The genetics of domestication of rice bean, Vigna umbellata.

BACKGROUND AND AIMS: The Asian genus Vigna, to which four cultivated species (rice bean, azuki bean, mung 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 BC(1)F(1) 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 phenotypic 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 improvement of rice bean; interchange of major QTLs between rice bean and azuki bean might be useful for broadening the genetic variation of both species.

INTRODUCTION

The genus Vigna subgenus Ceratotropis consists of 21 species that are distributed across a wide region of Asia. Six cultivated species belong to this subgenus (Tomooka ). Among these, mung bean (Vigna radiata), rice bean (V. umbellata), black gram (V. mungo) and azuki bean (V. angularis) are economically important in Asian countries. Taxonomically, wild forms of species are usually recognized as varieties below the rank of species of the Asian Vigna. However, numerous differences in morphological and physiological traits associated with domestication are observed between the cultivated and wild forms. These differences, collectively called the domestication syndrome, result from selection over several thousands of years of adaptation to cultivated environments, human nutritional requirements and preferences (Hawkes, 1983). The four cultivated species listed above are therefore ideal material for improving our comparative genomics-based understanding of the gene evolution related to domestication within and among Vigna species and for characterizing useful traits as quantitative trait loci (QTLs) for use in breeding.

To compare the genomic structures and genomic regions associated with domestication among these four species, genetic linkage maps of azuki bean (Han ) and black gram (Chaitieng ) were constructed previously using simple sequence repeat (SSR), restriction fragment length polymorphism (RFLP) and amplified fragment length polymorphism (AFLP) markers; the QTLs for domestication-related traits of azuki bean have been identified (Isemura ) in populations derived from crosses between cultivated and wild forms. The order of common markers on the linkage groups (LGs) is highly conserved between the azuki bean map (Han ) and the black gram map (Chaitieng ), although differences, such as inversions, deletions, duplications and a translocation, were observed between the two genomes in several LGs.

This paper focuses on the rice bean genome as part of a comparative genome analysis among members of the subgenus Ceratotropis. Rice bean is a traditional crop grown across south, south-east and east Asia, and its wild form is distributed across a wide area of the tropical monsoon forest climatic zone from eastern India, Nepal, Myanmar (Burma), Thailand, Laos and southern China to East Timor (Arora ; Tomooka ; Bisht ; Seehalak ; Gautam ; Tomooka, 2009). The dried mature seeds of rice bean are usually eaten with rice or in soups, and the leaves, flowers, shoots and young pods are eaten as a vegetable in the upland areas of south-east Asia and southern China. Rice bean is also important as a fodder and a green manure (Tomooka ). Recently, with 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 ; Bajracharya ; Muthusamy ). The domesticated accessions from south-east Asia showed the largest genetic variations in AFLP (Seehalak ) and SSR (J. Tian et al., Institute of Cereal and Oil Crops, Hebei Academy of Agricultural and Forestry Sciences, China, unpubl. res.) markers and the largest phenotypic variations in seed coat colour and seed size (Tomooka, 2009). These results suggest that south-east Asia was the centre of the origin and genetic diversity of this species (Tomooka, 2009).

To date, rice bean has not been subjected to systematic breeding, despite the species' many useful characteristics, including bruchid resistance (Tomooka ; Kashiwaba ; Somta ); disease resistance, particularly to yellow mosaic virus (Arora ; Borah ) and bacterial leaf spot (Arora ), and the highest potential grain yield among Ceratotropis species (Smartt, 1990). Therefore, rice bean can best be described as a scientifically neglected crop of great potential.

Genetic linkage maps using rice bean as one parent have been constructed for populations derived from interspecific crosses [rice bean × azuki bean (Kaga ) and rice bean × V. nakashimae (Somta )]. These linkage maps have been used to localize genes for several qualitative traits (Kaga ) and QTLs for resistance to bruchid beetles and for seed weight (Somta ). However, a precise genetic linkage map from a large population derived from an intraspecific cross has not been constructed.

The objectives in this study were (a) to construct a precise genetic linkage map of rice bean using SSR markers from related grain legumes in an intraspecific population derived from cultivated and wild forms; (b) to examine genomic synteny between rice bean and azuki bean on the basis of the linkage map; (c) to identify QTLs for domestication-related traits in rice bean; and (d) to compare the QTLs detected in rice bean with QTLs in azuki bean.

MATERIALS AND METHODS

Mapping population

Plant materials were obtained from the Genebank (http://www.gene.affrc.go.jp/index_en.php) collection of the National Institute of Agrobiological Sciences (NIAS), Tsukuba, Japan. A single F1 hybrid between a cultivated rice bean (JP217439, Col. No. 2002M21) and a wild rice bean accession (JP210639, Col. No. CED99T-2) was backcrossed to cultivated rice bean (JP217439) as a male parent to construct a BC1F1 population consisting of 198 plants. The wild parent accession was chosen because AFLP analysis had revealed that it was highly differentiated from other accessions (Seehalak ). It was collected by one of us (N.T.) from Kanchanaburi province in Thailand; its habitat was a moist deciduous forest along a river (Tomooka ). The cultivated parent accession, a landrace from Myanmar (Tomooka ), was selected because it had the largest seeds among the germplasm conserved in the NIAS Genebank.

Growth condition and trait measurement

Thirty-one traits related to domestication were evaluated (Table 1). Of these, 30 were treated as quantitative traits and one (hilum colour) as a qualitative trait. The BC1F1 population of 198 plants, together with ten plants of each parent, were grown in a vinyl house at NIAS, Tsukuba, Japan (36 °2′N, 140 °8′E), from September 2006 to March 2007 under natural day length. The natural day length gradually decreased from 12 h 28 min (14 September 2006) to 9 h 42 min (22 December) and thereafter increased to 12 h 18 min (25 March). From October, the vinyl house was heated to maintain a minimum temperature of 15 °C.

Table 1.

