A snp based consensus genetic map for synteny based

Plant Biotechnology Journal (2015), pp. 1–9

doi: 10.1111/pbi.12371

A SNP-based consensus genetic map for synteny-based trait targeting in faba bean (Vicia faba L.) Anne Webb1,†, Amanda Cottage1,†, Thomas Wood1, Khalil Khamassi1,‡, Douglas Hobbs1, Krystyna Gostkiewicz1, Mark White2, Hamid Khazaei3,‡, Mohamed Ali4,§, Daniel Street5, G erard Duc6, Fred L. Stoddard3, Fouad Maalouf7, Francis C. Ogbonnaya7,#, Wolfgang Link4, Jane Thomas1 and Donal M O’Sullivan1,8,* 1

National Institute of Agricultural Botany, Cambridge, UK

2

PGRO, Thornaugh, Peterborough, UK

3

Department of Agricultural Sciences, University of Helsinki, Helsinki, Finland Department of Crop Sciences, Georg-August-Universita€t, Go€ttingen, Germany

4 5 6

LGC Ltd (Genomics Division), Hoddesdon, Herts, UK INRA, UMR1347 Agroe cologie, Dijon, France

7

ICARDA, Terbol, Lebanon

8

School of Agriculture, Policy and Development, University of Reading, Whiteknights, UK

Received 8 December 2014; revised 12 February 2015; accepted 3 March 2015. *Correspondence (Tel +44 118 3786729; fax +44 118 926 1244; email d.m.osullivan@reading.ac.uk) † These authors made an equal contribution. ‡ Present address: Plant science Department, University of Saskatchewan, Agriculture Building, 51 Campus Drive, S7N 5A8 Saskatoon, SK, Canada. § Present address: Agronomy Department, College of Agriculture, Assiut University, Assiut 71526, Egypt # Present address: Grains Research and Development Corporation (GRDC) Level 4 East Building, 4 National Circuit, Barton, ACT 2600, Australia.

Keywords: legume, faba bean, KASP genotyping, single nucleotide

Summary Faba bean (Vicia faba L.) is a globally important nitrogen-fixing legume, which is widely grown in a diverse range of environments. In this work, we mine and validate a set of 845 SNPs from the aligned transcriptomes of two contrasting inbred lines. Each V. faba SNP is assigned by BLAST analysis to a single Medicago orthologue. This set of syntenically anchored polymorphisms were then validated as individual KASP assays, classified according to their informativeness and performance on a panel of 37 inbred lines, and the best performing 757 markers used to genotype six mapping populations. The six resulting linkage maps were merged into a single consensus map on which 687 SNPs were placed on six linkage groups, each presumed to correspond to one of the six V. faba chromosomes. This sequence-based consensus map was used to explore synteny with the most closely related crop species, lentil and the most closely related fully sequenced genome, Medicago. Large tracts of uninterrupted colinearity were found between faba bean and Medicago, making it relatively straightforward to predict gene content and order in mapped genetic interval. As a demonstration of this, we mapped a flower colour gene to a 2-cM interval of Vf chromosome 2 which was highly colinear with Mt3. The obvious candidate gene from 78 gene models in the collinear Medicago chromosome segment was the previously characterized MtWD40-1 gene controlling anthocyanin production in Medicago and resequencing of the Vf orthologue showed a putative causative deletion of the entire 50 end of the gene.

polymorphism, synteny.

Introduction Faba bean is a globally significant grain legume, with 4–5 million tonnes annual production worldwide in recent years (FAO, 2014), providing the predominant affordable dietary source of protein to subsistence farmers and urban populations across North Africa, Horn of Africa (including Ethiopia) and the Middle and Near East as well as parts of China. The use of faba bean is deeply embedded in the cuisine and culture of these countries and the great agronomic and morphological diversity of the crop genepool has permitted it to become adapted to many agroecological settings and different end uses (Cubero, 1974; Duc et al., 2010).

As a nitrogen-fixing legume, it has an important role in

maintaining soil fertility in rotational systems (Jensen et al., €pke and Nemecek, 2010). However, faba bean produc2010; Ko tion and consumption are becoming increasingly geographically disjointed, with alarming reductions in output in precisely those countries with rapidly growing populations that depend most heavily on faba beans as a dietary protein source. For example, Egypt’s self-sufficiency in faba bean production fell from 96% to 52% between 1998 and 2009 due to widespread infestation of the Nile Delta region by Orobanche crenata Forsk. and the near abandonment of faba bean production in the middle Nile Valley region due to the prevalence of faba bean necrotic virus (Mahmoud Zeid, pers. commun.). Moreover, the almost unchecked spread of parasitic weeds of the genus Orobanche

Please cite this article as: Webb, A., Cottage, A., Wood, T., Khamassi, K., Hobbs,D., Gostkiewicz, K., White, M., Khazaei, H., Ali, M., Street, D., Duc, G., Stoddard, F.L., Maalouf, F., Ogbonnaya, F.C., Link, W., Thomas, J. and O’Sullivan, D.M. (2015) A SNP-based consensus genetic map for synteny-based trait targeting in faba bean (Vicia faba L.). Plant Biotechnol. J., doi: 10.1111/pbi.12371

ª 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd

1

2 Anne Webb et al. has led to abandonment for faba bean growing of entire provinces not only in Egypt but across the entire Mediterranean basin (Maalouf et al., 2009). These as well as other production constraints such as drought and extreme temperature sensitivity could potentially be addressed by more effective genomicsenabled genetic analysis/breeding strategies.

Despite the global significance of the crop and the urgency of researchable challenges it faces, faba bean genetic and genomic resources have only started to be developed in the last few years, due in part at least to its outcrossing habit and large (approx.13 Gb) genome size. Initially, mapping studies have been based on random amplified polymorphic DNA (RAPD) marker technology and delivered small numbers of cross-specific trait-linked sequence characterized amplified region (SCAR) markers (Avila et al., 2003, 2004; Diaz-Ruiz et al., 2010; Gutierrez et al., 2006, 2007). Later, the development of simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) marker sets potentially more amenable to deployment in genetic studies and breeding have been reported (Ellwood et al., 2008; Gong et al., 2011; Kaur et al., 2012; Ma et al., 2011; Wang et al., 2011; Zeid et al., 2009). The recent development of the gene sequence-based maps of V. faba (CruzIzquierdo et al., 2012; Ellwood et al., 2008; El-Rodeny et al., 2014; Kaur et al., 2014a; Satovic et al., 2013) has been a big step forward for faba bean genetics. However, so far, gene-based linkage maps have either utilized multiple low-throughput marker systems to achieve assignment of most major linkage groups to one of the six chromosomes (Satovic et al., 2013) or generated multiple linkage groups per chromosome, mostly unassigned (El-Rodeny et al., 2014; Kaur et al., 2014a,b), and therefore, there is not yet a single genotyping platform for V. faba which permits genomewide alignment to a comprehensive consensus map providing coverage of all six chromosomes and supports systematic targeting of marker development to regions of interest exploiting synteny with sequenced legumes.

We previously demonstrated conversion of set of mapped cleaved amplified polymorphic sequence (CAPS) and SNAPshot assays to Kompetitive Allele Specific PCR (KASP) format in faba bean (Cottage et al., 2012). Here, we significantly increase the SNP coverage of the faba bean genome by discovering, validating and mapping a much larger number of SNP markers, and further pursue the objective of making all useful SNPs readily accessible by opting for the low-cost, easy-to-use, KASP genotyping platform. We find extensive colinearity of the V. faba linkage map with the sequenced model legume M. truncatula and demonstrate the potential for application of this new syntenically anchored SNP map in trait dissection and breeding via the identification of a candidate gene/causative polymorphism for white-coloured flowers.

Polish variety ‘Albus’ and NV648-1, a derivative of the ICARDA Bean Pure Line ‘BPL100 , a coloured flower, black hilum inbred line – yielded 17 397 and 16 041 contigs >200 bp in length for NV643-4 and NV648-1, respectively. From amongst these 5562 and 5534 contigs of NV643-4 and NV648-1, respectively, aligned to contigs from the other V. faba line as well as to at least one putative gene of M. truncatula. These transcript assemblies are available from the GenBank Transcript Sequence Assembly (TSA) repository under the accession names GARZ00000000 (NV643-4) and GASA00000000 (NV648-1). The versions described in this paper are the first versions, GARZ01000000 and GASA01 000000, respectively. Tables 1 and 2 show some metrics of the sequencing runs and assembly results.

