Annals of Botany 106: 927–944, 2010 doi:10.1093/aob/mcq188, available online at www.aob.oxfordjournals.org

The genetics of domestication of , umbellata

Takehisa Isemura1,†, Akito Kaga1,†, Norihiko Tomooka1,*, Takehiko Shimizu2 and Duncan Alexander Vaughan1,‡ 1National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan and 2Institute of Society for Techno-Innovation of Agriculture, Forestry and Fisheries, Kamiyokoba Ippaizuka 446-1, Tsukuba, Ibaraki 305-0854, Japan †These two authors contributed equally to this paper. ‡Present address: FAO Regional Office for Asia and the Pacific, Maliwan Mansion, 39 Phra Atit Road, Bangkok 10200, Thailand. * For correspondence. E-mail [email protected]

Received: 10 June 2010 Returned for revision: 5 August 2010 Accepted: 25 August 2010 Published electronically: 29 September 2010

† Background and Aims The Asian genus Vigna, to which four cultivated species (rice bean, azuki bean, and black gram) belong, is suitable for comparative genomics. The aims were to construct a genetic linkage map of rice bean, to identify the genomic regions associated with domestication in rice bean, and to compare these regions with those in azuki bean. † Methods A genetic linkage map was constructed by using simple sequence repeat and amplified fragment length polymorphism markers in the BC1F1 population derived from a cross between cultivated and wild rice bean. Using this map, 31 domestication-related traits were dissected into quantitative trait loci (QTLs). The genetic linkage map and QTLs of rice bean were compared with those of azuki bean. † Key Results A total of 326 markers converged into 11 linkage groups (LGs), corresponding to the haploid number of rice bean chromosomes. The domestication-related traits in rice bean associated with a few major QTLs distributed as clusters on LGs 2, 4 and 7. A high level of co-linearity in marker order between the rice bean and azuki bean linkage maps was observed. Major QTLs in rice bean were found on LG4, whereas major QTLs in azuki bean were found on LG9. † Conclusions This is the first report of a genetic linkage map and QTLs for domestication-related traits in rice bean. The inheritance of domestication-related traits was so simple that a few major QTLs explained the pheno- typic variation between cultivated and wild rice bean. The high level of genomic synteny between rice bean and azuki bean facilitates QTL comparison between species. These results provide a genetic foundation for improve- ment of rice bean; interchange of major QTLs between rice bean and azuki bean might be useful for broadening the genetic variation of both species.

Key words: Rice bean, , azuki bean, comparative genomics, SSR, domestication, wild species, QTL.

INTRODUCTION to domestication within and among Vigna species and for char- The genus Vigna subgenus Ceratotropis consists of 21 species acterizing useful traits as quantitative trait loci (QTLs) for use that are distributed across a wide region of Asia. Six cultivated in breeding. species belong to this subgenus (Tomooka et al., 2002). To compare the genomic structures and genomic regions Among these, mung bean (Vigna radiata), rice bean associated with domestication among these four species, (V. umbellata), black gram (V. mungo) and azuki bean genetic linkage maps of azuki bean (Han et al., 2005) and (V. angularis) are economically important in Asian countries. black gram (Chaitieng et al., 2006) were constructed pre- Taxonomically, wild forms of species are usually recognized viously using simple sequence repeat (SSR), restriction frag- as varieties below the rank of species of the Asian Vigna. ment length polymorphism (RFLP) and amplified fragment However, numerous differences in morphological and physio- length polymorphism (AFLP) markers; the QTLs for logical traits associated with domestication are observed domestication-related traits of azuki bean have been identified between the cultivated and wild forms. These differences, col- (Isemura et al., 2007) in populations derived from crosses lectively called the domestication syndrome, result from selec- between cultivated and wild forms. The order of common tion over several thousands of years of adaptation to cultivated markers on the linkage groups (LGs) is highly conserved environments, human nutritional requirements and preferences between the azuki bean map (Han et al., 2005) and the (Hawkes, 1983). The four cultivated species listed above are black gram map (Chaitieng et al., 2006), although differences, therefore ideal material for improving our comparative such as inversions, deletions, duplications and a translocation, genomics-based understanding of the gene evolution related were observed between the two genomes in several LGs. # The Author 2010. Published by Oxford University Press on behalf of the Annals of Botany Company. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 928 Isemura et al. — Rice bean domestication

This paper focuses on the rice bean genome as part of a A single F1 hybrid between a cultivated rice bean comparative genome analysis among members of the subgenus (JP217439, Col. No. 2002M21) and a wild rice bean accession Ceratotropis. Rice bean is a traditional crop grown across (JP210639, Col. No. CED99T-2) was backcrossed to culti- south, south-east and east Asia, and its wild form is distributed vated rice bean (JP217439) as a male parent to construct a across a wide area of the tropical monsoon forest climatic zone BC1F1 population consisting of 198 . The wild parent from eastern , , Myanmar (Burma), Thailand, Laos accession was chosen because AFLP analysis had revealed and southern to East Timor (Arora et al., 1980; that it was highly differentiated from other accessions Tomooka et al., 2002; Bisht et al., 2005; Seehalak et al., (Seehalak et al., 2006). It was collected by one of us (N.T.) 2006; Gautam et al., 2007; Tomooka, 2009). The dried from Kanchanaburi province in Thailand; its habitat was a mature seeds of rice bean are usually eaten with rice or in moist deciduous forest along a river (Tomooka et al., , and the leaves, flowers, shoots and young pods are 2000a). The cultivated parent accession, a landrace from eaten as a vegetable in the upland areas of south-east Asia Myanmar (Tomooka et al., 2003), was selected because it and southern China. Rice bean is also important as a had the largest seeds among the germplasm conserved in the and a (Tomooka et al., 2002). Recently, with NIAS Genebank. the aim of using these genetic resources efficiently, researchers studied genetic diversity in cultivated and wild rice bean from Thailand, India and Nepal using molecular markers (Seehalak Growth condition and trait measurement et al., 2006; Bajracharya et al., 2008; Muthusamy et al., 2008). The domesticated accessions from south-east Asia showed the Thirty-one traits related to domestication were evaluated largest genetic variations in AFLP (Seehalak et al., 2006) and (Table 1). Of these, 30 were treated as quantitative traits and SSR (J. Tian et al., Institute of and Oil Crops, Hebei one (hilum colour) as a qualitative trait. The BC1F1 population Academy of Agricultural and Forestry Sciences, China, of 198 plants, together with ten plants of each parent, were unpubl. res.) markers and the largest phenotypic variations in grown in a vinyl house at NIAS, Tsukuba, Japan (36 82′N, seed coat colour and seed size (Tomooka, 2009). These 140 88′E), from September 2006 to March 2007 under results suggest that south-east Asia was the centre of the natural day length. The natural day length gradually decreased origin and genetic diversity of this species (Tomooka, 2009). from 12 h 28 min (14 September 2006) to 9 h 42 min (22 To date, rice bean has not been subjected to systematic December) and thereafter increased to 12 h 18 min (25 breeding, despite the species’ many useful characteristics, March). From October, the vinyl house was heated to maintain including bruchid resistance (Tomooka et al., 2000b; a minimum temperature of 15 8C. Kashiwaba et al., 2003; Somta et al., 2006); disease resistance, Seedling traits – leaf primary length (LFPL), leaf primary particularly to yellow mosaic virus (Arora et al., 1980; Borah width (LFPW) and epicotyl length (ECL) – were recorded et al., 2001) and bacterial leaf spot (Arora et al., 1980), and the when the first trifoliate leaf opened, and vegetative traits – highest potential grain yield among Ceratotropis species leaflet maximum length (LFML), leaflet maximum width (Smartt, 1990). Therefore, rice bean can best be described as (LFMW), stem internode length (1st to 10th: ST1I to ST10I), a scientifically neglected crop of great potential. stem length (STL) and stem thickness (STT) – were recorded Genetic linkage maps using rice bean as one parent have when the 10th trifoliate leaf was fully developed. After all pods been constructed for populations derived from interspecific had been harvested, branch number (BRN) and position of the crosses [rice bean × azuki bean (Kaga et al., 2000) and rice first branch on the main stem (BRP) were recorded. These × bean V. nakashimae (Somta et al., 2006)]. These linkage traits were evaluated in the BC1F1 generation. maps have been used to localize genes for several qualitative Seed-related traits were investigated by using the seeds from traits (Kaga et al., 2000) and QTLs for resistance to bruchid BC1F1 plants. Seed water absorption (SDWA) was investigated beetles and for seed weight (Somta et al., 2006). However, a as an index of seed dormancy. Fifteen unscarified seeds were precise genetic linkage map from a large population derived placed on wet filter paper and incubated in the dark at 15 8C from an intraspecific cross has not been constructed. for 24 h; the number of seeds that absorbed water was then The objectives in this study were (a) to construct a precise recorded. Seed dimensions – seed length (SDL), seed width genetic linkage map of rice bean using SSR markers from (SDW) and seed thickness (SDT) – were averages of ten related grain in an intraspecific population derived seeds. The 100-seed weight (SD100WT) was evaluated from cultivated and wild forms; (b) to examine genomic using intact seeds. synteny between rice bean and azuki bean on the basis of Pod traits – pod length (PDL), pod width (PDW) and the linkage map; (c) to identify QTLs for domestication- number of twists along the length of the dehisced pod when related traits in rice bean; and (d) to compare the QTLs kept at room temperature (PDT) – were based on ten pods. detected in rice bean with QTLs in azuki bean. PDT was used as an index of pod dehiscence. The number of days from sowing to first flowering (FLD) and number of days from first flowering to harvesting of first MATERIALS AND METHODS pod (PDDM) were recorded in the BC1F1 population. The number of seeds per pod (SDNPPD) was measured in Mapping population ten pods. materials were obtained from the Genebank (http://www. Seed hilum colour (SDHC) was evaluated in seeds from BC1F1 gene.affrc.go.jp/index_en.php) collection of the National plants, and the seed hilum of each plant was classified as either Institute of Agrobiological Sciences (NIAS), Tsukuba, Japan. cultivated parent type (white) or wild parent type (pale red). TABLE 1. Domestication-related traits examined in the BC1F1 or BC1F1:2 population derived from the cross between cultivated and wild rice bean