Domestication-related traits examined in the BC1F1 or BC1F1:2 population derived from the cross between cultivated and wild rice bean

General attribute	Organ	Trait	Trait abbreviation	QTL/gene	Evaluation method	Evaluated population
Seed dormancy	Seed	Seed – water absorption (%)	SDWA	Sdwa	Percentage of seeds that had absorbed water 1 d after sowing at 15 °C 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
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
	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
	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 (cm)	LFML	Lfml	Length of the largest terminal leaflet on leaves between node on 1st trifoliate leaf and node on 10th trifoliate leaf	BC1F1
		Leaf – maximum leaflet width (cm)	LFMW	Lfmw	Width of the largest terminal leaflet on leaves between node on 1st trifoliate leaf and node on 10th trifoliate leaf	BC1F1
Plant type	Epicotyl	Epicotyl length (cm)	ECL	Ecl	Length from cotyledon to primary leaf	BC1F1
	Stem	Stem – internode length (1st to 10th) (cm)	ST1I-ST10I	St1i–St10i	Length from node on primary leaf to each node	BC1F1
		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 (ith node)	BRP	Brp	Position of first branch on main stem	BC1F1
Phenology	Flower	Flower – days to first flower (day)	FLD	Fld	Number of days from sowing to flowering of 1st flower	BC1F1
	Pod	Pod – days to maturity of all pods (day)	PDDM	Pddm	Number of days from flowering of first flower to harvesting of first pods	BC1F1
Yield potential	Seed	Seed – number of seeds per pod (count)	SDNPPD	Sdnppd	Number of seeds per pod	BC1F1:2
Pigmentation	Seed	Seed – hilum colour	SDHC	Sdhc	Pale red or white	BC1F1:2

Seedling traits – leaf primary length (LFPL), leaf primary width (LFPW) and epicotyl length (ECL) – were recorded when the first trifoliate leaf opened, and vegetative traits – leaflet maximum length (LFML), leaflet maximum width (LFMW), stem internode length (1st to 10th: ST1I to ST10I), stem length (STL) and stem thickness (STT) – were recorded when the 10th trifoliate leaf was fully developed. After all pods had been harvested, branch number (BRN) and position of the first branch on the main stem (BRP) were recorded. These traits were evaluated in the BC1F1 generation.

Seed-related traits were investigated by using the seeds from BC1F1 plants. Seed water absorption (SDWA) was investigated as an index of seed dormancy. Fifteen unscarified seeds were placed on wet filter paper and incubated in the dark at 15 °C for 24 h; the number of seeds that absorbed water was then recorded. Seed dimensions – seed length (SDL), seed width (SDW) and seed thickness (SDT) – were averages of ten seeds. The 100-seed weight (SD100WT) was evaluated using intact seeds.

Pod traits – pod length (PDL), pod width (PDW) and number of twists along the length of the dehisced pod when kept at room temperature (PDT) – were based on ten pods. 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 pod (PDDM) were recorded in the BC1F1 population. The number of seeds per pod (SDNPPD) was measured in ten pods.

Seed hilum colour (SDHC) was evaluated in seeds from BC1F1 plants, and the seed hilum of each plant was classified as either cultivated parent type (white) or wild parent type (pale red).

DNA extraction

Total genomic DNA in BC1F1 plants was extracted from 200 mg of fresh leaf tissue using a DNeasy plant mini kit (Qiagen, Valencia, CA, USA). The DNA concentration was adjusted to 5 ng μL−1 for the SSR analysis and to 25 ng μL−1 for the AFLP analysis by comparison with known concentrations of standard λDNA in 1 % agarose gel.

SSR analysis

The SSR analysis in the BC1F1 population was performed by the method of Han . Three hundred and twenty-five SSR primer pairs were screened from azuki bean (Wang ), 163 from cowpea (Vgina unguiculata; Li ) and 40 from common bean (Phaseolus vulgaris; Yu ; Gaitan-Solis ; Blair ; Guerra-Sanz, 2004) to determine whether there was polymorphism between the two parents. By using the read2Marker program (Fukuoka ), another 156 cowpea SSR primer pairs (Table S1 in Supplementary Data, available online) on cowpea genomic sequences from the Cowpea Genomics Knowledge Base (Chen ; http://cowpeagenomics.med.virginia.edu/CGKB/) were designed and screened for parental polymorphisms.

AFLP analysis

AFLP analysis was performed with an AFLP Core Reagent Kit (Invitrogen, Carlsbad, CA, USA). The steps of DNA digestion, ligation of adaptor and pre-selective amplification were performed in accordance with the method of Han . Selective amplification and detection of AFLP bands were performed using the method of Kaga . On the basis of information on the AFLP primer combinations used to construct several linkage maps of Asian Vigna species (Han ; Chaitieng ; Kaga ), 28 primer combinations were selected for the AFLP analysis in the BC1F1 population.

Map construction

The linkage map was constructed using the method of Han using JoinMap v. 4·0 (Van Ooijen, 2006). Marker segregation was analysed by a chi-squared test for goodness-of-fit to the expected Mendelian ratio (1 : 1). The recombination frequencies were converted into map distance (cM) by using the Kosambi mapping function (Kosambi, 1944). After a framework map had been built using codominant SSR markers, dominant SSR and AFLP markers were integrated into the framework. Numbering of the LGs followed that for azuki bean (Han ).

Data analysis

For the quantitative traits, the mean, standard error minimum value, maximum value and broad-sense heritability were calculated, and the frequency distributions of phenotypes in the BC1F1 (or BC1F1:2) population were examined for each. The correlations between pairs of traits were also calculated. The hilum colour segregation pattern was investigated in each BC1F1 individual and the frequency distribution was analysed by chi-squared test for goodness-of-fit to the expected Mendelian ratio (1 : 1). The segregation pattern data were used to identify the map position of the gene controlling this trait.

QTL analysis

Mean data for each trait (SDWA data were arcsine-transformed first) were used in the QTL analysis. QTLs were identified by means of multiple interval mapping using the software package MultiQTL v. 3·0, as described by Kaga . QTL nomenclature followed Somta . To test for randomness of the genomic distribution of QTLs for domestication-related traits, chi-squared tests were calculated as described by Isemura . To test whether or not QTLs were randomly distributed along an LG, a Poisson distribution function was calculated as described by Isemura .

RESULTS

Genetic linkage map of rice bean

SSR primer pairs developed from azuki bean, cowpea and common bean were used to construct the genetic linkage map of rice bean. The percentage fragment amplification in rice bean was high, with values between 73·6 % (cowpea) and 96·6 % (azuki bean) (Table 2). Of the 325 azuki bean, 163 cowpea and 40 common bean SSR primer pairs screened, 167 azuki bean (51·4 %), 44 cowpea (27·0 %) and 7 common bean (17·5 %) primer pairs showed clear polymorphism between the cultivated and wild rice bean parents (Table 2). All 223 marker loci derived from the 218 polymorphic SSR primer pairs were used for mapping; five azuki bean SSR primer pairs (CEDG018, CEDG081, CEDG154, CEDG251 and CEDG302) detected two loci.