Information content of validated KASP SNP assays Of 1001 selected target sequences (data not shown) containing putative NV643-4/NV648-1 SNPs, assays were successfully designed for 845 SNPs (for details of individual target sequences, Table S1). To provide continuity with previous work (Cottage et al., 2012) and a link to the previously published Vf6 9 Vf27 maps (Cruz-Izquierdo et al., 2012; Ellwood et al., 2008), the 48 most informative SNP assays developed from the Vf6 9 Vf27 map (Cottage et al., 2012) were included in the subsequent analyses. With the exception of the aforementioned set of 48 previously described SNPs, all SNPs described here were discovered by alignment of sequences from just a single pair of genotypes. Therefore, we wished to assess allele frequencies in a diverse germplasm sample so that assays could be scored for information content and to quantify any ascertainment bias towards rare alleles, peculiar to one or other of the ‘discovery’ genotypes. A second, overlapping objective was to screen a set of parents of mapping populations that already existed or are under development, so that users of these populations could quickly extend coverage of their linkage maps to encompass polymorphic SNPs from the new set. To fulfil these complementary purposes, a set of 37 inbred lines (the ‘validation panel’) was assembled and genotyped (Table 3). A total of 891 SNP assays were validated and assigned to one of four quality classes based on call rate and tightness of clustering of the three possible genotypic classes.

Figure S1 shows raw genotyping data for two SNPs assigned to quality classes I and IV, respectively. For the remainder of analyses, only the 757 SNPs, henceforth referred to as ‘NV648/ NV6430 SNPs, belonging to quality classes I and II are considered further. Allele calls for only 67.6% 512 of 757 of the high-quality ‘NV648/NV6430 SNP assays matched the SNP allele predicted from sequencing (Table S1). Of the 245 mismatched allele calls, Table 1 Statistics for 454 sequencing of V. faba ‘Albus’ and ‘BPL10’

Results

Average raw

Transcriptome resequencing for SNP discovery Pyrosequencing and de novo transcript assembly of RNA from seedling tissues of two contrasting faba bean lines – NV643-4, a partly inbred line derived from the white flowered, white hilum

Genotype

Tissue

Total reads

read length

NV643-4 (Albus)

10-day-old seedling

788 918

525

NV648-1 (BPL10)

10-day-old seedling

751 023

527

Table 2 Statistics for transcriptome assembly of V. faba ‘Albus’ and ‘BPL10’ in gsAssembler, implemented in Newbler v. 2.3 (Roche) Genotype

Total contigs

N50

Total coverage %

Contigs in TSA submission

GenBank TSA accession

NV634-4

26 709

1152

90.10

17 397

GARZ00000000

NV648-1

21 591

1119

89.72

16 041

GASA00000000

ª 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd, Plant Biotechnology Journal, 1–9

A faba bean SNP-based consensus genetic map 3 Table 3 Inbred lines of V. faba, used to validate panel of SNP markers. The original accessions were provided by National Institute of Agricultural Botany (NIAB), United Kingdom, The Georg-August€ ttingen (GAUG), Germany; the International Center for University, Go Agricultural Research in Dry Areas (ICARDA), Syria; the Instituto de n y Formacio n Agroalimentaria (IFAPA), Spain; the Investigacio University of Helsinki (UoH) Finland; and the Institut National de la R echerche Agronomique (INRA), France Accession

Other

code

reference

NV153-1

ig12658

NIAB

98.73

1.27

NV639-1

Hedin

GAUG

98.61

1.39

NV643-3

Albus

NIAB

97.85

2.15

NV644-1

Kasztelan

NIAB

92.56

7.44

NV648-2

BPL10

ICARDA

98.87

1.13

NV656-3

ig101942

NIAB

99.37

0.63

NV657-2

INRA-29H

IFAPA

99.11

0.89

NV658-2

CGN07715 cf-3

GAUG

99.24

0.76

NV662-1

IFAPA

97.22

2.78

NV713-1

VF136 ^te d’Or Co

GAUG

98.36

1.64

NV714-1

Hiverna

GAUG

99.25

0.75

Towards a consensus SNP-based genetic linkage map

NV715-1

Webo

GAUG

99.10

0.90

NV716-1

Wibo

GAUG

99.36

0.64

NV717-1

L79-79

GAUG

99.50

0.50

NV718-1

L977-88

GAUG

99.75

0.25

NV719-1

L979-S1

GAUG

99.62

0.38

NV720-1

Bourdon

GAUG

99.24

0.76

NV721-1

Arrisot

GAUG

98.99

1.01

NV722-1

Banner

GAUG

98.99

1.01

NV723-1

Bulldog

GAUG

99.12

0.88

NV724-1

Pietranera

GAUG

99.24

0.76

NV725-1

GIZA3-2

GAUG

99.36

0.64

NV726-1

GIZA402

GAUG

99.11

0.89

NV727-1

BPL4628

ICARDA

98.86

1.14

NV728-1

TW

ICARDA

99.11

0.89

NV729-1

VF6

ICARDA

98.58

1.42

NV730-1

ILB4347-4

ICARDA

99.24

0.76

NV731-1

ILB4347-3

ICARDA

99.50

0.50

NV732-1

BPL710

ICARDA

97.83

2.17

NV733-1

NA112

ICARDA

99.12

0.88

NV734-1

ILB938 lodie Me

UoH

99.62

0.38

NV735-1

UoH

98.99

1.01

NV736-1

Aurora

UoH

98.22

1.78

NV737-1

CRB285

INRA

97.11

2.89

NV738-1

CRB2516

INRA

99.21

0.79

The six mapping populations listed in Table 4 were used to construct first individual linkage maps and in a second step, a consensus map for V. faba. The framework for the consensus map was provided by the NV648-1 9 NV643-4 F2 linkage map, where 470 of the 757 ‘NV648/NV6430 and 10 of the ‘Vf6/Vf270 SNP markers could be placed in just six linkage groups. The other five maps were more fragmented with between 12 and 20 linkage groups and less well populated with between 109 and 231 SNPs. Nonetheless, 207 SNPs not placed in the framework NV648-1 9 NV643-4 F2 map were added to the consensus map through the integration of these additional populations. The final consensus map constructed using Merge Map (Wu et al., 2011), referred to as the Vf 2014 Consensus, consisted of 687 SNPs (653 ‘NV646/NV6430 and 34 ‘Vf6/Vf270 SNPs) corresponding to 542 unique, that is non-co-segregating, loci on six linkage groups and spanning a total length of 1403.8 cM (Figure 1, Table S2). The average gap size in this map is 2.6 cM, and the largest single gap is 24.7 cM. We postulate that these six linkage groups correspond to the six physical chromosomes. Based on the distribution of ‘Vf6/Vf270 SNPs from linkage groups previously assigned to physical chromosomes (Cruz-Izquierdo et al., 2012; Ruiz-Rodriguez et al., 2014), we have numbered these six linkage groups I-VI to reflect the physical assignments already made (Table S3).

NV739-1

CRB2702

INRA

94.63

5.37

NV740-1

CRB100107

INRA

99.60

0.40

Donor

% Homo-

% Hetero-

zygosity

zygosity

both alleles were observed amongst the 35 nondiscovery genotypes, but amongst the remaining 73 SNPs, the clear majority (60) of minor alleles were private to the NV648-1 discovery genotype, indicating that this line is the more ‘exotic’ of the discovery genotypes with respect to the diversity sampled in the validation panel. Figure S2 shows the genetic distance relationships between lines of the validation panel. As expected, BPL10 is on the longest branch, reflecting the ascertainment bias of the SNP discovery. However, polymorphism information content (PIC) values were high with an average of 0.316 amongst the 656 nonprivate SNPs. To add SNPs discovered using different discovery genotypes and markers which could be polymorphic both in the previously reported Vf6 9 Vf27 gene-based linkage map and maps created using the new SNPs described here, 40 of the markers from the Vf6 9 Vf27 map (Cottage et al., 2014), henceforth referred to as ‘Vf6/Vf270 SNPs were genotyped on the validation panel. The average PIC of this subset of SNPs was 0.329. Heterozygosity amongst the inbred lines using the combined set of 837 class I–II SNPs averaged 1.35%, consistent with the status of these as relatively pure research lines used for genetic analyses.

