Long None D1 T n

1659 green round None A2 1659 green round None D2 T n

1792 yellow round None A3 1792 yellow round None D3 C a

710 green round None A4 C A T n 710 green round None D4

195 green round None A5 T A T a 195 green round None D5

1597 yellow long None A6 C A T n 1597 yellow long None D6 C a

693 purple round None A7 C A T n 693 purple round None D7

1571 yellow round None A8 C A T n 1571 yellow round None D8

1581 purple round None A9 C A T n 1581 purple round None D9 T n

1637 purple long None A10 C A T n 1637 purple long None D10

706 green round Spines A11 C A T n 706 green round Spines D11

1567 green round None A12 C A T n 1567 green round None D12 T n

1298 Purple Long None B1 C A T n 1298 Purple Long None E1 T n

1290 green round None B2 C G T b 1290 green round None E2 T n

356 yellow round None B3 C A T n 356 yellow round None E3 T n

415 green round None B4 C G T b 415 green round None E4

197 green round None B5 C A G c 197 green round None E5 T n

1838 yellow long None B6 C A T n 1838 yellow long None E6

Individual 5GT Colour Long? Spines? Sample name position position position position position position position position position position position allele

187 204 205 222 237 264 348 369 420 430 470

1654 purple round None E7 A G T C T A A A C A C

1659 yellow round None

1792 purple round None E9 A G T C T A A A C A C n

710 purple long None E10 A G T C T A A A C T C a

195 green round Spines E11 A A T C T A G G T A T b

1597 green round None E12 A G T C T A A A C A C n

693 Purple Long None F1 A A T C T A A G T A T c

1571 green round None F2 A G T C T A A A C A C n

1581 yellow round None F3 A G T C T A A A C A C n

1637 green round None

706 green round None F5 A A T C T A A G T A T c

1567 yellow long None F6 A G T C T A A A C A C n

1298 purple round None F7 A G T C T A A A C A C n

1290 yellow round None F8 A G T C T A A A C A C n

356 purple round None F9 A G T C T A A A C A C n

415 purple long None

197 green round Spines F11 A G T T T A A A C A C d

1838 green round None F12 C G G C G C A A C A C e

Individual OV Colour Long? Spines? Sample name position position position position position allele

98 100 103 413 472

1654 Purple Long None G1 T G T C G n


1792 yellow round None G3 G A A T G b

710 green round None G4 T C T C G c

195 green round None G5 G A A C A d

1597 yellow long None G6 T G A C G e

693 purple round None G7 T C A C G a

1571 yellow round None G8 T C A C G a

1581 purple round None G9 T A A C G f

1637 purple long None G10 T C A C G a

706 green round Spines G11 T C A C G a


1298 Purple Long None H1 T A A C G f


356 yellow round None H3 T A A C G f


197 green round None H5 T A A C G f

1838 yellow long None H6 T C T C G c

Documents

Dissertation Jessica Denning 25300792