General Trait QTL/ Evaluated attribute Organ Trait abbreviation gene Evaluation method population

Seed dormancy Seed Seed–water absorption (%) SDWA Sdwa Percentage of seeds that had absorbed water 1 d after sowing at 15 8C in incubator BC1F1:2

Pod dehiscence Pod Pod – number of twist (count) PDT Pdt Number of twists along the length of the shattered pod BC1F1 Isemura Gigantism Seed Seed–100 seed-weight (g) SD100WT Sd100wt Weight of 100 seeds BC1F1:2 Seed length (mm) SDL Sdl Longest distance from top to bottom of the seed BC1F1:2 Seed width (mm) SDW Sdw Longest distance from hilum to its opposite side BC1F1:2

Seed thickness (mm) SDT Sdt Longest distance between both sides of the hilum BC1F1:2 al. et Pod Pod length (cm) PDL Pdl Overall length in straight pod BC1F1 Pod width (mm) PDW Pdw Distance of the widest part BC1F1 Stem Stem thickness (mm) STT Stt Stem diameter under the primary leaf BC1F1 domestication bean Rice — Leaf Leaf–primary leaf length (cm) LFPL Lfpl Distance from pulvinus to top BC1F1 Leaf–primary leaf width (cm) LFPW Lfpw Distance of the Widest part BC1F1 Leaf–maximum leaflet length LFML Lfml Length of the largest terminal leaflet on leaves between node on 1st trifoliate leaf BC1F1 (cm) and node on 10th trifoliate leaf Leaf–maximum leaflet width LFMW Lfmw Width of the largest terminal leaflet on leaves between node on 1st trifoliate leaf BC1F1 (cm) and node on 10th trifoliate leaf Plant type Epicotyl Epicotyl length (cm) ECL Ecl Length from cotyledon to primary leaf BC1F1 Stem Stem – internode length (1st to ST1I-ST10I St1i– Length from node on primary leaf to each node BC1F1 10th) (cm) St10i Stem length (cm) STL Stl Length from node on primary leaf to node on 10th trifoliate leaf BC1F1 Branch Branch – number (count) BRN Brn Number of branches on main stem BC1F1 Branch – position of 1st branch BRP Brp Position of first branch on main stem BC1F1 (ith node) Phenology Flower – days to first flower FLD Fld Number of days from sowing to flowering of 1st flower BC1F1 (day) Pod Pod–days to maturity of all PDDM Pddm Number of days from flowering of first flower to harvesting of first pods BC1F1 pods (day) Yield potential Seed Seed–number of seeds per pod SDNPPD Sdnppd Number of seeds per pod BC1F1:2 (count) Pigmentation Seed Seed–hilum colour SDHC Sdhc Pale red or white BC1F1:2 929 930 Isemura et al. — Rice bean domestication

DNA extraction hilum colour segregation pattern was investigated in each BC F individual and the frequency distribution was analysed Total genomic DNA in BC F plants was extracted from 1 1 1 1 by chi-squared test for goodness-of-fit to the expected 200 mg of fresh leaf tissue using a DNeasy plant mini kit Mendelian ratio (1 : 1). The segregation pattern data were used (Qiagen, Valencia, CA, USA). The DNA concentration was 2 to identify the map position of the gene controlling this trait. adjusted to 5 ng mL 1 for the SSR analysis and to 25 ng mL21 for the AFLP analysis by comparison with known con- centrations of standard lDNA in 1 % agarose gel. QTL analysis Mean data for each trait (SDWA data were arcsine- SSR analysis transformed first) were used in the QTL analysis. QTLs were

The SSR analysis in the BC1F1 population was performed identified by means of multiple interval mapping using the by the method of Han et al. (2005). Three hundred and software package MultiQTL v. 3.0, as described by Kaga twenty-five SSR primer pairs were screened from azuki bean et al. (2008). QTL nomenclature followed Somta et al. (Wang et al., 2004), 163 from (Vgina unguiculata; (2006). To test for randomness of the genomic distribution Li et al., 2001) and 40 from common bean ( vul- of QTLs for domestication-related traits, chi-squared tests garis; Yu et al., 2000; Gaitan-Solis et al., 2002; Blair et al., were calculated as described by Isemura et al. (2007).To 2003; Guerra-Sanz, 2004) to determine whether there was test whether or not QTLs were randomly distributed along polymorphism between the two parents. By using the an LG, a Poisson distribution function was calculated as read2Marker program (Fukuoka et al., 2005), another 156 described by Isemura et al. (2007). cowpea SSR primer pairs (Table S1 in Supplementary Data, available online) on cowpea genomic sequences from the Cowpea Genomics Knowledge Base (Chen et al., 2007; http:// RESULTS cowpeagenomics.med.virginia.edu/CGKB/) were designed Genetic linkage map of rice bean and screened for parental polymorphisms. SSR primer pairs developed from azuki bean, cowpea and common bean were used to construct the genetic linkage AFLP analysis map of rice bean. The percentage fragment amplification in rice bean was high, with values between 73.6 % (cowpea) AFLP analysis was performed with an AFLP Core Reagent . Kit (Invitrogen, Carlsbad, CA, USA). The steps of DNA diges- and 96 6 % (azuki bean) (Table 2). Of the 325 azuki bean, tion, ligation of adaptor and pre-selective amplification were 163 cowpea and 40 common bean SSR primer pairs screened, 167 azuki bean (51.4 %), 44 cowpea (27.0 %) and 7 common performed in accordance with the method of Han et al. . (2005). Selective amplification and detection of AFLP bands bean (17 5 %) primer pairs showed clear polymorphism were performed using the method of Kaga et al. (2008).On between the cultivated and wild rice bean parents (Table 2). the basis of information on the AFLP primer combinations All 223 marker loci derived from the 218 polymorphic SSR used to construct several linkage maps of Asian Vigna primer pairs were used for mapping; five azuki bean SSR species (Han et al., 2005; Chaitieng et al., 2006; Kaga et al., primer pairs (CEDG018, CEDG081, CEDG154, CEDG251 2008), 28 primer combinations were selected for the AFLP and CEDG302) detected two loci. analysis in the BC F population. In the AFLP analysis, 1380 bands were detected by 28 1 1 primer combinations. The total number of bands per primer pair ranged from 39 to 59, and the average number was Map construction 49.3. Out of 1380 bands, 103 (range 1–9 per primer combi- nation, average 3.7) showed clear polymorphism between the The linkage map was constructed using the method of Han . cultivated and wild parents (Table 3). et al. (2005) using JoinMap v. 4 0(Van Ooijen, 2006). Marker A total of 326 loci (172 azuki bean SSR loci, 44 cowpea segregation was analysed by a chi-squared test for SSR loci, 7 common bean SSR loci and 103 AFLP loci) goodness-of-fit to the expected Mendelian ratio (1 : 1). The could be assigned to 11 LGs covering a total of 796.1cMof recombination frequencies were converted into map distance the rice bean genome at an average marker density of 2.5 (cM) by using the Kosambi mapping function (Kosambi, cM (Fig. 1 and Table 4). The genomic region in this map 1944). After a framework map had been built using codomi- nant SSR markers, dominant SSR and AFLP markers were integrated into the framework. Numbering of the LGs followed TABLE 2. Summary of amplification and polymorphic rates of that for azuki bean (Han et al., 2005). SSR primer pairs from three legumes in rice bean

No. of SSR primer pairs Data analysis Screened Amplified (%) Polymorphic (%) For the quantitative traits, the mean, standard error minimum value, maximum value and broad-sense heritability were calcu- Azuki bean 325 314 (96.6) 167 (51.4) lated, and the frequency distributions of phenotypes in the Cowpea 163 120 (73.6) 44 (27.0) . . BC F (or BC F ) population were examined for each. The Common bean 40 37 (92 5) 7 (17 5) 1 1 1 1:2 Total 528 471 (89.2) 218 (41.3) correlations between pairs of traits were also calculated. The Isemura et al. — Rice bean domestication 931