223 were associated with NV643, where unfortunately, individuals from distinct sublines of this accession had been used for transcriptome sequencing and crossing, respectively. For 241 of 245 of the class I-II SNPs where discovery and genotyped calls were discordant, both alleles were present in the validation panel despite the lack of the predicted NV643-3 (discovery sequence) allele in NV643-4 (genotyped) DNA, and therefore, we attribute the majority of mismatches in predicted versus observed allele calls to NV643 stock heterogeneity rather than sequencing errors or flaws in the assay design. Amongst the 512 class I-II SNPs where discovery sequence and SNP genotype were concordant, 438 were nonprivate with respect to the discovery genotypes as

Colinearity of the faba bean genetic map with the sequenced M. truncatula genome and the first SNP map of Lens culinaris Synteny between the ‘Vf 2014 Consensus’ linkage map and the M. truncatula Mt3.5 pseudomolecule assembly, and the first comprehensive SNP map for lentil (Sharpe et al., 2013) and M. truncatula was investigated by assigning orthologous links to reciprocal best BLAST hits between the Medicago genome, SNPflanking sequence from V faba and SNP-containing contigs assembled from several lines of lentil. The comparative map view shown in Figure S3 represents the full set of links thus obtained at a BLAST cut-off of E 30 and portrays a relatively simple pattern of colinearity between the respective genomes. Mt1 is colinear in a single, virtually uninterrupted block with Vf Chr III and in lentil to the central part of Linkage group 1 (LC_1) which is thought to represent

ª 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd, Plant Biotechnology Journal, 1–9

4 Anne Webb et al.

Table 4 Mapping populations genotyped with SNP markers developed in this study Population

Male parent

Female parent

progeny analysed

1

NV643-1

NV648-1

136 F2

2

NV643-1

NV657

165 F2

3

NV639

NV658

52 F2

4

NV153-1

5

NV644-1 lodie/2 Me

6

^te D’Or/1 Co

mapped SNPs

linkage groups

map length (cM)

Segregating traits

481

6

1166.5

Flower colour (white/wild type)

231

13

983.3

Flower colour (white/wild type)

204

18

1198.9

Flower opening (closed/open)

125 F2

192

12

1164.9

Plant height (dwarf/tall)

ILB938/2

194 F5

200

13

818.2

Vicine-convicine (high/low),

BPL4628/1521

101 RILs

108

20

158.1

Frost hardiness

stipule spot (present/absent)

chromosome 1 of L. culinaris. Similarly, Mt2 appears colinear to Chr 1 in bean and Lc2 in lentil. Also, Mt3 and Mt6 chromosomes appear colinear to substantial blocks of Vf Chr II and I in faba bean and Lc3 and Lc2 in lentil, respectively. Mt7 is colinear to blocks of Vf Chr V and I in faba bean and Lc 8in lentil, and Mt8 is colinear with large blocks in Vf Chr VI and Chr IV in bean and smaller areas of Lc1, 4 and 7 in lentil. Large parts of Mt4 are syntenous to Vf Chr II,IV and VI and Lc4 and Lc7. This broad picture of high degree of conservation of gene order over few, extensive blocks of colinearity suggests that the gene content and order of V. faba genetic intervals currently lacking SNP coverage can be relatively straightforwardly and accurately predicted from aligned stretches of the M. truncatula reference genome sequence.

Mapping of the ZT1 zero-tannin end-use quality trait Condensed tannins have long been recognized as antinutritional factors when faba bean is used as the main protein source in feed formulations for monogastric livestock and poultry (Bond, 1976).

Previous work has shown that the absence of the precursors of condensed tannins, the anthocyanins, in floral tissues, resulting in a highly recognizable white flower phenotype, is pleiotropic to the absence of condensed tannins in the seed coat (Bond, 1976; Picard, 1976). The zero-tannin (zt) trait as it is called, and has been genetically defined as a simple character, governed independently by two complementary recessive genes zt-1 and zt-2 (Cabrera and Martin, 1989; Picard, 1976). SCAR markers linked to the more common zt-1 source of zero tannin/white flower were identified by bulked segregant analysis (Gutierrez et al., 2007) but the resulting linkage group was not associated with any particular chromosome and the sequence of the markers gives no clue as to their location in the V. faba genome. The segregation of the white flower phenotype in the NV648 (♀) 9 NV643 (♂) F2 population showed a simple 3 : 1 coloured-to-white ratio, indicating a single dominant gene was responsible, with the dominant allele coming from the coloured parent. The trait mapped to Vf chromosome 2, in a region of highly conserved synteny with Medicago chromosome 3, between markers Vf_Mt3g092810_001 and Vf_Mt3g094760_001 (Figure S4A and B).

Use of synteny to target marker development to regions of interest and identify candidate genes The Medicago chromosome segment lying between flanking markers Vf_Mt3g092810_001 and Vf_Mt3g094760_001 corresponds to approx. 0.57 Mb of DNA with 78 predicted gene models (Figure S4B). Most of these encode hypothetical proteins associated with various retroelements and just two of these were considered as positional candidates for regulating the expression of flower colour in V. faba.

A gene ortholog of the Medicago WD-40 transcription factors, Transparent Testa Glabra 1 (TTG1) (Medtr3g092830, Medtr

3g092840), was identified in the interval between Vf_Mt3g92810_001 and Vf_Mt3g094760_001. Roles in master regulation of the anthocyanin biosynthesis pathway for TTG1 orthologs have been suggested in both Arabidopsis thaliana (Walker et al., 1999) and M. truncatula (Pang et al., 2009), whereas a deletion in the A2 gene, an ortholog of Medtr3g092840 in Pisum sativum, has been demonstrated to be responsible for a loss of flower colour (Hellens et al., 2010).

Their known conserved role in regulation of flower and seed anthocyanin production across Arabidopsis, Medicago and pea therefore made these compelling biological candidates for regulating anthocyanin production and thus flower pigmentation in V. faba. We therefore proceeded to investigate whether sequence variation at the Vf_Mt3g092840 locus, which we will henceforth refer to as VfTTG1, could be responsible for the loss of flower colour in NV643. The coding region of VfTTG1 was cloned and sequenced from NV648 (Figure S4C). However, the 50 end of the VfTTG1 sequence could not be amplified in the white-flowered NV643 line, suggesting a sequence difference or deletion affecting the 50 priming site. Genome walking was utilized to obtain the sequence of the 50 region of the VfTTG1 locus in NV643. This showed that the entire 50 end of VfTTG1 gene and upstream promoter elements were missing. The 50 flanking sequence instead showing high levels of homology to the VfENOD11 gene (a 213 bp region) and an adjacent region of 388 bp with homology to the PsIPT1 locus, encoding adenylate isopentenyltransferase) as illustrated in Figure S4D. Primers designed to amplify across the putative deletion amplified a fragment of the expected size (data not shown).

An allelism test conducted by crossing NV643 to known sources of zt-1 and zt-2 allowed us to conclude that VfTTG1 is in fact, ZT1 (data not shown). Taken together, the allelic variation, positional and functional data from related taxa strongly indicate that a deletion in the coding region of VfTTG1/ZT1 in V. faba is responsible for the loss of floral pigmentation in NV643 and demonstrates how translational functional genomics exploiting well-described synteny between Medicago and V. faba can be used to quickly and accurately identify causative genes contributing to specific phenotypes in a crop.

Discussion Emergence of V. faba from ‘orphan legume’ status In the space of just over 1 year, faba bean entries in GenBank have gone from 5000 EST reads (Ray and Georges, 2010) to multiple transcriptomes (Kaur et al., 2014a,b; this study). Like pigeon pea, groundnut, lentil and other legumes, there is now a linkage map available with a density of marker coverage appropriate to most currently available RIL mapping populations.