Table 2.

Summary of amplification and polymorphic rates of SSR primer pairs from three legumes in rice bean

	No. of SSR primer pairs
	Screened	Amplified (%)	Polymorphic (%)
Azuki bean	325	314 (96·6)	167 (51·4)
Cowpea	163	120 (73·6)	44 (27·0)
Common bean	40	37 (92·5)	7 (17·5)
Total	528	471 (89·2)	218 (41·3)

In the AFLP analysis, 1380 bands were detected by 28 primer combinations. The total number of bands per primer pair ranged from 39 to 59, and the average number was 49·3. Out of 1380 bands, 103 (range 1–9 per primer combination, average 3·7) showed clear polymorphism between the cultivated and wild parents (Table 3).

Table 3.

Polymorphism levels of AFLP primer combinations used to construct the rice bean linkage map

	No. of bands
Primer pair	Total	Polymorphic	Percentage polymorphism
E32M39	53	2	3·8
E32M44	41	2	4·9
E32M45	56	6	10·7
E33M49	45	3	6·7
E41M54	48	1	2·1
E42M42	41	1	2·4
E43M75	57	5	8·8
E44M44	52	5	9·6
E44M59	43	4	9·3
E45M33	53	4	7·5
E46M34	53	2	3·8
E47M47	59	8	13·6
E51M89	47	3	6·4
E63M55	41	6	14·6
E63M63	55	3	5·5
E66M66	44	3	6·8
E80M46	39	1	2·6
E81M81	54	4	7·4
E82M82	52	4	7·7
E83M83	52	2	3·8
E86M54	56	9	16·1
E87M87	55	5	9·1
E88M88	48	3	6·3
E90M35	46	3	6·5
E90M90	51	3	5·9
E91M71	42	3	7·1
E91M91	45	3	6·7
E93M93	52	5	9·6
Total	1380	103
Average	49·3	3·7	7·5

A total of 326 loci (172 azuki bean SSR loci, 44 cowpea SSR loci, 7 common bean SSR loci and 103 AFLP loci) could be assigned to 11 LGs covering a total of 796·1 cM of the rice bean genome at an average marker density of 2·5 cM (Fig. 1 and Table 4). The genomic region in this map covered 95·7 % of the azuki bean genome (832·1 cM) reported by Han . The number of markers on each LG ranged from 14 (LG7) to 42 (LG8). The length of each LG ranged from 105·8 (LG1) to 39·4 cM (LG11). The average distance between two adjacent markers ranged from 1·58 (LG11) to 5·04 cM (LG7). All LGs except LG5 had gaps greater than 10 cM between markers. LGs 1 and 3 had gaps greater than 15 cM. Sixty-one markers (18·7 %) showed segregation distortion (P < 0·05). The ratio of plants with heterozygous genotypes was high for all 61 markers which were found on LGs 5, 7, 8 and 11. Interestingly, all of the markers on LG11 showed segregation distortion at significant levels.

Fig. 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.

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	105·8	22	4	1	10	37	2·94
2	76·3	18	5	0	9	32	2·46
3	56·7	9	3	0	9	21	2·84
4	97·1	20	4	1	9	34	2·94
5	50·3	18	2	1	11	32	1·62
6	83·1	16	7	0	7	30	2·87
7	65·5	8	0	0	6	14	5·04
8	68·6	21	9	1	11	42	1·67
9	95·3	9	3	2	12	26	3·81
10	58·0	17	4	0	11	32	1·87
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.

Variation in domestication-related traits

The means, minima, maxima, standard errors and broad-sense heritabilities of traits in the parental lines and BC1F1 or BC1F1:2 populations were determined (Table 5). The mean SDWA of the cultivated parent was higher than that of the wild parent. PDT of the wild parent was greater than that of the cultivated parent. The sizes of the leaf, stem, seed and pod were larger in the cultivated parent. Among the stem-length-related traits, ECL, ST1I to ST10I and STL were longer in the cultivated parent.