TABLE 3. Polymorphism levels of AFLP primer combinations traits of wild parent precisely. In December 2007, pods and used to construct the rice bean linkage map seeds were obtained from wild plants sown again in April 2007 and were used to evaluate the pod- and seed-related No. of bands traits of the wild parent. SDNPPD and BRN were greater in the cultivated parent than in the wild parent. Primer pair Total Polymorphic Percentage polymorphism The BC1F1 plants and BC1F1:2 lines showed a high degree E32M39 53 2 3.8 of morphological and physiological variation (Table 5). E32M44 41 2 4.9 Seed- and pod-size-related traits showed high heritability E32M45 56 6 10.7 (.80 %), whereas stem- and leaf-related traits showed low E33M49 45 3 6.7 heritability (,70 %) except in the case of ST4I, ST10I and E41M54 48 1 2.1 E42M42 41 1 2.4 STL. The means of the BC1F1 plants and BC1F1:2 lines fell E43M75 57 5 8.8 between the means of the cultivated and wild parents for all E44M44 52 5 9.6 traits except SDNPPD, PDT and BRP. Most traits in these E44M59 43 4 9.3 populations showed a nearly normal distribution among . E45M33 53 4 7 5 parents (Fig. S1 in Supplementary data, available online). E46M34 53 2 3.8 E47M47 59 8 13.6 Transgressive segregation was observed in SDNPPD, PDT, E51M89 47 3 6.4 FLD, STT and BRN. E63M55 41 6 14.6 In general, there were significant positive correlations (P , E63M63 55 3 5.5 0.05) between related traits, such as between stem length and E66M66 44 3 6.8 E80M46 39 1 2.6 each internode length, and between seed-size-related traits E81M81 54 4 7.4 and pod-size-related traits (Table S2 in Supplementary Data). E82M82 52 4 7.7 PDT was negatively correlated with seed-size-related traits, E83M83 52 2 3.8 each internode length (ST1I to ST10I) and stem length . E86M54 56 9 16 1 (STL). Water absorption by seeds (SDWA) was positively cor- E87M87 55 5 9.1 E88M88 48 3 6.3 related with epicotyl length and lower internode length (ST1I E90M35 46 3 6.5 and ST2I). SDNPPD was highly correlated with E90M90 51 3 5.9 seed-size-related traits and PDL. Seed- and pod-size-related E91M71 42 3 7.1 traits and PDDM were positively correlated. E91M91 45 3 6.7 E93M93 52 5 9.6 Total 1380 103 Average 49.33.77.5 QTLs for domestication-related traits QTL analyses were performed for each trait in the popu- . . lation (Table 6 and Fig. 2; for details see Fig S2 in covered 95 7 % of the azuki bean genome (832 1 cM) reported Supplementary Data). In total, 73 QTLs and one morphologi- by Han et al. (2005). The number of markers on each LG cal marker gene are reported for the 31 domestication-related ranged from 14 (LG7) to 42 (LG8). The length of each LG . . traits. The number of QTLs may have been overestimated as ranged from 105 8 (LG1) to 39 4 cM (LG11). The average a result of measurement of related traits such as 100-seed distance between two adjacent markers ranged from 1.58 . weight, seed length, seed width and seed thickness. (LG11) to 5 04 cM (LG7). All LGs except LG5 had gaps Generally, one to seven QTLs were detected for each trait at greater than 10 cM between markers. LGs 1 and 3 had gaps a significant level (P , 0.001), except in the case of FLD, greater than 15 cM. Sixty-one markers (18.7 %) showed segre- . LFPL, LFPW, ST3I and BRP, for which no QTLs were gation distortion (P , 0 05). The ratio of plants with heterozy- detected. gous genotypes was high for all 61 markers which were found on LGs 5, 7, 8 and 11. Interestingly, all of the markers on Water absorption by seeds (SDWA). One of the most important LG11 showed segregation distortion at significant levels. changes that occurred during domestication was a reduction in seed dormancy. The SDWA assay revealed that the water Variation in domestication-related traits absorption rate of the cultivated parent (98 %) was higher than that of the wild parent (33 %) (Table 5). Five QTLs The means, minima, maxima, standard errors and broad- were detected, on LG2, LG3, LG4, LG8 and LG10, and sense heritabilities of traits in the parental lines and BC1F1 QTL Sdwa4.4.1 on LG4 explained 25.1 % of the phenotypic or BC1F1:2 populations were determined (Table 5). The variation. The alleles of the cultivated parent increased mean SDWA of the cultivated parent was higher than that of SDWA at Sdwa4.3.1, Sdwa4.4.1 and Sdwa4.8.1 and decreased the wild parent. PDT of the wild parent was greater than that water absorption at Sdwa4.2.1 and Sdwa4.10.1. The allele of of the cultivated parent. The sizes of the leaf, stem, seed and the cultivated parent at Sdwa4.4.1 had an especially large pod were larger in the cultivated parent. Among the effect, increasing the SDWA by 10.5%. stem-length-related traits, ECL, ST1I to ST10I and STL were longer in the cultivated parent. Pod dehiscence (PDT). Loss (reduction) of pod dehiscence is FLD in the cultivated parent was 91 d and PDDM was 64 advantageous for harvesting seeds. The number of twists d. Because only one plant of the wild parent flowered, and it along the length of the shattered pod was used as an index produced only one pod, it was not possible to evaluate the of pod dehiscence. The cultivated parent had fewer twists. 932 Isemura et al. — Rice bean domestication

LG1 LG2 LG3 LG4 LG5 LG6

0·0 CEDG133 CEDG293 CEDG029 0·0 0·0 cp08204 0·0 CEDG211 0·0 CEDG205 0·0 CEDG020 2·1 CEDG149 CEDG285 2·1 E90M90-352 1·0 cp01354 3·7 E93M93-263 3·7 E63M55-097 4·7 CEDG144 2·1 cp03853 6·4 CEDG169 7·7 E32M45-225 8·1 CEDC035 10·1 E93M93-128 9·6 cp00420 12·2 CEDG014 10·1 CEDG136 15·1 E91M71-168 13·3 CEDG124 CEDG060 12·7 CEDG187 12·9 16·7 CEDG208 CEDG006 15·9 CEDGI84 14·4 cp03852 18·2 E82M82-141 E87M87-073 19·1 CEDG067 18·7 E47M47-262 17·4 CEDG139 20·1 CEDG264* 18·8 E43M75-177 19·3 CEDG186 21·2 BM170* cp04508 cp04320 24·4 22·4 CEDC028 22·7 E46M34-107 CEDG268 23·6 E44M44-297 22·0 E90M90-196 CEDAAG004 22·9 CEDC055 24·3 cp02271 25·2 E91M91-207 27·2 CEDG250 26·4 E32M45-098 25·4 CEDG027* 26·9 E86M54-128 30·7 CEDG272 28·8 CEDG050 E66M66-205 25·9 CEDG253 CEDG037 29·3 E91M91-109 27·4 CEDG084 E33M49-164 29·2 29·9 CEDG065 cp11079 30·2 cp00416 26·4 E33M44-143* 33·2 CEDAAT002 33·0 CEDG003 28·4 CEDG080 CEDC031 27·5 E51M89-213* 34·3 CEDC018 E81M81-097 CEDG185 CEDG154b 33·5 CEDG053 29·0 E44M44-324 34·6 30·7 CEDG236* 33·6 CEDG191 35·3 CEDG244 E86M54-222 33·3 CEDG008* 34·6 cp04111 cp06180 cp10882 cp01038 36·7 CEDC009 34·8 CEDGI54a E44M59-258 35·0 CEDG302b CEDG225 37·0 36·0 CEDG018a* 34·1 E63M63-315 CEDG086 39·2 E63M55-154 37·2 E44M59-091 38·4 37·1 CEDG114* E86M54-180 CEDG101 E46M34-124** CEDG290** 34·7 39·7 E44M44-255 E44M59-111 38·9 E33M44-245 cp00680 cp10093 44·3 CEDG145** CEDG009** 35·2 43·1 E42M42-151 37·8 40·0 cp01953 42·3 CEDG088 E93M93-114** 38·7 45·1 CEDG109 44·8 E83M83-171** CEDG023** CEDG118 46·9 E86M54-313 45·6 CEDG025 CEDG251b 45·3 E88M88-230* E86M54-378* CEDG245 47·5 CEDG091 47·3 47·2 CEDG221 48·3 E63M55-475 CEDG018b CEDC033 47·7 CEDG288 49·4 CEDG261 48·0 50·3 cp09102 49·0 E44M44-082 E32M45-181 CEDG146 52·2 E41M54-127 51·9 E63M55-083 53·4 CEDG232 53·3 E82M82-292 CEDG176 E90M90-381 54·6 55·5 CEDG107 54·5 55·7 cp08299 56·7 CEDG010 57·6 E86M54-082 cp06216 62·7 CEDG128 CEDG231 60·5 CEDG103 CEDG062 61·6 CEDG015 CEDG057 62·5 E66M66-125 63·8 E63M55-263 64·3 CEDC039 65·6 CEDG026 64·3 cp00080 CEDC030 66·1 CEDG275 64·8 CEDG102 67·1 CEDG207 69·0 CEDG248 66·9 cp05137 CEDC050 69·7 CEDG175 CEDG119 69·2 CEDAAG002 70·0 cp09781 70·2 E90M35-148 72·1 74·3 AYI 76·3 E32M45-250 79·4 VM32

83·1 CEDG137 85·5 BM181 87·1 CEDG242 88·3 CEDG181 87·6 CEDG019 89·4 CEDG011 89·7 CEDG254 91·4 E81M81-436 92·3 cp02468 91·9 E43M75-122 92·8 E45M33-117 92·4 CEDG127 94·3 CEDG189 97·1 CEDG074

101·1 CEDG283 LG7 LG8 LG9 LG10 LG11 105·8 E90M35-283 0·0 E43M75-165 0·0 CEDG302a 0·0 BM157 0·0 E32M39-195 0·0 E63M55-470*