ª 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd, Plant Biotechnology Journal, 1–9

A faba bean SNP-based consensus genetic map 5

I 0.0 0.4 1.3 1.7 2.1 2.9 3.8 4.5 4.9 5.3 5.9 8.3 9.4 10.5 11.3 12.4 14.6 15.0 26.1 26.5 27.2 28.0 37.2 38.3 39.0 40.9 42.2 43.0 44.2 45.9 48.2 48.6 48.7 51.2 56.6 59.6 60.7 62.4 64.0 64.4 71.3 73.9 75.7 76.0 77.6 77.9 78.3 78.5 78.8 80.0 80.6 83.7 84.4 86.6 88.7 91.6 92.0 94.2 94.5 95.8 96.9 100.6 106.1 110.2 111.0 112.1 114.0 116.0 116.8 118.1 118.5 118.9 120.0 121.0 122.2 124.0 126.6 128.3 132.5 137.5 138.7 142.0 146.4 153.9 155.4 159.1 167.5 168.2 170.9 171.5 179.1 180.2 180.9 183.4 184.1 184.5 184.9 189.2 189.7 190.4 200.0 201.5 205.3 205.6 212.5 213.4 214.8 218.1 219.6 220.0 220.4 223.1 224.2 224.9 226.2 229.0 229.4 230.3 232.1 236.7 238.0 244.9 247.1 247.5 249.8 250.1 250.5 251.8 256.4 256.8 267.3 270.4 271.6 275.0 277.0 280.9 284.0 284.4 284.8 288.7 294.2 304.8 311.9 315.3 326.5 331.1 335.5 338.5 338.9 350.0 358.5 359.0 359.9 363.6 364.2 379.0 386.7 388.4 390.5 391.8 393.0 396.4 397.9 405.2 407.4 410.0 412.4 416.0 417.7

II Vf_Mt2g005590_001 Vf_Mt2g005930_001 Vf_Mt2g005900_001 Vf_Mt2g006070_001 Vf_Mt1g083330_001 Vf_Mt2g007220_001 Vf_Mt2g007780_001 Vf_Mt2g007930_001 Vf_Mt2g008150_001 Vf_Mt2g008440_001 Vf_Mt2g008610_001 Vf_Mt2g009320_001 Vf_Mt2g010540_001 Vf_Mt2g010740_001 Vf_Mt2g010880_001 Vf_Mt2g010980_001 Vf_Mt2g010970_001 Vf_Mt2g011080_001 Vf_Mt2g012470_001 Vf_Mt2g012810_001 Vf_Mt2g013220_001 Vf_Mt2g013690_001 Vf_Mt2g013900_001 Vf_Mt2g015010_001 Vf_Mt2g015370_001 Vf_Mt2g015390_001 Vf_Mt2g019790_001 Vf_Mt2g020160_001 Vf_Mt2g021130_001 Vf_Mt5g084030_001 Vf_Mt2g025490_001 Vf_Mt2g026180_001 Vf_Mt2g026550_001 Vf_Mt2g027240_001 Vf_Mt2g027860_001 Vf_Mt2g028000_001 Vf_Mt2g027100_001 Vf_Mt2g028620_001 Vf_Mt2g030200_001 Vf_Mt2g030240_001 Vf_Mt2g029180_001 Vf_Mt2g036810_001 Vf_Mt2g033460_001 Vf_Mt2g034610_001 Vf_Mt2g035830_001 Vf_Mt2g037970_001 Vf_Mt2g038390_001 Vf_Mt2g042700_001 Vf_Mt8g062520_001 Vf_Mt5g007030_001 Vf_Mt5g005990_001 Vf_Mt5g007360_001 Vf_Mt5g008210_001 Vf_Mt5g008450_001 Vf_Mt5g008680_001 Vf_Mt5g009720_001 Vf_Mt5g009120_001 Vf_Mt5g009240_001 Vf_Mt5g010190_001 Vf_Mt5g011550_001 Vf_Mt5g010620_001 Vf_Mt5g011520_001 Vf_Mt5g012030_001 Vf_Mt5g013300_001 Vf_Mt5g013540_001 Vf_Mt5g013590_001 Vf_Mt5g013980_001 Vf_Mt5g014410_001 Vf_Mt5g015050_001 Vf_Mt5g015280_001 Vf_Mt5g015500_001 Vf_Mt5g016250_001 Vf_Mt5g016600_001 Vf_Mt5g017090_002* Vf_Mt1g016750_001 Vf_Mt5g018600_001 Vf_Mt5g021170_001 Vf_Mt5g022700_001 Vf_Mt5g022580_001 Vf_Mt5g023210_001 Vf_Mt5g024730_001 Vf_Mt5g024900_001 19a15_3 Vf_Mt5g025470_001 Vf_Mt5g026550_001 Vf_Mt5g025960_001 Vf_Mt5g026650_001 Vf_Mt5g026780_001 Vf_Mt5g027460_003 Vf_Mt5g026840_001 Vf_Mt5g027530_001 Vf_Mt5g027980_001 Vf_Mt5g044970_001 Vf_Mt5g044990_001 Vf_Mt5g045630_001 Vf_Mt5g046030_001 Vf_Mt5g044980_001 Vf_Mt5g043020_001 Vf_Mt5g041750_001 Vf_Mt2g088750_001 Vf_Mt5g039210_001 Vf_Mt5g035810_001 Vf_Mt5g037120_001 Vf_Mt5g038280_001 Vf_Mt5g039000_001 Vf_Mt5g037960_001 Vf_Mt5g033880_001 Vf_Mt5g033220_001 Vf_Mt2g063510_001 Vf_Mt5g005120_001 Vf_Mt5g005710_001 Vf_Mt5g098420_001 Vf_Mt5g098060_001 Vf_Mt5g097510_001 Vf_Mt5g097360_001 Vf_Mt5g097200_001 Vf_Mt5g095650_001 Vf_Mt5g093880_001 Vf_Mt5g093580_001 Vf_Mt5g092970_001 Vf_Mt5g091110_001 Vf_Mt5g089870_001 Vf_Mt5g090520_001 Vf_Mt5g090770_001 Vf_Mt5g091200_001 Vf_Mt5g087700_001 Vf_Mt5g086350_001 Vf_Mt5g083370_001 Vf_Mt5g083490_001 Vf_Mt5g083150_001 Vf_Mt5g080450_001 Vf_Mt5g079730_001 Vf_Mt5g076620_001 Vf_Mt5g076470_001 Vf_Mt5g076950_001 Vf_Mt5g077780_001 Vf_Mt5g078030_001 Vf_Mt5g076180_001 Vf_Mt5g076250_001 Vf_Mt5g076080_001 Vf_Mt5g075490_001 Vf_Mt5g075180_001 Vf_Mt5g074880_001 Vf_Mt5g072620_001 Vf_Mt5g073210_001 Vf_Mt5g068760_001 Vf_Mt5g067470_001 Vf_Mt5g066100_001 Vf_Mt5g063390_001 Vf_Mt5g059210_001 Vf_Mt5g055940_001 Vf_Mt5g047260_001 Vf_Mt5g032070_001 Vf_Mt5g032550_001 Vf_Mt2g064880_001 Vf_Mt6g032890_001 Vf_Mt2g071620_001 Vf_Mt2g064620_001 Vf_Mt2g069030_001 Vf_Mt6g013720_001 Vf_Mt6g071280_001 Vf_Mt6g071210_003 Vf_Mt6g082480_001 Vf_Mt6g084190_001 Vf_Mt6g071210_002 Vf_Mt6g088460_001 Vf_Mt6g087000_001 Vf_Mt6g090520_001 Vf_Mt6g092790_001 Vf_Mt6g092820_001 Vf_Mt7g100410_001 Vf_Mt6g005800_001 Vf_Mt6g008170_001 Vf_Mt6g012630_001 Vf_Mt6g018430_001 Vf_Mt6g023070_001 Vf_Mt6g054890_001 Vf_Mt2g075860_001 Vf_Mt6g059930_001 Vf_Mt2g075820_001 Vf_Mt2g075840_001 Vf_Mt2g075910_001 Vf_Mt2g075950_001 Vf_Mt2g076340_001 Vf_Mt2g082430_001 Vf_Mt2g086560_001 Vf_Mt2g086880_001 Vf_Mt2g086950_001 Vf_Mt2g086970_001 LG018SNP Vf_Mt2g089140_001 Vf_Mt2g093280_001 Vf_Mt2g098470_001 Vf_Mt2g097740_002 Vf_Mt2g097950_001 Vf_Mt2g098240_001 Vf_Mt2g098890_001 Vf_Mt2g099480_001 Vf_Mt7g021530_001 Vf_Mt8g017240_001 Vf_Mt7g020850_001 PGDH Vf_Mt7g016650_001 Vf_Mt7g013300_001 Vf_Mt7g010100_001 Vf_Mt7g013160_001 Vf_Mt7g080730_001