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
Unit	Trait	Mean	Standard error	Minimum	Maximum	Mean	Standard error	Minimum	Maximum	Mean	Standard error	Minimum	Maximum	Heritability (%)
SD100WT*	g	27·4	0·314	25·8	28·5	1·8	0·039	1·7	1·9	14·6	0·216	7·4	23·5	96·1
SDL*	mm	10·3	0·073	10·1	10·7	4·3	0·024	4·3	4·4	8·0	0·039	6·6	9·6	92·5
SDW*	mm	6·0	0·036	5·8	6·1	2·2	0·034	2·1	2·2	4·8	0·027	3·7	5·8	94·9
SDT*	mm	7·0	0·030	6·9	7·2	2·5	0·017	2·4	2·5	5·5	0·030	4·4	6·6	97·7
SDWA*	%	98·0	1·225	95·0	100·0	33·0	2·550	25·0	40·0	63·8	1·957	0·0	100·0	97·2
SDNPPD*	count	7·1	0·197	6·4	7·8	6·7	0·108	6·4	6·9	4·9	0·094	1·5	8·8	90·6
SDHC*	–	White				Pale red				Pale red : white = 96 : 97 (χ2 = 0·005)				–
PDL	cm	11·8	0·258	10·6	12·6	4·5	0·078	4·4	4·7	8·3	0·083	5·4	11·2	81·5
PDW	mm	12·3	0·074	12·0	12·6	4·1	0·014	4·0	4·1	9·1	0·053	7·0	11·2	96·0
PDT	count	1·9	0·163	1·5	2·9	4·3	0·127	4·1	4·7	4·3	0·112	1·2	6·9	94·3
FLD	day	90·5	0·845	86·0	94·0	98·0	–	98·0	98·0	89·4	0·382	74·0	109·0	–
PDDM	day	63·9	0·934	61·0	69·0	–	–	–	–	57·1	0·272	49·0	66·0	–
ECL	cm	12·2	0·179	11·2	12·7	2·4	0·053	2·2	2·7	8·5	0·065	5·8	11·1	83·1
ST1I	cm	0·7	0·018	0·6	0·7	0·1	0·011	0·1	0·2	0·3	0·008	0·1	0·6	85·3
ST2I	cm	0·8	0·042	0·6	1·0	0·2	0·026	0·1	0·3	0·5	0·011	0·2	1·2	57·1
ST3I	cm	1·2	0·049	1·0	1·4	0·4	0·033	0·3	0·5	0·9	0·012	0·5	1·4	52·1
ST4I	cm	2·0	0·053	1·8	2·2	0·6	0·024	0·5	0·7	1·5	0·020	0·8	2·3	83·1
ST5I	cm	2·6	0·096	2·2	3·0	0·9	0·053	0·7	1·1	2·1	0·028	1·2	3·2	67·8
ST6I	cm	3·6	0·156	3·0	4·4	1·1	0·045	0·9	1·3	2·6	0·035	1·6	4·1	55·8
ST7I	cm	4·6	0·228	3·4	5·4	2·0	0·068	1·7	2·3	3·5	0·049	2·0	5·6	51·7
ST8I	cm	6·7	0·400	5·3	8·3	2·8	0·073	2·4	3·1	4·2	0·101	2·4	8·3	50·7
ST9I	cm	13·3	1·001	9·2	17·7	3·2	0·084	2·8	3·5	8·0	0·282	2·9	17·8	61·8
ST10I	cm	19·0	0·330	17·5	19·8	3·6	0·083	3·2	4·0	15·4	0·416	3·6	28·3	98·0
STL	cm	66·5	1·604	59·6	72·9	17·2	0·204	16·5	18·4	47·9	0·917	25·7	78·2	90·7
STT	mm	7·9	0·158	7·3	8·5	4·6	0·115	4·1	5·0	7·2	0·045	5·5	8·5	41·6
LFPL	cm	10·0	0·287	8·7	10·9	4·0	0·083	3·6	4·4	8·5	0·053	6·1	10·0	34·4
LFPW	cm	3·6	0·111	3·2	3·9	1·5	0·045	1·4	1·8	2·5	0·022	1·6	3·3	39·1
LFML	cm	18·5	0·202	17·9	19·4	11·1	0·277	9·9	12·7	17·0	0·113	12·7	20·8	79·7
LFMW	cm	12·1	0·194	11·3	13·1	6·1	0·172	5·5	7·2	10·8	0·080	7·6	14·2	77·3
BRN	count	7·1	0·295	6·0	8·0	2·7	0·236	2·0	4·0	6·4	0·131	3·0	11·0	82·5
BRP	ith	3·6	0·263	2·0	4·0	1·8	0·147	1·0	2·0	3·7	0·061	2·0	7·0	49·2

* These traits were evaluated in the BC1F1:2 generation.

FLD in the cultivated parent was 91 d and PDDM was 64 d. Because only one plant of the wild parent flowered, and it produced only one pod, it was not possible to evaluate the traits of wild parent precisely. In December 2007, pods and seeds were obtained from wild plants sown again in April 2007 and were used to evaluate the pod- and seed-related traits of the wild parent. SDNPPD and BRN were greater in the cultivated parent than in the wild parent.

The BC1F1 plants and BC1F1:2 lines showed a high degree of morphological and physiological variation (Table 5). Seed- and pod-size-related traits showed high heritability (>80 %), whereas stem- and leaf-related traits showed low heritability (<70 %) except in the case of ST4I, ST10I and STL. The means of the BC1F1 plants and BC1F1:2 lines fell between the means of the cultivated and wild parents for all traits except SDNPPD, PDT and BRP. Most traits in these populations showed a nearly normal distribution among parents (Fig. S1 in Supplementary data, available online). Transgressive segregation was observed in SDNPPD, PDT, FLD, STT and BRN.

In general, there were significant positive correlations (P < 0·05) between related traits, such as between stem length and each internode length, and between seed-size-related traits and pod-size-related traits (Table S2 in Supplementary Data). PDT was negatively correlated with seed-size-related traits, each internode length (ST1I to ST10I) and stem length (STL). Water absorption by seeds (SDWA) was positively correlated with epicotyl length and lower internode length (ST1I and ST2I). SDNPPD was highly correlated with seed-size-related traits and PDL. Seed- and pod-size-related traits and PDDM were positively correlated.

QTLs for domestication-related traits

QTL analyses were performed for each trait in the population (Table 6 and Fig. 2; for details see Fig S2 in Supplementary Data). In total, 73 QTLs and one morphological marker gene are reported for the 31 domestication-related traits. The number of QTLs may have been overestimated as a result of measurement of related traits such as 100-seed weight, seed length, seed width and seed thickness. Generally, one to seven QTLs were detected for each trait at a significant level (P < 0·001), except in the case of FLD, LFPL, LFPW, ST3I and BRP, for which no QTLs were detected.