3·4 E51M89-231 3·8 CEDG075 5·8 E33M49-081 9·0 E45M33-410 6·3 CEDG182 6·8 E63M63-193 10·9 CEDG180 11·6 E83M83-222 11·9 CEDG198 10·4 PV-ctt001** 10·6 CEDG064 10·8 CEDG156 13·0 CEDG280 12·9 CEDG168*** 12·2 CEDG070 13·5 CEDG298 E90M35-210*** CEDG170*** 17·6 CEDG113 19·2 18·8 E44M59-320 CEDGAA002***E32M39-291*** CEDG216 CEDG223 20·2 CEDG013*** cp00464*** 17·3 PV-atcc001 19·9 20·7 CEDC014*** 19·0 cp10971 cp02661 E88M88-163 20·0 cp06190 cp00451 E81M81-113 21·2 CEDG042*** 21·1 CEDG143 22·5 cp10211*** E47M47-079*** E86M54-170 21·7 CEDG033 23·5 E43M75-086 CEDG172*** CEDG002*** 23·9 CEDG271 CEDG081b 25·3 E47M47-107*** 27·8 cp06377 24·0 cp05325 26·3 E93M93-162*** CEDG044*** 32·5 CEDG230 24·3 27·7 CEDG215 25·9 CEDG116 26·9 E45M33-289*** 38·0 E86M54-238 29·4 E66M66-230 27·9 CEDG287*** cp02470 CEDG265 26·4 CEDG081a 27·1 CEDG068 30·0 E80M46-105*** 38·9 CEDC016 33·6 E32M45-160 33·0 CEDG111 30·3 CEDG097 32·0 CEDG098*** VM24 E91M71-179 33·1 CEDG135 41·1 35·6 35·2 CEDG076*** 41·7 CEDG269 37·9 E88M88-099 34·2 cp05914 CEDG021 36·8 CEDG273** 45·6 cp00200 38·3 CEDG024 36·1 E87M87-082 38·9 cp05096* 49·0 E87M87-137 39·3 CEDG259 39·4 CEDG066** 41·0 E33M49-235 50·1 CEDG073* 40·4 CEDG166 E91M71-240 42·5 E47M47-305 50·6 42·6 E44M44-270 51·1 CEDG016* cp04824* CEDG092* 44·1 E32M45-392 51·6 CEDG202* CEDG030* 48·1 CEDG041 45·6 GATS11 53·4 E47M47-179* E45M33-069* 48·9 E82M82-159 49·7 E91M91-185 54·1 CEDG040* CEDG257* 51·1 E47M47-201 54·6 CEDG286 CEDG059 52·6 E43M75-100 cp08513 CEDG130 55·5 CEDC038 E93M93-121 55·8 E87M87-088 57·0 CEDG218* 57·1 E63M63-185 58·0 CEDG243 57·6 E81M81-121 E47M47-145 CEDG260 59·2 CEDC011 E86M54-094 60·9 VM27 63·3 CEDG200* 65·4 CEDG251a* 65·5 CEDG085* 66·4 E51M89-202* 67·5 cp10549* 68·6 cp00226* 72·7 E47M47-141 73·9 E87M87-077 75·0 cp01225

81·7 CEDG267

88·0 CEDG173 CEDG228 88·5 E82M82-184

95·3 CEDG022 Isemura et al. — Rice bean domestication 933

TABLE 4. Numbers of markers and average distances between markers in each linkage group on the rice bean linkage map

No. of markers

SSR

Linkage group (LG) Length (cM) Azuki bean Cowpea Common bean AFLP Total Distance between two maker loci (cM)

1 105.8224 1 10372.94 276.3 18 5 0 9 32 2.46 356.7930 9212.84 497.1 20 4 1 9 34 2.94 550.3182 1 11321.62 683.1 16 7 0 7 30 2.87 765.5800 6145.04 868.6219 1 11421.67 995.3 9 3 2 12 26 3.81 10 58.0174 0 11321.87 11 39.4 14 3 1 8 26 1.58 Total 796.1 172* 44 7 103 326 2.45

* The number of polymorphic markers differed from the number of primers, because on each of five of the primers (CEDG018, CEDG081, CEDG154, CEDG251 and CEDG302) two loci were detected.

One QTL, Pdt4.7.1, with a relatively high contribution to this seed- and pod-size-related traits. However, in contrast to the trait (42.4 %) was found on LG7. latter, the alleles from the cultivated parent had a negative effect on leaf size at these QTLs. The QTLs for these traits Seed size (SD100WT, SDL, SDW, SDT). Domestication of rice on LG4 explained 31.8 % (LFML) and 25.6 % (LFMW) of bean has resulted in a 15-fold increase in seed weight the phenotypic variation. (Table 5). Six or seven QTLs were detected for seed-size-related traits (SD100WT, SDL, SDW and SDT) on Stem length (STL), epicotyl length (ECL), internode length (ST1I LG1, LG2, LG3, LG4, LG5, LG7 and LG11. The QTLs to ST10I) and stem thickness (STT). In the cultivated parent, the with the highest contribution to these traits (25.2–33.4%) epicotyl, all internodes and the stem were longer, and stem was were found on LG4. The QTLs on LG2 explained a further thicker, than in the wild parent (Table 5). Three QTLs 14.9–17.6 % of the phenotypic variation. (Stl4.1.1, Stl4.5.1 and Stl4.7.1) for STL were found, on LG1, Pod size (PDL, PDW). Domestication of rice bean has resulted LG5 and LG7 (Table 6). Alleles from the cultivated parent in a 2.5-fold increase in pod length (Table 5). Three QTLs for at all QTLs had the effect of increasing stem length. PDL and six QTLs for PDW were found (Table 6). Those However, stem growth at the early, middle and late stages affecting these two traits were found mainly on LG2, LG3 was controlled by different QTLs. Stem length from the and LG4. The QTLs with the highest contribution for these primary to the 10th node QTLs (Stl4.1.1 on LG1, Stl4.7.1 on traits (23.1 % for PDL and 27.6 % for PDW) were found on LG7) and from the middle to the upper internode length LG4. As expected, the alleles from the cultivated parent QTLs (4th to 10th; St7i4.1.1 to St10i4.1.1 on LG1 and increased both PDL and PDW at all QTL positions. The St4i4.7.1 to St10i4.7.1 on LG7) were consistently located in QTLs for both PDL and PDW on LG2, LG3 and LG4 were similar map positions on LG1 and LG7. On the other hand, located close to the QTLs for seed-size-related traits on each QTLs for the lower (1st and 2nd) internode lengths respective LG (Fig. 2). (St1i4.7.1 and Stl2i4.7.1) in the early stem-growth stage were found mainly on LG7 (at different QTL positions from those Leaf size (LFPL, LFPW, LFML, LFMW). The primary leaf of the middle and upper internode lengths on LG7). Four length in many BC1F1 plants was close to that in the cultivated QTLs for epicotyl length (Ecl4.4.1, Ecl4.7.1, Ecl4.8.1 and parent. Primary leaf width of most of the BC1F1 plants was Ecl4.11.1) were found, on LG4, LG7, LG8 and LG11. distributed within a narrow range (about 0.9 cm), as deter- Among these, a QTL on LG7 was located close to QTLs for mined by the frequency distribution (Fig. S1 in the first and second internode length (St1i4.7.1 and Supplementary Data). The values of heritability for both St2i4.7.1). QTLs for epicotyl length (Ecl4.4.1 on LG4 and traits were low (,40 %; Table 5). As a result, no QTLs for Ecl4.7.1 on LG7) and first and second internode length these traits were detected. (St1i4.7.1 and St2i4.7.1 on LG7) were detected relatively Major QTLs for maximum leaf size (LFML, LFMW) were close to the QTLs for seed-size-related traits on LG4 and detected on LG4. These QTLs were located close to QTLs for LG7 (Fig. S2 in Supplementary Data). Only one stem diameter

F IG. 1. A genetic linkage map of rice bean based on SSR and AFLP markers. This map was constructed from 198 BC1F1 individuals of (cultivated rice bean × wild rice bean) × cultivated rice bean. Map distances are shown to the left of the linkage groups and marker names are shown on the right. SSR markers with the prefix ‘CED’ are from azuki bean; those with ‘cp’ and ‘VM’ are from cowpea. SSR markers with the prefix ‘BM’ or ‘PV’ and the SSR markers AY1 (LG4) and GATS11 (LG9) are from common bean. Lower-case letters in SSR markers are suffixed to markers with multiple loci. SSR markers in italics indicate dominant loci. AFLP markers, with the prefix ‘E‘, are named according to the primer combination followed by the estimated fragment size. Markers showing significant deviation from the expected segregation ratios at P levels of 0.05, 0.01 and 0.001 are indicated with *, ** and ***, respectively. 934

TABLE 5. Means, standard errors, minima, maxima and heritability values for parents and for the BC1F1 or BC1F1:2 populations derived from a cross between cultivated rice bean and wild rice bean

Cultivated rice bean Wild rice bean BC1F1 or BC1F1:2

Trait Unit Mean Standard error Minimum Maximum Mean Standard error Minimum Maximum Mean Standard error Minimum Maximum Heritability (%)

SD100WT* g 27.40.314 25.828.51.80.039 1.71.914.60.216 7.423.596.1 SDL* mm 10.30.073 10.110.74.30.024 4.34.48.00.039 6.69.692.5 SDW* mm 6.00.036 5.86.12.20.034 2.12.24.80.027 3.75.894.9 SDT* mm 7.00.030 6.97.22.50.017 2.42.55.50.030 4.46.697.7 Isemura SDWA* % 98.01.225 95.0 100.033.02.550 25.040.063.81.957 0.0 100.097.2 SDNPPD* count 7.10.197 6.47.86.70.108 6.46.94.90.094 1.58.890.6 SDHC* – White Pale red Pale red : white ¼ 96 : 97 (x2 ¼ 0.005) – PDL cm 11.80.258 10.612.64.50.078 4.44.78.30.083 5.411.281.5 al. et PDW mm 12.30.074 12.012.64.10.014 4.04.19.10.053 7.011.296.0