0.0 1.4 3.6 4.8 5.2 8.9 14.2 14.8 21.3 27.3 33.2 34.0 37.4 38.4 42.2 45.7 47.7 48.7 48.9 50.1 50.4 61.8 63.7 66.0 67.7 71.8 72.1 73.4 73.7 74.2 77.6 78.6 80.1 82.5 84.9 85.7 91.4 92.8 94.7 96.7 97.1 97.4 97.8 98.2 99.0 101.6 106.7 107.2 110.9 112.8 113.6 114.7 115.9 116.3 116.7 117.3 118.1 118.8 121.2 121.6 122.1 124.8 126.4 126.8 127.6 129.4 129.5 131.0 131.3 133.3 138.0 138.4 139.3 144.6 146.1 147.2 147.8 151.3 151.6 159.3 160.3 162.5 164.1 167.2 168.9 172.8 173.8 182.0 183.4 187.3 188.4 190.5 192.1 195.0 198.0 200.8 205.1 213.7 225.9 226.3 226.7 228.6 229.4 229.7 230.6

III Vf_Mt4g006200_001 Vf_Mt4g005830_001 LUP108SNP Vf_Mt4g007030_001 Vf_Mt4g009920_001 Vf_Mt4g010330_001 Vf_Mt4g014710_001 Vf_Mt4g014430_001 Vf_Mt4g016930_001 Vf_Mt4g021340_001 Vf_Mt4g021340_002* Vf_Mt4g025120_001* Vf_Mt4g029440_001 Vf_Mt4g031820_001 Vf_Mt4g035200_001 Vf_Mt4g033790_001 Vf_Mt3g117120_001 Vf_Mt3g116500_001 Vf_Mt3g115990_001 Vf_Mt3g116050_001 Vf_Mt3g116080_001 Vf_Mt3g115870_001 AIGPa Vf_Mt3g114780_001 Vf_Mt3g114850_001 Vf_Mt5g024090_001 Vf_Mt3g110600_001 Vf_Mt3g110630_001 Vf_Mt3g111210_001 LG102 Vf_Mt3g109330_001 Vf_Mt3g109200_001 Vf_Mt3g108190_001 Vf_Mt3g108320_001 Vf_Mt3g108460_001 Vf_Mt3g108790_001 Vf_Mt3g108630_001 Vf_Mt3g108690_001 Vf_Mt3g107970_001 Vf_Mt3g108160_001 Vf_Mt3g106820_001 Vf_Mt3g106510_001 Vf_Mt3g106240_001 Vf_Mt3g104850_001 Vf_Mt3g104700_001 Vf_Mt3g104310_001 Vf_Mt3g102180_001 Vf_Mt3g101630_001 Vf_Mt3g101740_001 Vf_Mt3g101420_001 Vf_Mt3g100500_001 Vf_Mt3g100220_001 Vf_Mt3g100050_001 Vf_Mt3g099460_001 Vf_Mt3g099130_001 Vf_Mt3g098730_001 Vf_Mt3g098530_001 Vf_Mt3g095660_001 Vf_Mt3g096560_001 Vf_Mt3g094760_001 Vf_Mt3g092810_001 Vf_Mt3g092990_001 Vf_Mt3g092310_001 Vf_Mt3g092460_001 Vf_Mt3g092090_001 Vf_Mt3g091840_001 Vf_Mt3g091770_001 Vf_Mt3g091780_001 Vf_Mt3g091460_001 Vf_Mt3g088610_001 Vf_Mt3g087760_001 Vf_Mt3g090490_001 Vf_Mt3g086980_001 Vf_Mt3g090670_001 Vf_Mt3g091190_001 Vf_Mt3g091380_001 Vf_Mt8g095930_001 Vf_Mt3g087030_001 Vf_Mt3g087150_001 Vf_Mt3g087150_002 Vf_Mt3g086910_001 Vf_Mt3g086600_001 Vf_Mt3g086210_001 Vf_Mt3g086230_001 Vf_Mt3g085570_001 Vf_Mt3g085280_001 Vf_Mt3g085270_001 Vf_Mt3g084040_001 Vf_Mt3g084090_001 Vf_Mt3g083770_001 Vf_Mt3g083180_001 Vf_Mt3g079200_001 Vf_Mt3g079210_001 Vf_Mt3g079330_001 Vf_Mt2g014220_001 Vf_Mt2g014220_003 Vf_Mt3g078630_002 Vf_Mt3g062540_001 Vf_Mt3g077490_001 Vf_Mt3g077670_001 Vf_Mt3g076650_001 Vf_Mt3g076660_001 Vf_Mt3g076590_001 GLIP337SNP Vf_Mt3g072030_001 Vf_Mt3g072360_001 Vf_Mt3g072390_001 Vf_Mt3g072080_001 Vf_Mt3g071620_001 Vf_Mt3g070310_001 RBPC_0SNP Vf_Mt3g065190_001 Vf_Mt3g064740_001 Vf_Mt3g031380_001 Vf_Mt3g031280_001 Vf_Mt3g061590_001 Vf_Mt3g061640_001 Vf_Mt3g060850_001 AnMtL8 Vf_Mt3g055690_001 Vf_Mt3g055920_001 Vf_Mt3g052760_001 Vf_Mt3g051280_001 Vf_Mt5g075540_001 Vf_Mt3g026020_001 Vf_Mt3g020670_001 Vf_Mt3g019330_001 Vf_Mt3g017610_001 GLIP135 Vf_Mt3g008180_001 Vf_Mt3g008540_001 Vf_Mt3g005050_001 Vf_Mt3g010000_001 Vf_Mt3g005360_001 Vf_Mt3g010290_001

IV

0.0

Vf_Mt1g116810_001

4.7

Vf_Mt1g116230_001

10.9

Vf_Mt1g050730_001

16.7 17.1 20.5

Vf_Mt1g114560_001 Vf_Mt1g116110_001 Vf_Mt5g042440_001 Vf_Mt1g045800_001

26.7 28.9

Vf_Mt1g044570_001 Vf_Mt4g080370_001

35.5

Vf_Mt1g056180_001

46.3

cgP137FSNP

51.4 51.8

Vf_Mt1g064060_001 Vf_Mt1g061800_001 Vf_Mt7g100500_001

66.4 66.7 67.7 68.1 71.9 72.6 74.1 77.3 77.7 81.8 83.3 83.6 85.1 85.9 88.1 91.5 93.8 93.9 95.8 96.5 97.5 102.0 114.7 118.6 119.4 123.9 125.4 125.8 130.2 131.0 133.3 136.3 141.9 145.8 149.8 150.9

Vf_Mt1g071110_001 GLIP245SNP Vf_Mt1g072640_001 Vf_Mt1g072740_001 Vf_Mt1g073000_001 Vf_Mt1g075140_001 Vf_Mt1g075610_001 Vf_Mt7g035110_001 Vf_Mt1g079520_001 GLPSNP Vf_Mt1g079870_001 Vf_Mt1g079930_001 Vf_Mt1g080150_001 Vf_Mt1g081290_001 Vf_Mt1g082210_001 Vf_Mt1g082420_001 Vf_Mt1g082580_001 Vf_Mt1g083230_001 Vf_Mt1g083450_001 Vf_Mt1g083460_001 Vf_Mt1g083960_001 Vf_Mt1g085040_001 Vf_Mt1g087900_001 GLIP133SNP Vf_Mt1g086810_001 LG031SNP LG038SNP Vf_Mt1g088190_001 Vf_Mt1g089980_001 REPSNP Vf_Mt1g094870_001 Vf_Mt1g095140_001 Vf_Mt1g095530_001 Vf_Mt1g099390_001 Vf_Mt1g099390_002 Vf_Mt1g100180_001 Vf_Mt1g100980_001 Vf_Mt1g101520_001 Vf_Mt1g101840_001 Vf_Mt1g102430_001 Vf_Mt1g105040_001 Vf_Mt3g049970_001 Vf_Mt1g108280_001 Vf_Mt1g110320_001 Vf_Mt1g110690_001