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 +	1	7·13	0·0001	83·96	4·9	1·345
	Sd100wt4·2.1 +	2	20·97	0·0001	54·10	17·4	2·525
	Sd100wt4·3.1 +	3	5·51	0·0001	23·35	3·7	1·167
	Sd100wt4·4.1 +	4	29·97	0·0001	64·65	33·4	3·495
	Sd100wt4·5.1 +	5	9·54	0·0001	7·66	4·0	1·035
	Sd100wt4·5.2 +				35·41	3·4	0·871
	Sd100wt4·7.1 +	7	6·34	0·0001	34·94	5·0	1·357
	Total					71·8
SDL	Sdl4·1.1 +	1	11·73	0·0001	78·50	10·1	0·355
	Sdl4·2.1 +	2	19·06	0·0001	52·28	16·5	0·455
	Sdl4·4.1 +	4	25·08	0·0001	63·86	28·9	0·602
	Sdl4·5.1 +	5	10·53	0·0001	7·52	3·1	0·140
	Sdl4·5.2 +				35·41	5·1	0·229
	Sdl4·7.1 +	7	7·23	0·0001	29·32	5·6	0·264
	Total					69·3
SDW	Sdw4·1.1 +	1	5·01	0·0001	70·58	4·4	0·156
	Sdw4·2.1 +	2	15·83	0·0001	55·61	14·9	0·287
	Sdw4·3.1 +	3	7·99	0·0001	23·35	6·3	0·186
	Sdw4·4.1 +	4	21·26	0·0001	63·60	26·3	0·381
	Sdw4·5.1 +	5	4·01	0·0001	1·37	3·2	0·132
	Sdw4·7.1 +	7	4·95	0·0001	36·71	4·4	0·156
	Sdw4·11·1 +	11	7·18	0·0001	27·42	6·0	0·182
	Total					65·5
SDT	Sdt4·1.1 +	1	4·65	0·0001	83·96	3·4	0·158
	Sdt4·2.1 +	2	19·59	0·0001	53·32	17·6	0·359
	Sdt4·3.1 +	3	10·92	0·0001	23·35	8·6	0·251
	Sdt4·4.1 +	4	20·75	0·0001	63·72	25·2	0·430
	Sdt4·5.1 +	5	8·82	0·0001	6·26	4·1	0·148
	Sdt4·5.2 +				35·41	3·3	0·122
	Sdt4·7.1 +	7	5·50	0·0001	41·69	4·1	0·173
	Total					66·3
SDWA	Sdwa4·2.1–	2	4·10	0·0003	68·11	5·5	–2·24
	Sdwa4·3.1 +	3	4·33	0·0002	33·36	6·0	2·45
	Sdwa4·4.1 +	4	14·04	0·0001	82·52	25·1	10·54
	Sdwa4·8.1 +	8	3·15	0·0010	61·37	4·3	1·73
	Sdwa4·10·1–	10	3·23	0·0009	24·28	4·3	–1·75
	Total					45·2
SDNPPD	Sdnppd4·4.1 +	4	3·59	0·0008	62·68	7·4	0·696
	Sdnppd4·9.1 +	9	7·49	0·0001	9·35	5·4	0·461
	Sdnppd4·9.2 +				72·24	9·3	0·801
	Total					22·1
PDL	Pdl4·2.1 +	2	10·51	0·0001	57·06	16·7	0·920
	Pdl4·3.1 +	3	5·06	0·0001	22·37	6·9	0·592
	Pdl4·4.1 +	4	13·65	0·0001	63·59	23·1	1·082
	Total					46·7
PDW	Pdw4·1.1 +	1	9·44	0·0001	66·77	8·7	0·441
	Pdw4·2.1 +	2	12·30	0·0001	56·61	11·9	0·518
	Pdw4·3.1 +	3	7·21	0·0001	23·35	5·9	0·364
	Pdw4·4.1 +	4	21·86	0·0001	62·94	27·6	0·789
	Pdw4·5.1 +	5	7·75	0·0001	29·34	6·6	0·385
	Pdw4·7.1 +	7	5·19	0·0001	32·23	4·4	0·313
	Total					65·1
PDT	Pdt4·7.1–	7	20·27	0·0001	14·52	42·4	–1·994
	Total					42·4
FLD	Not detected
PDDM	Pddm4·4.1 +	4	6·96	0·0001	78·19	17·7	3·143
	Total					17·7
ECL	Ecl4·4.1 +	4	5·79	0·0001	73·40	9·8	0·565
	Ecl4·7.1 +	7	3·61	0·0004	48·78	5·8	0·436
	Ecl4·8.1 +	8	6·06	0·0001	44·72	10·4	0·583
	Ecl4·11·1 +	11	4·31	0·0001	0·00	7·2	0·484
	Total					33·2
ST1I	St1i4·5.1 +	5	3·55	0·0008	21·76	7·3	0·061
	St1i4·7.1 +	7	4·75	0·0001	41·69	9·8	0·071
	Total					17·1
ST2I	St2i4·7.1 +	7	3·28	0·0009	48·16	7·5	0·081
	Total					7·5
ST3I	Not detected
ST4I	St4i4·1.1 +	1	3·66	0·0005	85·99	7·4	0·155
	St4i4·7.1 +	7	4·26	0·0001	14·61	9·3	0·174
	Total					16·7
ST5I	St5i4·7.1 +	7	5·54	0·0001	16·47	12·9	0·281
	Total					12·9
ST6I	St6i4·7.1 +	7	4·80	0·0001	10·62	10·8	0·295
	Total					10·8
ST7I	St7i4·1.1 +	1	4·39	0·0001	99·10	9·6	0·421
	St7i4·7.1 +	7	4·36	0·0001	10·62	8·8	0·404
	Total					18·4
ST8I	St8i4·1.1 +	1	4·42	0·0001	97·14	12·0	0·795
	St8i4·7.1 +	7	5·79	0·0001	10·72	16·3	0·926
	Total					28·3
ST9I	St9i4·1.1 +	1	4·92	0·0001	92·58	14·5	2·440
	St9i4·7.1 +	7	3·76	0·0003	10·62	10·4	2·068
	Total					24·9
ST10I	St10i4·1.1 +	1	4·85	0·0001	95·93	14·4	3·620
	St10i4·7.1 +	7	4·11	0·0002	0·00	11·2	3·199
	Total					25·6
STL	Stl4·1.1 +	1	4·75	0·0001	93·50	12·9	7·392
	Stl4·5.1 +	5	3·36	0·0009	24·29	8·3	5·922
	Stl4·7.1 +	7	4·60	0·0001	11·13	11·6	7·023
	Total					32·8
STT	Stt4·9.1 +	9	3·59	0·0007	47·45	13·0	0·377
	Total					13·0
LFPL	Not detected
LFPW	Not detected
LFML	Lfml4·3.1 +	3	4·27	0·0001	21·92	6·7	0·824
	Lfml4·4.1–	4	15·93	0·0001	64·58	31·8	–1·802
	Total					38·5
LFMW	Lfmw4·4.1–	4	11·38	0·0001	64·32	25·6	–1·130
	Total					25·6
BRN	Brn4·4.1 +	4	5·18	0·0001	64·82	12·9	1·321
	Total					12·9
BRP	Not detected

Fig. 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.

Water absorption by seeds (SDWA)

One of the most important changes that occurred during domestication was a reduction in seed dormancy. The SDWA assay revealed that the water absorption rate of the cultivated parent (98 %) was higher than that of the wild parent (33 %) (Table 5). Five QTLs were detected, on LG2, LG3, LG4, LG8 and LG10, and QTL Sdwa4.4.1 on LG4 explained 25·1 % of the phenotypic variation. The alleles of the cultivated parent increased SDWA at Sdwa4·3.1, Sdwa4.4.1 and Sdwa4·8.1 and decreased water absorption at Sdwa4.2.1 and Sdwa4.10.1. The allele of the cultivated parent at Sdwa4.4.1 had an especially large effect, increasing the SDWA by 10·5 %.