PDT count 1.90.163 1.52.94.30.127 4.14.74.30.112 1.26.994.3 domestication bean Rice — FLD day 90.50.845 86.094.098.0– 98.098.089.40.382 74.0 109.0– PDDM day 63.90.934 61.069.0– – – – 57.10.272 49.066.0– ECL cm 12.20.179 11.212.72.40.053 2.22.78.50.065 5.811.183.1 ST1I cm 0.70.018 0.60.70.10.011 0.10.20.30.008 0.10.685.3 ST2I cm 0.80.042 0.61.00.20.026 0.10.30.50.011 0.21.257.1 ST3I cm 1.20.049 1.01.40.40.033 0.30.50.90.012 0.51.452.1 ST4I cm 2.00.053 1.82.20.60.024 0.50.71.50.020 0.82.383.1 ST5I cm 2.60.096 2.23.00.90.053 0.71.12.10.028 1.23.267.8 ST6I cm 3.60.156 3.04.41.10.045 0.91.32.60.035 1.64.155.8 ST7I cm 4.60.228 3.45.42.00.068 1.72.33.50.049 2.05.651.7 ST8I cm 6.70.400 5.38.32.80.073 2.43.14.20.101 2.48.350.7 ST9I cm 13.31.001 9.217.73.20.084 2.83.58.00.282 2.917.861.8 ST10I cm 19.00.330 17.519.83.60.083 3.24.015.40.416 3.628.398.0 STL cm 66.51.604 59.672.917.20.204 16.518.447.90.917 25.778.290.7 STT mm 7.90.158 7.38.54.60.115 4.15.07.20.045 5.58.541.6 LFPL cm 10.00.287 8.710.94.00.083 3.64.48.50.053 6.110.034.4 LFPW cm 3.60.111 3.23.91.50.045 1.41.82.50.022 1.63.339.1 LFML cm 18.50.202 17.919.411.10.277 9.912.717.00.113 12.720.879.7 LFMW cm 12.10.194 11.313.16.10.172 5.57.210.80.080 7.614.277.3 BRN count 7.10.295 6.08.02.70.236 2.04.06.40.131 3.011.082.5 BRP ith 3.60.263 2.04.01.80.147 1.02.03.70.061 2.07.049.2

* These traits were evaluated in the BC1F1:2 generation. Isemura et al. — Rice bean domestication 935

TABLE 6. QTLs detected in a population derived from a cross between cultivated rice bean and wild rice bean

Trait QTL name LG LOD P Loci (cM) PVE (%) Substitution effect

SD100WT Sd100wt4.1.1 + 17.13 0.0001 83.96 4.91.345 Sd100wt4.2.1 + 220.97 0.0001 54.10 17.42.525 Sd100wt4.3.1 + 35.51 0.0001 23.35 3.71.167 Sd100wt4.4.1 + 429.97 0.0001 64.65 33.43.495 Sd100wt4.5.1 + 59.54 0.0001 7.66 4.01.035 Sd100wt4.5.2 + 35.41 3.40.871 Sd100wt4.7.1 + 76.34 0.0001 34.94 5.01.357 Total 71.8 SDL Sdl4.1.1 + 111.73 0.0001 78.50 10.10.355 Sdl4.2.1 + 219.06 0.0001 52.28 16.50.455 Sdl4.4.1 + 425.08 0.0001 63.86 28.90.602 Sdl4.5.1 + 510.53 0.0001 7.52 3.10.140 Sdl4.5.2 + 35.41 5.10.229 Sdl4.7.1 + 77.23 0.0001 29.32 5.60.264 Total 69.3 SDW Sdw4.1.1 + 15.01 0.0001 70.58 4.40.156 Sdw4.2.1 + 215.83 0.0001 55.61 14.90.287 Sdw4.3.1 + 37.99 0.0001 23.35 6.30.186 Sdw4.4.1 + 421.26 0.0001 63.60 26.30.381 Sdw4.5.1 + 54.01 0.0001 1.37 3.20.132 Sdw4.7.1 + 74.95 0.0001 36.71 4.40.156 Sdw4.11.1 + 11 7.18 0.0001 27.42 6.00.182 Total 65.5 SDT Sdt4.1.1 + 14.65 0.0001 83.96 3.40.158 Sdt4.2.1 + 219.59 0.0001 53.32 17.60.359 Sdt4.3.1 + 310.92 0.0001 23.35 8.60.251 Sdt4.4.1 + 420.75 0.0001 63.72 25.20.430 Sdt4.5.1 + 58.82 0.0001 6.26 4.10.148 Sdt4.5.2 + 35.41 3.30.122 Sdt4.7.1 + 75.50 0.0001 41.69 4.10.173 Total 66.3 SDWA Sdwa4.2.1– 24.10 0.0003 68.11 5.5–2.24 Sdwa4.3.1 + 34.33 0.0002 33.36 6.02.45 Sdwa4.4.1 + 414.04 0.0001 82.52 25.110.54 Sdwa4.8.1 + 83.15 0.0010 61.37 4.31.73 Sdwa4.10.1– 10 3.23 0.0009 24.28 4.3–1.75 Total 45.2 SDNPPD Sdnppd4.4.1 + 43.59 0.0008 62.68 7.40.696 Sdnppd4.9.1 + 97.49 0.0001 9.35 5.40.461 Sdnppd4.9.2 + 72.24 9.30.801 Total 22.1 PDL Pdl4.2.1 + 210.51 0.0001 57.06 16.70.920 Pdl4.3.1 + 35.06 0.0001 22.37 6.90.592 Pdl4.4.1 + 413.65 0.0001 63.59 23.11.082 Total 46.7 PDW Pdw4.1.1 + 19.44 0.0001 66.77 8.70.441 Pdw4.2.1 + 212.30 0.0001 56.61 11.90.518 Pdw4.3.1 + 37.21 0.0001 23.35 5.90.364 Pdw4.4.1 + 421.86 0.0001 62.94 27.60.789 Pdw4.5.1 + 57.75 0.0001 29.34 6.60.385 Pdw4.7.1 + 75.19 0.0001 32.23 4.40.313 Total 65.1 PDT Pdt4.7.1– 720.27 0.0001 14.52 42.4–1.994 Total 42.4 FLD Not detected PDDM Pddm4.4.1 + 46.96 0.0001 78.19 17.73.143 Total 17.7 ECL Ecl4.4.1 + 45.79 0.0001 73.40 9.80.565 Ecl4.7.1 + 73.61 0.0004 48.78 5.80.436 Ecl4.8.1 + 86.06 0.0001 44.72 10.40.583 Ecl4.11.1 + 11 4.31 0.0001 0.00 7.20.484 Total 33.2 ST1I St1i4.5.1 + 53.55 0.0008 21.76 7.30.061 St1i4.7.1 + 74.75 0.0001 41.69 9.80.071 Total 17.1 Continued 936 Isemura et al. — Rice bean domestication

TABLE 6. Continued

Trait QTL name LG LOD P Loci (cM) PVE (%) Substitution effect

ST2I St2i4.7.1 + 73.28 0.0009 48.16 7.50.081 Total 7.5 ST3I Not detected ST4I St4i4.1.1 + 13.66 0.0005 85.99 7.40.155 St4i4.7.1 + 74.26 0.0001 14.61 9.30.174 Total 16.7 ST5I St5i4.7.1 + 75.54 0.0001 16.47 12.90.281 Total 12.9 ST6I St6i4.7.1 + 74.80 0.0001 10.62 10.80.295 Total 10.8 ST7I St7i4.1.1 + 14.39 0.0001 99.10 9.60.421 St7i4.7.1 + 74.36 0.0001 10.62 8.80.404 Total 18.4 ST8I St8i4.1.1 + 14.42 0.0001 97.14 12.00.795 St8i4.7.1 + 75.79 0.0001 10.72 16.30.926 Total 28.3 ST9I St9i4.1.1 + 14.92 0.0001 92.58 14.52.440 St9i4.7.1 + 73.76 0.0003 10.62 10.42.068 Total 24.9 ST10I St10i4.1.1 + 14.85 0.0001 95.93 14.43.620 St10i4.7.1 + 74.11 0.0002 0.00 11.23.199 Total 25.6 STL Stl4.1.1 + 14.75 0.0001 93.50 12.97.392 Stl4.5.1 + 53.36 0.0009 24.29 8.35.922 Stl4.7.1 + 74.60 0.0001 11.13 11.67.023 Total 32.8 STT Stt4.9.1 + 93.59 0.0007 47.45 13.00.377 Total 13.0 LFPL Not detected LFPW Not detected LFML Lfml4.3.1 + 34.27 0.0001 21.92 6.70.824 Lfml4.4.1– 415.93 0.0001 64.58 31.8–1.802 Total 38.5 LFMW Lfmw4.4.1– 411.38 0.0001 64.32 25.6–1.130 Total 25.6 BRN Brn4.4.1 + 45.18 0.0001 64.82 12.91.321 Total 12.9 BRP Not detected

QTL, Stt4.9.1, was detected, on LG9. The alleles from the cul- near the QTLs for seed- and pod-size-related traits. The tivated parent increased stem thickness. This QTL explained alleles from the cultivated parent delayed maturity. 13.0 % of the phenotypic variation. Seed number per pod (SDNPPD). The cultivated parent pro- Branch number (BRN) and first branch position (BRP). The cul- duced slightly more seeds per pod than the wild parent tivated parent produced more branches on the main stem and (Table 5). Three QTLs with small effects (5.4–9.3%) on developed the first branch at a higher internode than did the SDNPPD were identified, on LG4 and LG9 (two of them on wild parent (Table 5). Only one significant QTL, Brn4.4.1, LG9). The QTL on LG4 was detected close to the QTLs for was identified for BRN. This QTL was detected close to the seed- and pod-size-related traits. At all QTLs, alleles from QTLs for seed- and pod-size-related traits. No significant the cultivated parent had a positive effect on SDNPPD. QTL was found for BRP. Seed hilum colour (SDHC). The pale red hilum from the wild