165.6 165.9 167.8 173.9 174.7 177.4 180.0 180.3 181.4 186.9 188.3 189.7

Vf_Mt3g076630_001 Vf_Mt1g030550_001* Vf_Mt1g031620_001 Vf_Mt1g007160_001 Vf_Mt1g008140_001 Vf_Mt1g009200_001 Vf_Mt1g007370_001 Vf_Mt1g007490_001 Vf_Mt1g012440_001 Vf_Mt1g012610_001 Vf_Mt1g014230_001 Vf_Mt1g013400_001

195.5

Vf_Mt1g016390_001

202.1 203.3 206.3 210.4 213.2 216.5

BGALSNP Vf_Mt1g017950_001 Vf_Mt1g018180_001 Vf_Mt1g018320_001 Vf_Mt1g018790_001 Vf_Mt1g019770_001 Vf_Mt1g019810_001 Vf_Mt1g021670_001 Vf_Mt1g021760_001 Vf_Mt7g059170_001 Vf_Mt1g025550_001 Vf_Mt1g025570_001 Vf_Mt1g025950_001 Vf_Mt1g025950_002 Vf_Mt1g026130_001

78.6

216.9 217.2

0.0 1.6 3.0 5.3 6.6 8.5 8.6 14.7 17.7 19.6 20.0

Vf_Mt4g133070_001 Vf_Mt3g118200_00 Vf_Mt3g118430_001 Vf_Mt3g117800_001 Vf_Mt3g118320_001 Vf_Mt4g132020_001 Vf_Mt4g131830_001 Vf_Mt4g132300_001 Vf_Mt4g130700_001 Vf_Mt4g128750_001 Vf_Mt4g128840_001 Vf_Mt4g128220_001 Vf_Mt4g127860_001 Vf_Mt4g127690_001

30.3 31.6 32.0 33.7 36.4 39.5 40.9 45.6 49.1

Vf_Mt4g125100_001 Vf_Mt4g125110_001 Vf_Mt4g124930_001 Vf_Mt4g124600_001 Vf_Mt4g124640_001 Vf_Mt4g124070_001 GLIP137 Vf_Mt4g122670_001 Vf_Mt4g121600_001 Vf_Mt4g121960_001 Vf_Mt4g118420_001 LG085SNP

55.9 62.4 62.8 63.2 63.5 66.7 67.4 74.6 78.0 78.8 82.3 84.3 86.3 86.9 88.2 91.4 100.2 105.1 111.0 111.9 112.2

146.4 146.8 148.7 158.7 159.9 161.8 163.3 168.2 171.0 175.3 176.0 176.4 177.4

Vf_Mt4g114900_001 Vf_Mt4g113280_001 Vf_Mt4g113270_001 Vf_Mt8g077000_001 Vf_Mt4g112900_001 Vf_Mt4g112530_001 Vf_Mt4g113090_001 Vf_Mt7g036900_001 Vf_Mt4g104240_001 Vf_Mt4g103550_001 Vf_Mt4g101920_001 Vf_Mt4g101130_001 Vf_Mt4g100760_001 GLIP139SNP Vf_Mt4g100510_001 Vf_Mt4g100930_001 Vf_Mt4g100920_001 Vf_Mt4g098740_001 Vf_Mt4g101620_001 Vf_Mt8g076060_001 Vf_Mt8g076780_001 Vf_Mt4g095360_001* Vf_Mt4g098710_001 Vf_Mt8g073110_001 Vf_Mt8g074530_001 Vf_Mt8g078230_001 Vf_Mt8g067490_001 Vf_Mt8g066730_001 Vf_Mt8g075260_001 Vf_Mt8g066630_001 Vf_Mt3g077990_001 Vf_Mt4g095440_001 Vf_Mt8g066180_001 Vf_Mt8g073580_001 Vf_Mt8g070510_001 Vf_Mt8g074010_001 Vf_Mt8g061890_001 Vf_Mt7g070290_001 Vf_Mt8g011470_001 GLIP253SNP Vf_Mt8g012400_001 Vf_Mt8g012380_001 Vf_Mt8g014660_001 Vf_Mt8g018770_001 13n10_1 PPHSNP Vf_Mt8g020800_001 Vf_Mt8g022050_001 Vf_Mt8g022290_001 Vf_Mt8g022770_001 Vf_Mt8g023310_001 CNGC4 Vf_Mt4g090120_001 Vf_Mt8g087440_001 Vf_Mt7g038120_001 Vf_Mt8g030430_001 Vf_Mt7g076160_001 Vf_Mt8g038880_001 Vf_Mt8g039690_001 Vf_Mt8g040150_001 Vf_Mt8g040550_001

187.5 188.2 192.6 194.1 194.5 199.8 203.1 205.2 206.0 208.9 209.9

PRATSNP Vf_Mt8g030600_001 Vf_Mt8g030620_001 Vf_Mt8g032450_001 Vf_Mt8g041410_001 Vf_Mt8g043510_001 Vf_Mt8g061230_001 Vf_Mt8g021140_001 Vf_Mt8g061350_001 Vf_Mt8g058270_001 GLIP089 Vf_Mt8g006370_001 Vf_Mt8g008870_001

115.3 116.6 117.4 118.9 120.8 121.4 126.2 128.2 129.4 130.7 134.3 138.3 142.4

V 0.0 1.1 2.5 5.0 8.0 9.5 10.6 12.7 13.2 14.4 17.3 20.0 22.5 33.6 35.1 36.1 48.7 56.8 62.1 62.9 64.1 65.6 66.3 67.2 67.5 67.9 68.8 69.2 72.2 73.3 76.7 77.1 79.3 80.5 84.5 88.2 90.5 92.0 92.3 98.0 99.9 100.1 100.6 103.9 111.4 113.6 114.8 115.2 115.6 116.2 124.5 126.4 128.0 129.1 133.0 141.3 143.6 144.7 145.3 146.1 147.2 151.0 152.2

VI Vf_Mt2g101430_001 Vf_Mt7g031900_001 Vf_Mt2g104010_001 Vf_Mt2g104110_001 Vf_Mt2g101870_001 Vf_Mt2g104100_001 Vf_Mt2g101350_001 Vf_Mt2g100090_001 Vf_Mt7g024320_001 Vf_Mt2g099510_001 Vf_Mt7g024540_001 Vf_Mt7g026340_001 Vf_Mt7g032240_001 Vf_Mt7g030010_001 Vf_Mt7g051360_001 Vf_Mt7g080890_001 Vf_Mt7g058910_001 Vf_Mt2g063200_001 Vf_Mt7g078800_001 Vf_Mt7g108490_001 Vf_Mt7g073340_001 Vf_Mt7g073970_001 Vf_Mt7g075940_001 AnMtS37SNP Vf_Mt7g078730_001 Vf_Mt7g079680_001 Vf_Mt7g079840_001 Vf_Mt7g080040_001 Vf_Mt7g080250_001 Vf_Mt7g081010_001 Vf_Mt7g086430_001 Vf_Mt7g081260_001 Vf_Mt7g084590_001 Vf_Mt7g083230_001 Vf_Mt7g085090_001 Vf_Mt7g084010_001 Vf_Mt7g088140_001 Vf_Mt7g086700_001 Vf_Mt7g088340_001 Vf_Mt7g088050_001 Vf_Mt7g088720_001 Vf_Mt7g090150_001 Vf_Mt7g090890_001 Vf_Mt7g090930_001 Vf_Mt7g092450_001 Vf_Mt7g092260_001 Vf_Mt7g093200_001 Vf_Mt7g093500_001 Vf_Mt7g104350_001 Vf_Mt4g076080_001 Vf_Mt8g078370_001 Vf_Mt7g098250_001 Vf_Mt7g099040_001 Vf_Mt7g099280_001 Vf_Mt7g098440_001 Vf_Mt7g099950_001 Vf_Mt7g100730_001 Vf_Mt7g100730_002* Vf_Mt7g101070_001 Vf_Mt7g101170_001 Vf_Mt7g102310_001 Vf_Mt7g104020_001 Vf_Mt7g104220_001 Vf_Mt7g104430_001 Vf_Mt7g104600_001 Vf_Mt7g104720_001 Vf_Mt7g104870_001 Vf_Mt7g105070_001 Vf_Mt7g108590_001 Vf_Mt7g109340_001 Vf_Mt7g109820_001 Vf_Mt7g110600_001 Vf_Mt7g111090_001 Vf_Mt7g112640_001 Vf_Mt7g112740_001 Vf_Mt7g113850_001 Vf_Mt7g114210_001 Vf_Mt7g114590_001 Vf_Mt7g114940_001 Vf_Mt7g115070_001 Vf_Mt7g116600_001 Vf_Mt7g117970_001 Vf_Mt7g118320_001