Pod dehiscence (PDT)

Loss (reduction) of pod dehiscence is advantageous for harvesting seeds. The number of twists along the length of the shattered pod was used as an index of pod dehiscence. The cultivated parent had fewer twists. One QTL, Pdt4·7.1, with a relatively high contribution to this trait (42·4 %) was found on LG7.

Seed size (SD100WT, SDL, SDW, SDT)

Domestication of rice bean has resulted in a 15-fold increase in seed weight (Table 5). Six or seven QTLs were detected for seed-size-related traits (SD100WT, SDL, SDW and SDT) on LG1, LG2, LG3, LG4, LG5, LG7 and LG11. The QTLs with the highest contribution to these traits (25·2–33·4 %) were found on LG4. The QTLs on LG2 explained a further 14·9–17·6 % of the phenotypic variation.

Pod size (PDL, PDW)

Domestication of rice bean has resulted in a 2·5-fold increase in pod length (Table 5). Three QTLs for PDL and six QTLs for PDW were found (Table 6). Those affecting these two traits were found mainly on LG2, LG3 and LG4. The QTLs with the highest contribution for these traits (23·1 % for PDL and 27·6 % for PDW) were found on LG4. As expected, the alleles from the cultivated parent increased both PDL and PDW at all QTL positions. The QTLs for both PDL and PDW on LG2, LG3 and LG4 were located close to the QTLs for seed-size-related traits on each respective LG (Fig. 2).

Leaf size (LFPL, LFPW, LFML, LFMW)

The primary leaf length in many BC1F1 plants was close to that in the cultivated parent. Primary leaf width of most of the BC1F1 plants was distributed within a narrow range (about 0·9 cm), as determined by the frequency distribution (Fig. S1 in Supplementary Data). The values of heritability for both traits were low (<40 %; Table 5). As a result, no QTLs for these traits were detected.

Major QTLs for maximum leaf size (LFML, LFMW) were detected on LG4. These QTLs were located close to QTLs for seed- and pod-size-related traits. However, in contrast to the latter, the alleles from the cultivated parent had a negative effect on leaf size at these QTLs. The QTLs for these traits on LG4 explained 31·8 % (LFML) and 25·6 % (LFMW) of the phenotypic variation.

Stem length (STL), epicotyl length (ECL), internode length (ST1I to ST10I) and stem thickness (STT)

In the cultivated parent, the epicotyl, all internodes and the stem were longer, and stem was thicker, than in the wild parent (Table 5). Three QTLs (Stl4.1.1, Stl4.5.1 and Stl4.7.1) for STL were found, on LG1, LG5 and LG7 (Table 6). Alleles from the cultivated parent at all QTLs had the effect of increasing stem length. However, stem growth at the early, middle and late stages was controlled by different QTLs. Stem length from the primary to the 10th node QTLs (Stl4.1.1 on LG1, Stl4.7.1 on LG7) and from the middle to the upper internode length QTLs (4th to 10th; St7i4.1.1 to St10i4.1.1 on LG1 and St4i4.7.1 to St10i4.7.1 on LG7) were consistently located in similar map positions on LG1 and LG7. On the other hand, QTLs for the lower (1st and 2nd) internode lengths (St1i4.7.1 and Stl2i4.7.1) in the early stem-growth stage were found mainly on LG7 (at different QTL positions from those of the middle and upper internode lengths on LG7). Four QTLs for epicotyl length (Ecl4.4.1, Ecl4.7.1, Ecl4.8.1 and Ecl4.11.1) were found, on LG4, LG7, LG8 and LG11. Among these, a QTL on LG7 was located close to QTLs for the first and second internode length (St1i4.7.1 and St2i4.7.1). QTLs for epicotyl length (Ecl4.4.1 on LG4 and Ecl4.7.1 on LG7) and first and second internode length (St1i4.7.1 and St2i4.7.1 on LG7) were detected relatively close to the QTLs for seed-size-related traits on LG4 and LG7 (Fig. S2 in Supplementary Data). Only one stem diameter QTL, Stt4.9.1, was detected, on LG9. The alleles from the cultivated parent increased stem thickness. This QTL explained 13·0 % of the phenotypic variation.

Branch number (BRN) and first branch position (BRP)

The cultivated parent produced more branches on the main stem and developed the first branch at a higher internode than did the wild parent (Table 5). Only one significant QTL, Brn4.4.1, was identified for BRN. This QTL was detected close to the QTLs for seed- and pod-size-related traits. No significant QTL was found for BRP.

Flowering time (FLD)

The frequency distribution in BC1F1 plants revealed that most of the plants flowered within a short period (about 10 d; Fig. S1 in Supplementary Data). As a result, no QTL was identified.

Days to pod maturity (PDDM)

As has been shown in azuki bean (Kaga ), it was expected that in rice bean it would take more time to maturity (translocation of dry matter to the seed) in the cultivated parent than in the wild parent, presumably because of the difference in seed size between the two parents. Although it was not possible to evaluate the PDDM of the wild parent precisely, one QTL was found, on LG4. This QTL explained 17·7 % of the phenotypic variation and was found near the QTLs for seed- and pod-size-related traits. The alleles from the cultivated parent delayed maturity.

Seed number per pod (SDNPPD)

The cultivated parent produced slightly more seeds per pod than the wild parent (Table 5). Three QTLs with small effects (5·4–9·3 %) on SDNPPD were identified, on LG4 and LG9 (two of them on LG9). The QTL on LG4 was detected close to the QTLs for seed- and pod-size-related traits. At all QTLs, alleles from the cultivated parent had a positive effect on SDNPPD.

Seed hilum colour (SDHC)

The pale red hilum from the wild parent was dominant to the white hilum. The segregation ratio for this trait fitted the expected ratio (1 : 1). The recessive gene for white hilum from the cultivated parent was tentatively named sdhc4.5.1. This gene was mapped between AFLP markers E63M55-097 and E93M93-128 on LG5.

Distribution of domestication-trait-related QTLs

QTLs with large effect were found on LGF2, LG4 and LG7 (Fig. 2) as described below.