Flowering time (FLD). The frequency distribution in BC1F1 parent was dominant to the white hilum. The segregation plants revealed that most of the plants flowered within a ratio for this trait fitted the expected ratio (1 : 1). The recessive short period (about 10 d; Fig. S1 in Supplementary Data). gene for white hilum from the cultivated parent was tentatively As a result, no QTL was identified. named sdhc4.5.1. This gene was mapped between AFLP markers E63M55-097 and E93M93-128 on LG5. Days to pod maturity (PDDM). As has been shown in azuki bean (Kaga et al., 2008), it was expected that in rice bean it would take more time to maturity (translocation of dry matter to the Distribution of domestication-trait-related QTLs seed) in the cultivated parent than in the wild parent, presumably QTLs with large effect were found on LGF2, LG4 and LG7 because of the difference in seed size between the two parents. (Fig. 2) as described below. Although it was not possible to evaluate the PDDM of the wild parent precisely, one QTL was found, on LG4. This QTL LG2. QTLs for seed- and pod-size-related traits were found in explained 17.7 % of the phenotypic variation and was found a narrow region. In the region near cowpea marker cp08299, Isemura et al. — Rice bean domestication 937

Seed size (+) Pod size (+) Upper internode and stem length (+) LG1

Seed size (+) Pod size (+) Water absorption by seed (–) LG2 cp08299 CEDG261 CEDG026 E63M55-083 E66M66-125

Leaf size (+) Pod size (+) Seed size (+) Water absorption by seed (+) LG3

Seed size (+) Water absorption by seed (+) Pod size (+) Epicotyl length (+) Leaf size (–) Pod maturity (+) Seed number/pod (+) Branch number (+) LG4 AY1 CEDG181 CEDG011 CEDG103 CEDG175

Hilum colour (white) Pod size (+) Seed size (+) Seed size (+) LG5

LG6

Pod dehiscence (–) Seed size (+) Upper internode and stem length (+) Epicotyl and lower internode length (+) LG7 CEDG064 CEDG143

Epicotyl length (+) Water absorption by seed (+) LG8

Stem thickness (+) Seed number / pod (+) LG9

Water absorption by seed (–) LG10

10 cM

Epicotyl length (+) Distribution regions of QTL with large effect LG11

F IG. 2. Summary of domestication-related QTLs with large effect detected on each linkage group in a population derived from a cross between cultivated and wild rice bean. The signs ‘ + ’ and ‘–’ after trait names indicate positive and negative effects of the allele from cultivated rice bean on the trait. Seed size, SD100WT, SDL, SDW and SDT; pod size, PDL and PDW; leaf size, LFML and LFMW; lower internode length, ST1I and ST2I; upper internode length, ST6I to ST10I.

QTLs for seed size (Sd100wt4.2.1, etc.) and pod size (Pdl4.2.1 LG4. Between SSR markers CEDG103 and CEDG175, QTLs and Pdw4.2.1) were found. At about 15 cM away from this with a strong effect on seed, pod and leaf sizes were found. region, a QTL for seed coat permeability (Sdwa4.2.1)was QTLs for seed number per pod and branch number were located. Interestingly, the cultivated parent alleles decreased also found in this region. At about 20 cM away from this seed coat permeability. region, a QTL strongly associated with seed coat permeability 938 Isemura et al. — Rice bean domestication

(Sdwa4.4.1) was located. QTLs for pod maturity (Pddm4.4.1) significantly lower than expected. Furthermore, x2 tests of the and epicotyl length (Ecl4.4.1) were also linked to this area. number of QTLs in each 10-cM interval indicated a non-random distribution on LG1, LG2, LG3, LG4 and LG7 (Table 8). LG7. QTLs were distributed in two regions. Near SSR marker CEDG064, QTLs for pod dehiscence (Pdt4.7.1), middle to upper internode length (St4i4.7.1, etc.) and stem length DISCUSSION (Stl4.7.1) were found. Between SSR marker CEDG215 and General features of the rice bean genetic linkage map AFLP marker E91M91-185 (an interval of about 20 cM), QTLs for seed size (Sdwt4.7.1, etc.), epicotyl length and lower internode The present rice bean linkage map is the first to be constructed length (Ecl4.7.1, St1i4.7.1 and St2i4.7.1) were detected. from a large population derived from an intraspecific cross. The number of LGs corresponded to the basic number of Non-random distribution of QTLs. Although it was unknown chromosomes in rice bean. Many codominant SSR markers whether all 73 QTLs identified for domestication-related traits from azuki bean, cowpea and common bean were integrated had independent gene actions on each trait, the observed into the genome, and no gap was .20 cM. Therefore, this number of QTLs was compared with the expected number of rice bean map is useful for understanding genomic synteny 2 QTLs based on each LG length (Table 7). The x value was among species within Ceratotropis, as well as for identifying 47.6; this value is significant at P ¼ 0.001, suggesting a depar- QTLs for useful traits. ture from random distribution across the rice bean genome. The numbers of QTLs on LG5 and LG7 were significantly higher than expected, whereas the number of QTLs on LG6 was Segregation distortion Two linkage maps had been constructed for populations TABLE 7. Observed and expected numbers of QTLs on each derived from interspecific crosses between rice bean and linkage group azuki bean (Kaga et al., 2000) and between rice bean and V. nakashimae (Somta et al., 2006). Markers showing segre- No. of QTLs gation distortion were observed on some LGs on both maps, Length 2 suggesting some interspecific genetic barriers. Although the LG (cM) Detected Expected x present rice bean linkage map was based on an intraspecific LG1 105.8119.70.17 BC1F1 cross population (rice bean/wild rice bean//rice bean), LG2 76.37 7.00.00 61 markers still showed segregation distortion (P , 0.05, LG3 56.77 5.20.62 18.7 %). Notably, all 26 markers located on LG11 were LG4 97.1138.91.88 highly skewed towards a heterozygous genotype, making the LG5 50.3104.66.29* number of wild rice bean alleles higher than expected. A LG6 83.10 7.67.62** LG7 65.5176.020.12*** similar phenomenon was observed in a BC1F1 population of LG8 68.62 6.32.93 rice (Oryza sativa/O. glaberrima//O. sativa): marked segre- LG9 95.33 8.73.77 gation distortion towards a heterozygous genotype (increased LG10 58.01 5.33.51 number of O. glaberrima alleles) was recorded (Doi et al., LG11 39.42 3.60.72 Total 796.1737347.64*** 1998; Koide et al., 2008b). It was proposed that the gamete eliminator gene S1 (on rice chromosome 6) induces abortion *, **, *** Significant at 5 %, 1 % and 0.1 % levels, respectively. of gametes carrying the opposite allele only in heterozygotes. The wild rice bean accession used here might possess a similar genetic factor that causes partial elimination of the opposite TABLE 8. Number of QTLs in each 10-cM interval allele in heterozygotes. A high level of marker segregation distortion on LG11 has No. of QTLs also been reported on a rice bean × V. nakashimae F2 map LG Detected Average† Range‡ x2§ (Somta et al., 2006). On rice chromosome 6, several genes affecting segregation distortion, such as cim (the cross- LG1 11 1.00 0–5 32.98*** incompatibility reaction in the male), Cif (the cross- LG2 7 1.88 0–6 35.26*** incompatibility reaction in the female) and S (gamete LG3 7 1.17 0–6 155.84*** 1 LG4 13 1.30 0–10 96594.29*** eliminator in O. rufipogon) have been detected (Koide et al., LG5 10 1.67 0–4 9.10 2008a). Because all the markers distributed along a distance LG6 0 0.00 0 0.00 of about 40 cM on LG11 of rice bean showed segregation dis- LG7 17 2.43 0–8 57.93*** tortion, several genes existing on LG11 might have played an . . LG8 2 0 29 0–1 0 18 important role in genetic differentiation, both within the rice LG9 3 0.30 0–1 0.29 LG10 1 0.17 0–1 0.03 bean and among Vigna species. LG11 2 0.50 0–1 0.59

*** Significant at 0.1 % level. Genome synteny between rice bean and azuki bean † Average QTL density on each linkage group. ‡ Range of the number of QTLs per 10-cM interval The rice bean linkage map was compared with an azuki § Departure from random distribution of QTLs in each 10-cM interval was bean linkage map (Han et al., 2005) by the distribution of tested under the null hypothesis in a Poisson goodness-of-fit. common azuki bean SSR markers (Fig. 3). Out of 172 azuki Isemura et al. — Rice bean domestication 939

LG1 LG2 LG3 LG4 LG5 LG6 CEDG029 CEDG133 CEDG285 CEDG149 CEDG144 CEDC035 CEDG020 CEDG006 CEDG205 CEDG139 CEDG014 CEDG187 CEDG124 CEDC028 CEDG184 CEDG154b CEDG050 CEDG037 CEDC055 CEDG067 CEDG065 CEDG264 CEDAAT002 CEDG018 CEDG186 CEDG191 CEDG003 CEDG268 CEDG244 CEDAAG004 CEDG154a CEDG118 CEDG053 CEDG253 CEDC009 CEDG084 CEDG080(9) CEDG088 CEDG225 CEDG245 CEDG091 CEDG008 CEDG018b CEDG109 CEDG261 CEDC033 CEDG018a CEDG114 CEDG025 CEDG232 CEDG146 CEDG251b CEDG107 CEDG290 (7,9,11) CEDG015 CEDG023 CEDG103 CEDG116b CEDG026 CEDG275 CEDG176 CEDG248 CEDC030 CEDC050 CEDG010 CEDC039 CEDAAG002 CEDG057 CEDG102 CEDG181 CEDG011