0.0 6.0 8.4 9.6 12.3 13.1 15.0 15.4 17.5 21.8 22.9

Vf_Mt8g107140_001 Vf_Mt8g107640_001 Vf_Mt8g106690_001 Vf_Mt8g104520_001 Vf_Mt8g105250_001 Vf_Mt8g103840_001 New_GLIP307SNP Vf_Mt8g102840_001 Vf_Mt8g102920_001 Vf_Mt8g102770_001 Vf_Mt8g102250_001 Vf_Mt8g101390_001 Vf_Mt3g034100_001

38.7 39.5

Vf_Mt8g100120_001 Vf_Mt8g095410_001

50.0 50.1 50.6 51.7 53.0 60.9 63.3 65.4 66.4 68.5 70.0

Vf_Mt8g091280_001 RNARSNP Vf_Mt8g093440_001 Vf_Mt8g092040_001 Vf_Mt8g092620_001 Vf_Mt8g093680_001 Vf_Mt8g088590_001 Vf_Mt8g087990_001 Vf_Mt8g085780_001 Vf_Mt8g085850_001 Vf_Mt8g086470_001 GLIP265SNP GLIP081SNP Vf_Mt8g083140_001 Vf_Mt4g085480_001 Vf_Mt4g085880_001 Vf_Mt4g085890_001 Vf_Mt4g085900_001 Vf_Mt4g087420_001 Vf_Mt4g087630_001 Vf_Mt4g086640_001 Vf_Mt4g087540_001 Vf_Mt4g087590_001 Vf_Mt4g088010_001 Vf_Mt4g091530_001 Vf_Mt4g092800_001 Vf_Mt4g092750_001 Vf_Mt4g092850_001 Vf_Mt4g093600_001 Vf_Mt4g093990_001

74.4 74.8 76.4 79.5 81.3 90.8 92.7 93.4 93.8

134.4 138.4 140.5 142.8 143.6 145.0 145.4 146.9 152.8 153.4

Vf_Mt4g081050_001 Vf_Mt4g077840_001 Vf_Mt4g077610_001 Vf_Mt4g078200_001 HYPTE3SNP Vf_Mt4g075050_001 Vf_Mt4g075200_001 Vf_Mt4g076990_001 Vf_Mt4g078660_001 LG068SNP Vf_Mt4g072160_001 Vf_Mt4g068110_001 Vf_Mt4g068130_001 Vf_Mt4g068480_001 Vf_Mt4g068550_001 Vf_Mt4g064820_001 Vf_Mt4g068010_001 Vf_Mt4g076870_001 Vf_Mt4g083970_001 Vf_Mt4g061670_001 Vf_Mt4g061070_001 Vf_Mt4g061010_001 Vf_Mt4g060940_001 Vf_Mt4g080100_001 Vf_Mt2g094640_001

163.4

Vf_Mt4g053880_001

171.5

Vf_Mt4g053250_001 Vf_Mt4g053270_001 Vf_Mt4g053520_001

176.2

Vf_Mt4g049540_001

118.5 121.1 122.8 128.3 129.9 130.3 132.5

Figure 1 A 687-locus consensus SNP-based map showing the six linkage groups thought to represent the six physical chromosomes of Vicia faba. Markers in red have been developed in previous studies. Graph created using MapChart (Voorrips, 2002).

Legume crop species now enjoying low-medium density SNP coverage of their genomes include cowpea (Muchero et al., 2009), common bean (Cort es et al., 2011), chickpea (Hiremath et al., 2012), lentil (Sharpe et al., 2013), pea (Deulvot et al., 2010) and faba bean (this work).

Uses and usability of Vicia faba SNP markers in markerassisted breeding Exploitation of molecular markers depends on many factors: their information content, entry cost, per data point cost,

ª 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd, Plant Biotechnology Journal, 1–9

6 Anne Webb et al. infrastructural requirements, ability to access information and coherence as a set of markers likely to provide reasonable genome coverage. We consider that we have attempted to address every single one of these bottlenecks. Our data show that PIC values are high, presumably as a consequence of high outcrossing rate, which suggests that these markers will be broadly useable in each of the main recognized seed size based gene pools (‘major’, ‘equina’ and ‘minor’). The KASP competitive PCR fluorescence assay chemistry was chosen with two particular criteria in mind: firstly, minimizing the cost to researchers of conducting low- to medium-resolution QTL scans and secondly, to cater for marker-assisted selection in real-world breeding programmes, where the cost per progeny eliminated on the basis of marker genotypes must be just pennies, ruling out any currently available genomewide genotyping technologies. Many mapping populations are available in the community and we hope that the lower cost barriers will make it possible for more groups to be involved in trait mapping than are currently. For example, SNP markers from this study found to be polymorphic in population 5 (Table 4) have been used to map QTL for stomatal traits (Khazaei et al., 2014a), low vicine–convicine (Khamassi et al., 2013; Khazaei et al., 2015) and a novel anthocyanin pigmentation locus, ssp1 (Khazaei et al., 2014b). Similarly, SNP markers from this study used to generate the linkage map for population 4 (Table 4) have been found linked to a novel dwarfing locus, Dwf1 (K. Khamassi, K. Gostkiewicz, and D.M. O’Sullivan, Unpublished data) and associated with frost tolerance (Sallam, 2014). Furthermore, to facilitate redesign of individual markers to suit existing laboratory set-ups and locations not easily served by KASP chemistry and commercial genotyping services, an interactive database-driven website (O’Sullivan and Green, 2012) has been set up to allow users to click from a marker name/ location through to a choice of viewing or downloading the original sequence alignment on which the SNP assay design was based as well as accessing clustering profiles, genotype scores and links to any associated references.

These data can be used to pick informative sets of SNPs discovered from four diverse genetic backgrounds and used to efficiently capture genomewide genetic relationships, measure diversity, map traits and once mapped, use marker-assisted selection schemes to breed for those traits.

Synteny-based mapping Our results broadly support, but refine and extend the conclusions of Cruz-Izquierdo et al. (2012) and Kaur et al. (2014a) regarding synteny between the eight M. truncatula chromosomes and the six V. faba chromosomes. The simple pattern of few, large blocks of colinearity between V. faba and M. truncatula reported here therefore greatly simplifies the task of translational functional genomics.

Synteny-based candidate gene discovery as practised for example in cowpea (Muchero et al., 2009) is now becoming a reality in faba bean. The identification of a candidate for the zerotannin trait illustrates this very well as the SNP markers most closely linked to the zero-tannin locus occur within a large block of colinearity between Mt3 and Vf LG2 in which a plausible candidate gene is found.

Towards isolation of causative genes for important trait variation Tannins (condensed polyphenols) are considered antinutritional compounds limiting the inclusion rates of faba bean in some

animal feed formulations. It has been established for a long time that tannins are absent from the testae of white-flowered bean lines, both phenotypes being manifestations of the absence of anthocyanin production. Recessive alleles at two separate complementary loci (zt1 and zt2) act independently to abolish anthocyanin production, testa tannins and flower colour markings. RAPD-based SCAR markers linked to zt1 were previously isolated using a bulked segregant analysis approach although these SCARs were not diagnostic for white flower colour in a panel of 37 European inbred lines and the linkage groups produced in this study did not contain any markers appearing on subsequent Vicia faba linkage maps (Cruz-Izquierdo et al., 2012).

Our results show that when the zero-tannin trait was placed on this syntenically anchored map, a rapid inspection of annotated gene content of the colinear region of the Mt3.5 genome revealed tandem copies of the WD40-repeat containing transcription factor, whose Arabidopsis orthologue had been functionally characterized as the TRANSPARENT TESTA GLABRA1 gene (Pang et al., 2009; Qi et al., 2011; Sagasser et al., 2002; Walker et al., 1999; Zhao et al., 2008). Although a causative polymorphism controlling determinate flowering trait in faba bean was previously isolated by amplification of the presumed functional ortholog of the TFL1 gene (Avila et al., 2007), this is, to our knowledge, the first time in faba bean that it has been possible to pinpoint a plausible causal polymorphism by combining a syntenically anchored SNP consensus map and sequence and gene function information from Medicago. The syntenic framework presented here provides a basis for the reconstruction of predicted Vf gene order encompassing the large majority of genes that fall within genetic intervals of nearly uninterrupted synteny. A ‘genome zipper’ of this nature is an essential stepping stone to exploit the power of population-based and bulk segregant mapping-by-sequencing approaches (Gardiner et al., 2014).