LG2

QTLs for seed- and pod-size-related traits were found in a narrow region. In the region near cowpea marker cp08299, QTLs for seed size (Sd100wt4.2.1, etc.) and pod size (Pdl4.2.1 and Pdw4.2.1) were found. At about 15 cM away from this region, a QTL for seed coat permeability (Sdwa4.2.1) was located. Interestingly, the cultivated parent alleles decreased seed coat permeability.

LG4

Between SSR markers CEDG103 and CEDG175, QTLs with a strong effect on seed, pod and leaf sizes were found. QTLs for seed number per pod and branch number were also found in this region. At about 20 cM away from this region, a QTL strongly associated with seed coat permeability (Sdwa4.4.1) was located. QTLs for pod maturity (Pddm4.4.1) and epicotyl length (Ecl4.4.1) were also linked to this area.

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 (Stl4.7.1) were found. Between SSR marker CEDG215 and AFLP marker E91M91-185 (an interval of about 20 cM), QTLs for seed size (Sdwt4.7.1, etc.), epicotyl length and lower internode length (Ecl4.7.1, St1i4.7.1 and St2i4.7.1) were detected.

Non-random distribution of QTLs

Although it was unknown whether all 73 QTLs identified for domestication-related traits had independent gene actions on each trait, the observed number of QTLs was compared with the expected number of QTLs based on each LG length (Table 7). The χ2 value was 47·6; this value is significant at P = 0·001, suggesting a departure 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 significantly lower than expected. Furthermore, χ2 tests of the number of QTLs in each 10-cM interval indicated a non-random distribution on LG1, LG2, LG3, LG4 and LG7 (Table 8).

Table 7.

Observed and expected numbers of QTLs on each linkage group

	Length	No. of QTLs
LG	(cM)	Detected	Expected	χ2
LG1	105·8	11	9·7	0·17
LG2	76·3	7	7·0	0·00
LG3	56·7	7	5·2	0·62
LG4	97·1	13	8·9	1·88
LG5	50·3	10	4·6	6·29*
LG6	83·1	0	7·6	7·62**
LG7	65·5	17	6·0	20·12***
LG8	68·6	2	6·3	2·93
LG9	95·3	3	8·7	3·77
LG10	58·0	1	5·3	3·51
LG11	39·4	2	3·6	0·72
Total	796·1	73	73	47·64***

*, **, *** Significant at 5 %, 1 % and 0·1 % levels, respectively.

Table 8.

Number of QTLs in each 10-cM interval

	No. of QTLs
LG	Detected	Average†	Range‡	χ2§
LG1	11	1·00	0–5	32·98***
LG2	7	1·88	0–6	35·26***
LG3	7	1·17	0–6	155·84***
LG4	13	1·30	0–10	96594·29***
LG5	10	1·67	0–4	9·10
LG6	0	0·00	0	0·00
LG7	17	2·43	0–8	57·93***
LG8	2	0·29	0–1	0·18
LG9	3	0·30	0–1	0·29
LG10	1	0·17	0–1	0·03
LG11	2	0·50	0–1	0·59

*** Significant at 0·1 % level.

† Average QTL density on each linkage group.

‡ Range of the number of QTLs per 10-cM interval

§ Departure from random distribution of QTLs in each 10-cM interval was tested under the null hypothesis in a Poisson goodness-of-fit.

DISCUSSION

General features of the rice bean genetic linkage map

The present rice bean linkage map is the first to be constructed from a large population derived from an intraspecific cross. The number of LGs corresponded to the basic number of chromosomes in rice bean. Many codominant SSR markers from azuki bean, cowpea and common bean were integrated into the genome, and no gap was >20 cM. Therefore, this rice bean map is useful for understanding genomic synteny among species within Ceratotropis, as well as for identifying QTLs for useful traits.

Segregation distortion

Two linkage maps had been constructed for populations derived from interspecific crosses between rice bean and azuki bean (Kaga ) and between rice bean and V. nakashimae (Somta ). Markers showing segregation distortion were observed on some LGs on both maps, suggesting some interspecific genetic barriers. Although the present rice bean linkage map was based on an intraspecific BC1F1 cross population (rice bean/wild rice bean//rice bean), 61 markers still showed segregation distortion (P < 0·05, 18·7 %). Notably, all 26 markers located on LG11 were highly skewed towards a heterozygous genotype, making the number of wild rice bean alleles higher than expected. A similar phenomenon was observed in a BC1F1 population of rice (Oryza sativa/O. glaberrima//O. sativa): marked segregation distortion towards a heterozygous genotype (increased number of O. glaberrima alleles) was recorded (Doi ; Koide ). It was proposed that the gamete eliminator gene S (on rice chromosome 6) induces abortion 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 allele in heterozygotes.

A high level of marker segregation distortion on LG11 has also been reported on a rice bean × V. nakashimae F2 map (Somta ). On rice chromosome 6, several genes affecting segregation distortion, such as cim (the cross-incompatibility reaction in the male), Cif (the cross-incompatibility reaction in the female) and S (gamete eliminator in O. rufipogon) have been detected (Koide ). Because all the markers distributed along a distance of about 40 cM on LG11 of rice bean showed segregation distortion, several genes existing on LG11 might have played an important role in genetic differentiation, both within the rice bean and among Vigna species.

Genome synteny between rice bean and azuki bean

The rice bean linkage map was compared with an azuki bean linkage map (Han ) by the distribution of common azuki bean SSR markers (Fig. 3). Out of 172 azuki bean SSR markers mapped on the rice bean linkage map, 129 were common to those used in the azuki bean map. The common SSR markers were present on all LGs, and the number of common markers per LG ranged from 7 (LG3 and LG7) to 15 (LG1). There was a high level of co-linearity of marker order between rice bean and azuki bean.

Fig. 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 ). 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.

However, a few differences, exemplified by genetic distance, inversions and duplications, were observed between the two linkage maps (Fig. 3). Of the 11 LGs, LG9 exhibited the most differences. The upper part of rice bean LG9 is considerably longer (about 1·4 times) than that in azuki bean. Three SSR markers, CEDG080, CEDG290 and CEDG172, on azuki bean LG9 are scattered to different rice bean LGs (LG3, LG5 and LG11, respectively). Inversions were usually found near the ends of LGs (on LG1, between CEDG133 and CEDG149; on LG3, between CED176 and CEDG010; on LG4, between CEDC028 and CEDC055 and between CEDG181 and CEDG011). In contrast, duplications of 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, duplicated on rice bean LG6 or in different positions on LG10, LG6 and LG1, whereas the rice bean single marker loci CEDG116 (LG10) and CEDG290 (LG5) were duplicated on azuki bean LG6 and LG9, respectively.