CEDG127

CEDG019 CEDG254 CEDG189 LG7 LG8 LG9 LG10 LG11

CEDG064 CEDG156 CEDG180 CEDG168 CEDG198 CEDC014 CEDG042 CEDG280 CEDG143 CEDG002 CEDG033 CEDG298 CEDG172(9) CEDG271 CEDG290c CEDG215 CEDG081b CEDG044 CEDG265 CEDG116a CEDG111 CEDC016 CEDG287 CEDG081a CEDG098 CEDG269 CEDG024 CEDG068 CEDG273 CEDG073 CEDG259 CEDG066 CEDG290b CEDG016 CEDG080 CEDG021 CEDG041 CEDG092 CEDG166 CEDG202 CEDG290a CEDG030 CEDG040 CEDG257 CEDG085 CEDG059 CEDG172 CEDG286 CEDC011 CEDG243 CEDG251a

CEDG173 10 cM CEDG228

CEDG022

F IG. 3. Comparative genetic linkage map of rice bean (left) and azuki bean (right), based on common azuki bean SSR markers. Linkage groups of the rice bean map were aligned with the corresponding LGs of the azuki linkage map (Han et al., 2005). The number of each LG corresponds to that used for the azuki linkage map. Lines between LGs connect the positions of common marker loci. Markers followed by a number in parenthesis indicate the positions of loci on other LGs. In the comparison of the rice bean and azuki bean maps, internal inversions were found between adjacent marker loci in italics. 940 Isemura et al. — Rice bean domestication bean SSR markers mapped on the rice bean linkage map, 129 remainder – LG1, LG3 and LG5 – were associated with were common to those used in the azuki bean map. The changes in plant size. common SSR markers were present on all LGs, and the Clustering of QTLs may be due to pleiotropy or close number of common markers per LG ranged from 7 (LG3 linkage of QTLs. Single mutations can have pleiotropic and LG7) to 15 (LG1). There was a high level of co-linearity effects on various organs. In , QTLs related to change of marker order between rice bean and azuki bean. in inflorescence sex and number and length of internodes in However, a few differences, exemplified by genetic dis- lateral branches and inflorescences are distributed within a tance, inversions and duplications, were observed between narrow genomic region (Doebley et al., 1995). Change in the two linkage maps (Fig. 3). Of the 11 LGs, LG9 exhibited these traits is explained by the pleiotropic effect of a single the most differences. The upper part of rice bean LG9 is con- tb1 gene. In arabidopsis, the seed testa colour mutant gene tt siderably longer (about 1.4 times) than that in azuki bean. causes a reduction in seed weight (Debeaujon et al., 2000). Three SSR markers, CEDG080, CEDG290 and CEDG172, Similarly, the seed testa colour mutant gene bks in tomato on azuki bean LG9 are scattered to different rice bean LGs decreases seed weight and increases fruit pH (Downie et al., (LG3, LG5 and LG11, respectively). Inversions were usually 2003). Further studies are required to determine whether pleio- found near the ends of LGs (on LG1, between CEDG133 tropy or close genetic linkage is responsible for the clustering and CEDG149; on LG3, between CED176 and CEDG010; of QTLs in rice bean. Near isogenic lines for 2 clustering on LG4, between CEDC028 and CEDC055 and between QTLs on LG4 are currently being developed to elucidate this CEDG181 and CEDG011). In contrast, duplications of question. marker loci were found near the centres of LGs. The single azuki bean marker loci CEDG018 (LG5), CEDG081 (LG10), CEDG154 (LG4) and CEDG251 (LG8) were, respectively, Comparison of domestication QTLs between rice bean and azuki duplicated on rice bean LG6 or in different positions on bean LG10, LG6 and LG1, whereas the rice bean single marker General features. In contrast to the high degree of conservation loci CEDG116 (LG10) and CEDG290 (LG5) were duplicated of the genomic structure between rice bean and azuki bean on azuki bean LG6 and LG9, respectively. (Isemura et al., 2007), the distribution of the main QTLs Even though the model Lotus japonicus has a small showed a marked difference (Table 9 and Fig. 4). For 28 dom- genome, large-scale segmental duplication at the sequence estication traits, 69 QTLs were found in rice bean and 76 in level has occurred within the genome (Sato et al., 2008). azuki bean, of which only 15 were common (Table 9 and Meiosis in hybrids between rice bean and azuki bean is Fig. 4). Most of the common QTLs were associated with normal (Ahn and Hartmann, 1978), and chromosomes of seed-size-related traits (SD100WT, SDL, SDW and SDT) both species have a similar C-banding pattern (Zheng et al., and pod dehiscence (PDT). 1991). However, the fluorescent banding patterns of rice In azuki bean, QTLs with a large effect [phenotypic vari- bean chromosomes, indicating the distributions of GC- and ation explained (PVE) . 20 %] were found on five LGs AT-rich genomic regions, have no similarity with those in (LG1, seed dormancy and internode length; LG2, seed size azuki bean (Zheng et al., 1993). The differences between and epicotyl and lower internode length; LG7, pod dehiscence rice bean and azuki bean observed here can be explained by and pod size; LG8, pod length; LG9, twining habit) (Fig. 4). In the occurrence of small chromosomal rearrangements or trans- rice bean, in contrast, they were detected on only two LGs locations during the evolutionary divergence of these species. (LG4, seed and pod size and seed dormancy; LG7, pod dehis- cence) (Fig. 4). In mung bean (T. Isemura et al., unpub. res.), Genomic regions and distributions of QTLs involved in rice bean 79 QTLs were found for 28 traits, and QTLs with a large effect domestication (PVE . 20 %) were found on seven LGs. These findings reveal that mutations that cause large changes in domestication Commonly, domestication-related traits are controlled by traits have occurred on fewer LGs in rice bean than in the two several major genes that are not randomly distributed across other species. Marked differences between rice bean and azuki crop genomes (Gepts, 2004) with the only one reported excep- bean were found on LG4 and LG9. Many QTLs with large tional example of sunflower domestication (Burke et al., 2007; effect were detected on LG4 of rice bean, whereas few Wills and Burke, 2007). This general rule, which is also appli- QTLs were detected on this LG in azuki bean. In contrast, cable to rice bean domestication, may be related to the major QTLs for domestication-related traits were abundant phenomenon called ‘cultivation magnetism’ and should be on LG9 in azuki bean, whereas only a few QTLs were detected considered under ‘protracted transition paradigm’ of crop dom- on LG9 of rice bean. estication (Allaby, 2010). Domestication QTLs of rice bean for various organs Seed size. The accession with the largest seed among germ- co-localized to several narrow genomic regions on LG1, plasm conserved in the NIAS Genebank was selected as the LG2, LG3, LG4, LG5 and LG7 (Tables 7 and 8 and Fig. 3 cultivated parent of rice bean. The 100-seed weight of this and Fig. S2 in Supplementary data). In particular, the distri- accession was 27.4 g (Table 5). This value is much larger bution of QTLs with large effects was limited to three LGs than those of commonly cultivated rice bean [4.89–6.68 g (LG2, LG4 and LG7). LG2 was associated with changes in (Bisht et al., 2005); 3.9g(Somta et al., 2006)]. There was a seed and pod size. LG4 was associated with changes in seed 15-fold weight difference between the cultivated and wild and pod size and water absorption by seeds. LG7 was associ- rice bean (1.8 g per 100 seeds) used here, and seven QTLs ated with changes in pod dehiscence and stem length. The were detected for 100-seed weight (Table 6). Two QTLs Isemura et al. — Rice bean domestication 941