Experimental procedures Plant material The faba bean inbred lines used in this study either for transcriptome resequencing or SNP validation and their sources are listed in Table 3, as well as all mapping population parents. Genetic linkage mapping was conducted using six biparental populations, further details of which are presented in Table 4.

RNA library preparation and sequencing RNA was extracted from complete 7-day-old seedlings of part inbred line NV643-4 (Albus) and inbred line NV648-1 (BPL10) using the Qiagen RNeasy Plant Mini Kit, and sequenced using Roche 454 GS-FLX Titanium chemistry. Raw reads were assembled in gsAssembler, based on Newbler v2.3, available free from Roche Life Sciences at http://454.com/products/analysis-software/ index.asp (Anonymous, 2010).

CDS, whole transcript and protein sequences from M. truncatula were obtained online from the M. truncatula Genome Sequencing Project, led by the International Medicago Genome Annotation Group (IMGAG). Version Mt3.5 (Data freeze: 12/31/ 2009, Assembly release date: 02/10/2010, annotation release date: 05/27/2010) of the annotated genome of M. truncatula, consisting of 8 chromosomes and some unanchored BACs was downloaded from http://www.medicagohapmap.org/downloads.php on 01 December 2010.

ª 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd, Plant Biotechnology Journal, 1–9

A faba bean SNP-based consensus genetic map 7

SNP discovery and validation

Linkage mapping

Assembled contigs from NV643-4 and NV648-1 were aligned to each other and to transcript sequences of M. truncatula using BLASTn. All contigs which gave unique reciprocal best hits over at least 100 bases at a BLASTn cut-off of E 30 in the respective faba bean transcriptome assemblies and to a single predicted M. truncatula gene over more than 100 bases were selected for further analysis.

The complete contigs of each NV643-4:NV648-1 pair were aligned using ClustalW (Chenna et al., 2003) and alignments containing SNPs more than 50 bp from either end of a contig were flagged for marker development. For each SNP, a consensus sequence with the target SNP and at least 50 bp either side of it was then searched against the database of M. truncatula pseudomolecules v.3.5, using the BLASTn function (http://www.medicagohapmap.org/advanced_search_page.php? seq). Any consensus which matched multiple chromosomes of M. truncatula or in multiple copies on the same chromosome or whose M. truncatula alignments contained introns located closer than 50 bp of either side of the SNP were excluded. Sequences with more than 50 bp between the target SNP and the nearest intron site on the corresponding M. truncatula contig were truncated at the nearest intron–exon junctions to avoid primers being designed across intron–exon splice sites which would not anneal to genomic (intron-containing) sequence. The remaining SNP-containing sequences were then searched in BLASTn against all contigs of NV643-4 and NV648-1. Any SNPcontaining alignments with hits to more than one contig from each line were excluded. A total of 887 KASP probes were designed by LGC Genomics Ltd. and each assay given an internal LGC Genomics ID as well as an assay name following the format Vf_MtXgYYYYYY_ZZZ where X indicates the Medicago chromosome number, YYYYYYY the predicted gene identifier of the reciprocal best BLAST hit M. truncatula orthologue as given in version Mt3.5 of the Medicago genome assembly (Young et al., 2011), and ZZZ the number of the assay (Table S1).

A panel of 37 lines of V. faba (Table 3) chosen to represent the parents/founders of key mapping population resources was assembled and DNA extracted from a seedling leaf of a single plant per line using a modified Tanksley method (Fulton et al., 1995). Designed KASP assays were genotyped across this panel according to the manufacturer’s instructions.

MapDisto 1.7.5.1 for Windows (Excel 2007 version) was used to find and refine linkage groups (Lorieux, 2012). The LOD was set to five and the map was generated using the procedure Automap. For calculation of all individual linkage maps, the maximum permitted recombination fraction, the parameter r in MapDisto, was set to 0.3, except for population four where the use of a more stringent setting of 0.2 was required to avoid spurious linkage. The consensus map was generated in MergeMap (Wu et al., 2008) from the linkage groups generated in MapDisto. Graphs were produced using MapChart (Voorrips, 2002).

SNP-marker quality filtering Clustering of alternate homozygous SNP alleles and heterozygous genotypes was manually inspected using LGC Genomics SNP viewer. Each SNP assay was scored on a I–IV scale according to tightness of clustering and the number of missing data calls. As an additional criterion on which to rank assays, gene diversity, often referred to as expected heterozygosity, a measure of how informative individual assays are, was calculated as 2pq where p is the frequency of the first allele and q (the frequency of the alternate allele). Lines or markers with more than 6% no-calls were excluded in an iterative process.

Markers were regarded as very skewed and removed, if either one of the homozygous parental alleles was absent or the homozygous to heterozygous ratio was >2.1 or <0.4 for F2 populations 1–4 and >0.25 or <0.045 for populations 5 and 6, respectively (Table 4).

Synteny analysis The flanking sequences of all mapped SNP markers and all lentil contigs were searched in BLASTn against M. truncatula CDS version Mt3.5 with an E-value cut-off E 37. The results were displayed in Strudel (Bayer et al., 2011). Discontinuous megablast was used to detect sequence homologies in the flanking region of the recessive VfWD40-1 allele from NV643.

Trait targeting by synteny The coding sequence of Vf_Mt3g092840 (VfTTG1) was amplified using standard PCR from NV648 using oligonucleotides: VfTTG1.F1: 50 -ATGAGATCTAAAACTACGCCTGTGG/VfTTG1.R1: 50 -TCAAACTCGAACCCTCAAAAGC (0.5 lM) using Phusion taq polymerase (N.E.B., Hitchin, UK). Thermal cycling conditions: 96 °C for 5 min, (30 9 96 °C/15 s, 58 °C/15 s, 72 °C/30 s) and 72 °C/10 min final extension. Products were cloned into a pGEMT Easy vector (Promega, Southampton, UK) and sequenced using BigDye V3.0 (Life Technologies, Paisley, UK).

A genome walking kit (Clontech, Mountain View (CA), USA) was used in conjunction with the VfTTG1.R1 primer to generate primary amplification products for walking. A nested primer (VfTTG1.N) 50 -TCAAACCCTCAAAAGCTGCATCTTGTTAG was then used for walking upstream generating a 982-bp product. This was cloned and sequenced as described previously.

Acknowledgements We would like to acknowledge funding from the Defra Pulse Crop Genetic Improvement Network and Technology Strategy Board grant TS/I001301 to DOS and JET. We are grateful to Peter Smith of Wherry & Sons Ltd. for access to mapping populations and to Melanie Febrer and Jane Rogers of The Genome Analysis Centre (TGAC), Norwich for generating the NV643 and NV648 transcriptome sequence data sets.

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Supporting information Additional Supporting information may be found in the online version of this article: Figure S1 Examples of SNP calls for the SNP validation panel of 37 inbred V. faba lines (Table 3) displayed in SNPViewer (LGC Ltd., UK) for a marker assigned to quality class I (top) and IV (bottom).

Figure S2 UPGMA dendrogram of the SNP-validation panel of 37 inbred V. faba lines, based on the panel of SNP markers developed in this study.

Figure S3 Display of synteny between chromosomes of M. truncatula (centre) and linkage groups likely to represent chromosomes of V. faba (left) and L. culinaris (Sharpe et al., 2013) (right).

Figure S4 A. A portion of the linkage map from the F2 population 1 (NV643-1 9 NV648-1) showing the map location of ZT1 between flanking markers Mt3g094760_001 and Vf_Mt3g092810_001.

Table S1 SNPs identified in V. faba lines Albus and BPL10, their flanking sequences and alleles, quality score and PIC values.

Table S2 Consensus linkage map created in MergeMap from from separate individual maps listed in Table S4.

Table S3 Markers mapped here and in previous studies and their assignment to linkage groups of three maps created for V. faba.

Table S4 Linkage groups from individual maps used to produce the consensus map in this study. Individual maps from populations listed in Table 2 were produced in MapDisto and given a weighting factor.

ª 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd, Plant Biotechnology Journal, 1–9