TABLE 9. Detected QTLs that were common to rice bean and azuki bean

Rice bean Azuki bean

Trait QTL LG Nearest marker QTL LG Nearest marker

SD100WT Sd100wt4.1.1 + 1 CEDG019 Sd100wt1.1.2 + 1 CEDG090 Sd100wt4.2.1 + 2 cp08299 Sd100wt1.2.1 + 2 CEDG261 SDL Sdl4.1.1 + 1 BM181 Sdl1.1.2 + 1 CEDG090 Sdl4.2.1 + 2 cp08299 Sdl1.2.1 + 2 CEDG261 Sdl4.7.1 + 7 CEDG111 Sdl1.7.1 + 7 CEDG111 SDW Sdw4.1.1 + 1 cp05137 Sdw1.1.1 + 1 CEDG090 Sdw4.2.1 + 2 cp08299 Sdw1.2.1 + 2 CEDC009 SDT Sdt4.1.1 + 1 CEDG019 Sdt1.1.1 + 1 CEDG090 Sdt4.2.1 + 2 cp08299 Sdt1.2.1 + 2 CEDC009 PDT Pdt4.7.1– 7 CEDG064 Pdt1.7.1– 7 CEDG064 ST04I St4i4.1.1 + 1 CEDG254 St4i1.1.1 + 1 CEDG090 ST07I St7i4.1.1 + 1 CEDG283 St7i1.1.1 + 1 CEDG090 STL Stl4.1.1 + 1 CEDG189 Stl1.1.1 + 1 CEDG090 Stl4.5.1 + 5 CEDG027 Stl1.5.1 + 5 CEDG067 STT Stt4.9.1 + 9 GATS11 Stt1.9.1 + 9 CEDG238 were involved in the 10-fold weight difference between culti- contribution (90.5 %) was detected at a similar map position vated (16 g per 100 seeds) and wild (1.7 g per 100 seeds) azuki (Isemura et al., 2007) and was considered common to the bean (Isemura et al., 2007). These two 100-seed weight QTLs two species (Table 9 and Fig. 4). were common to the two species and were identified on LG1 and LG2 (Table 9 and Fig. 4). In addition, a rice bean-specific 100-seed weight QTL with the largest effect (PVE ¼ 33.4%) Useful QTLs for breeding was detected on LG4 (Table 6). The differences in the large-effect domestication QTLs suggest that rice bean and azuki bean harbour specific useful Seed dormancy. Physical seed dormancy is generally caused by genes and can play important roles as new gene resources the presence of water-impermeable layers of palisade cells in for each other. For example, a QTL for twining habit with the seed coat (Finch-Savage and Leubner-Metzger, 2006). large effect on LG9 is specific to azuki bean, whereas the The water absorption occurs through the structure of the stro- 100-seed weight QTL with large effect on LG4 is specific to phiole (parenchymatous tissue) adjacent to the hilum in rice bean (Fig. 4). The seed-dormancy-related QTL with the Vigna species (Gopinathan and Babu, 1985). largest effect was detected on LG4 in rice bean and LG1 in Five seed-dormancy-related QTLs were detected in rice azuki bean. Similarly, the pod-size-related QTL with the bean (Table 6). Five were detected also in azuki bean largest effect was detected on LG4 in rice bean and LG7 in (Isemura et al., 2007). However, none is common to both azuki bean, and no common QTL was found. species. The seed-dormancy-related QTL with the largest Hybrids can be produced between rice bean and azuki bean effect was detected on LG4 in rice bean and on LG1 in when rice bean is used as the seed parent (Kaga et al., 2000). azuki bean. Therefore, it seems that the seed weight QTL on LG4 in rice A dominant gene controlling pale red hilum in the wild bean and the QTL for loss of twining habit on LG9 in azuki parent was mapped on LG5. However, no QTL for seed dor- bean are particularly useful for the breeding of larger-seeded mancy was found in this region. This result suggests that the azuki bean cultivars and determinate rice bean cultivars, mutation for change of hilum colour had no effect on physical respectively. dormancy in rice bean. The weak but positive effects on water absorption of the wild alleles for seed dormancy QTLs on LG2 and LG10 CONCLUSIONS might reflect the modest degree of seed dormancy in the wild parent (Table 5). Passport data revealed that the natural This is the first report of a genetic linkage map and QTLs for habitat of the wild parent was near a river in Kanchanaburi domestication-related traits in rice bean. A genetic linkage province, Thailand, where the soil may be moist and the temp- map of rice bean was constructed using 223 SSR markers erature high enough for growth throughout the year (Tomooka from related legume species and 103 AFLP markers. A total et al., 2000a). Therefore, strong seed dormancy during the dry of 326 markers converged into 11 LGs, corresponding to the season may not be important for this wild accession, and this haploid number of rice bean chromosomes. Using this map, may suggest why the wild accession possesses a QTL that 31 domestication-related traits in rice bean were dissected reduces seed dormancy. into 69 QTLs, but the differences between cultivated and wild parents were found to be controlled by only a few Pod dehiscence. A single QTL for pod dehiscence was detected major QTLs. Major QTLs for unrelated organs were distribu- in rice bean and azuki bean. In rice bean, a pod dehiscence ted as clusters on LGs 2, 4 and 7. The inheritance of QTL with a relatively high contribution to this trait (42.4%) domestication-related traits was so simple that the few major was found on LG7. In azuki bean, a QTL with a higher QTLs explained the phenotypic variation between cultivated 942

100-seed weight, Seed length, Seed width, Seed thickness + Pod dehiscence-

4th and 7th Internode lengths and Stem lengths +, Seed length + 8th–10th Internode lengths + 100-seed weight, Seed width, Seed thickness + Pod width + 6th–10th Internode lengths and Stem length + LG1 CEDC030 CEDC039 CEDC057 CEDC102 CEDG133 CEDG149 CEDG144 CEDG003 CEDG053 CEDG109 CEDG025 CEDG019 CEDG254 CEDG189 LG7 Seed dormancy - Seed dormancy - CEDG064 CEDG143 CEDG215 CEDG111 CEDG041 CEDG085 4th and 7th Internode lengths and Stem lengths +, 1st, 2nd, 3rd, 5th and 6th Internode lengths + Maximum leaf length, Maximum leaf width +

100-seed weight +, Seed length, Seed width, Seed thickness + Pod length, Pod width +

Seed length + 7th, 9th and 10th Internode lengths and Stem length -

Pod dehiscence + Twining habit - Pod length, Pod width + 100-seed weight, Seed length, Seed width, Seed thickness + Isemura Seed dormancy -

LG2

LG8 al. et CEDC018 CEDC009 CEDC050 CEDG029 CEDG285 CEDG006 CEDG050 CEDG065 CEDG244 CEDG225 CEDG261 CEDG026 CEDG275 CEDAAG002 CEDC016 CEDG156 CEDG033 CEDG271 CEDG265 CEDG269 CEDG073 CEDG016 CEDG092 CEDG202 CEDG030 CEDG040 CEDG257 CEDG059 CEDG286 CEDG251

100-seed weight, Seed length, Seed width, Seed thickness + domestication bean Rice — Pod length + Epicotyl, 1st - 3rd Internode lengths + Seed length + Primary leaf width +

100-seed weight, Seed length, Seed width, Seed thickness + Pod length, Pod width + Stem thickness + Seed dormancy - Branch number + Epicotyl length + LG9 CEDC011 CEDG024 CEDG259 CEDG166 CEDG173 CEDG228 CEDG022

LG4 Seed length CEDC035 CEDC028 CEDC055 CEDC033 CEDG139 CEDG154 CEDG088 CEDG091 CEDG232 CEDG107 CEDG103 CEDG181 CEDG011 CEDG127 Pod length +, Pod width +

Twining habit - Primary leaf width + Stem thickness + Maximum leaf width + 6th–10th Internode lengths and Stem length - Twinning habit -

Stem length + Stem thickness + 100-seed weight, Seed thickness +

LG5 CEDG020 CEDG014 CEDG124 CEDG184 CEDG067 CEDG264 CEDG268 CEDG253 CEDG008 CEDG018 CEDG114 CEDG023 10 cM

Stem length +

F IG. 4. Comparative QTL map of domestication-related traits in rice bean (upper) and azuki bean (lower; after Isemura et al., 2007). QTLs enclosed by rectangles are common to azuki bean and rice bean. QTLs in bold italics explain over 20 % of the phenotypic variation of the trait. Isemura et al. — Rice bean domestication 943 and wild rice bean. The high level of genome synteny between linkage map and its comparison with an azuki bean [Vigna angularis rice bean and azuki bean facilitated QTL comparison between (Willd.) Ohwi and Ohashi] linkage map. Theoretical and Applied Genetics 113: 1261–1269. the species. Major QTLs in rice bean were found on LG4, Chen X, Laudeman T, Rushton P, Spraggins T, Timko M. 2007. CGKB: an whereas major QTLs in azuki bean were found on LG9. The annotation knowledge base for cowpea (Vigna unguiculata L.) methyl- results suggest that the degree of domestication or divergence ation filtered genomic genespace sequences. BMC Bioinformatics 8: between wild and domesticated rice bean was lower than in 129. doi:10.1186/1471-2105-8-129. azuki bean. The results provide a genetic foundation for Debeaujon I, Leon-Kloosterziel KM, Koornneef M. 2000. Influence of the testa on seed dormancy, germination, and longevity in Arabidopsis. Plant improvement of rice bean. Interchanges of major QTLs Physiology 122: 403–413. between rice bean and azuki bean might be useful to Doebley J, Stec A, Gustus C. 1995. Teosinte branched 1 and the origin of broaden the genetic variation in both species. maize: evidence for epistasis and the evolution of dominance. Genetics 141: 333–346. Doi K, Yoshimura A, Iwata N. 1998. RFLP mapping and QTL analysis of SUPPLEMENTARY DATA heading date and pollen sterility using backcross populations between Oryza sativa L. and Oryza glaberrima Steud. Breeding Science 48:395–399. Supplementary data are available online at www.aob.oxford- Downie AB, Zhang D, Dirk LMA, et al. 2003. Communication between the journals.org/. Table S1: Information for cowpea SSR primer maternal testa and the embryo and/or endosperm affect testa attributes in pairs designed in the present study. Table S2: Correlation coef- tomato. Plant Physiology 133: 145–160. ficients between traits in the BC F (or BC F ) population of Finch-Savage WE, Leubner-Metzger G. 2006. Seed dormancy and the 1 2 1 1:2 control of germination. New Phytologist 171: 501–523. the cross between cultivated rice bean and wild rice bean. Fig. Fukuoka H, Nunome T, Minamiyama Y, Kono I, Namiki N, Kojima A. S1: Frequency distributions of all traits examined in the BC1F2 2005. Read2Marker: a data processing tool for microsatellite marker (or BC1F1:2) population. Fig. S2: Distributions of the QTLs development from a large data set. Biotechniques 39: 472–476. found on each LG in the populations analysed and the Gaitan-Solis E, Duque MC, Edwards KJ, Tohme J. 2002. 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