Even though the model legume Lotus japonicus has a small genome, large-scale segmental duplication at the sequence level has occurred within the genome (Sato ). Meiosis in hybrids between rice bean and azuki bean is normal (Ahn and Hartmann, 1978), and chromosomes of both species have a similar C-banding pattern (Zheng ). However, the fluorescent banding patterns of rice bean chromosomes, indicating the distributions of GC- and AT-rich genomic regions, have no similarity with those in azuki bean (Zheng ). The differences between rice bean and azuki bean observed here can be explained by the occurrence of small chromosomal rearrangements or translocations during the evolutionary divergence of these species.

Genomic regions and distributions of QTLs involved in rice bean domestication

Commonly, domestication-related traits are controlled by several major genes that are not randomly distributed across crop genomes (Gepts, 2004) with the only one reported exceptional example of sunflower domestication (Burke ; Wills and Burke, 2007). This general rule, which is also applicable to rice bean domestication, may be related to the phenomenon called ‘cultivation magnetism’ and should be considered under ‘protracted transition paradigm’ of crop domestication (Allaby, 2010).

Domestication QTLs of rice bean for various organs co-localized to several narrow genomic regions on LG1, LG2, LG3, LG4, LG5 and LG7 (Tables 7 and 8 and Fig. 3 and Fig. S2 in Supplementary data). In particular, the distribution of QTLs with large effects was limited to three LGs (LG2, LG4 and LG7). LG2 was associated with changes in seed and pod size. LG4 was associated with changes in seed and pod size and water absorption by seeds. LG7 was associated with changes in pod dehiscence and stem length. The remainder – LG1, LG3 and LG5 – were associated with changes in plant size.

Clustering of QTLs may be due to pleiotropy or close linkage of QTLs. Single mutations can have pleiotropic effects on various organs. In maize, QTLs related to change in inflorescence sex and number and length of internodes in lateral branches and inflorescences are distributed within a narrow genomic region (Doebley ). Change in these traits is explained by the pleiotropic effect of a single tb1 gene. In arabidopsis, the seed testa colour mutant gene tt causes a reduction in seed weight (Debeaujon ). Similarly, the seed testa colour mutant gene bks in tomato decreases seed weight and increases fruit pH (Downie ). Further studies are required to determine whether pleiotropy or close genetic linkage is responsible for the clustering of QTLs in rice bean. Near isogenic lines for 2 clustering QTLs on LG4 are currently being developed to elucidate this question.

Comparison of domestication QTLs between rice bean and azuki bean

General features

In contrast to the high degree of conservation of the genomic structure between rice bean and azuki bean (Isemura ), the distribution of the main QTLs showed a marked difference (Table 9 and Fig. 4). For 28 domestication traits, 69 QTLs were found in rice bean and 76 in azuki bean, of which only 15 were common (Table 9 and Fig. 4). Most of the common QTLs were associated with seed-size-related traits (SD100WT, SDL, SDW and SDT) and pod dehiscence (PDT).

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

Fig. 4.

Comparative QTL map of domestication-related traits in rice bean (upper) and azuki bean (lower; after Isemura ). 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.

In azuki bean, QTLs with a large effect [phenotypic variation explained (PVE) > 20 %] were found on five LGs (LG1, seed dormancy and internode length; LG2, seed size and epicotyl and lower internode length; LG7, pod dehiscence and pod size; LG8, pod length; LG9, twining habit) (Fig. 4). In rice bean, in contrast, they were detected on only two LGs (LG4, seed and pod size and seed dormancy; LG7, pod dehiscence) (Fig. 4). In mung bean (T. Isemura et al., unpub. res.), 79 QTLs were found for 28 traits, and QTLs with a large effect (PVE > 20 %) were found on seven LGs. These findings reveal that mutations that cause large changes in domestication traits have occurred on fewer LGs in rice bean than in the two other species. Marked differences between rice bean and azuki bean were found on LG4 and LG9. Many QTLs with large effect were detected on LG4 of rice bean, whereas few QTLs were detected on this LG in azuki bean. In contrast, major QTLs for domestication-related traits were abundant on LG9 in azuki bean, whereas only a few QTLs were detected on LG9 of rice bean.

Seed size

The accession with the largest seed among germplasm conserved in the NIAS Genebank was selected as the cultivated parent of rice bean. The 100-seed weight of this accession was 27·4 g (Table 5). This value is much larger than those of commonly cultivated rice bean [4·89–6·68 g (Bisht ); 3·9 g (Somta )]. There was a 15-fold weight difference between the cultivated and wild rice bean (1·8 g per 100 seeds) used here, and seven QTLs were detected for 100-seed weight (Table 6). Two QTLs were involved in the 10-fold weight difference between cultivated (16 g per 100 seeds) and wild (1·7 g per 100 seeds) azuki bean (Isemura ). These two 100-seed weight QTLs 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 = 33·4 %) was detected on LG4 (Table 6).

Seed dormancy

Physical seed dormancy is generally caused by the presence of water-impermeable layers of palisade cells in the seed coat (Finch-Savage and Leubner-Metzger, 2006). The water absorption occurs through the structure of the strophiole (parenchymatous tissue) adjacent to the hilum in Vigna species (Gopinathan and Babu, 1985).

Five seed-dormancy-related QTLs were detected in rice bean (Table 6). Five were detected also in azuki bean (Isemura ). However, none is common to both species. The seed-dormancy-related QTL with the largest effect was detected on LG4 in rice bean and on LG1 in azuki bean.

A dominant gene controlling pale red hilum in the wild parent was mapped on LG5. However, no QTL for seed dormancy was found in this region. This result suggests that the mutation for change of hilum colour had no effect on physical dormancy in rice bean.

The weak but positive effects on water absorption of the wild alleles for seed dormancy QTLs on LG2 and LG10 might reflect the modest degree of seed dormancy in the wild parent (Table 5). Passport data revealed that the natural habitat of the wild parent was near a river in Kanchanaburi province, Thailand, where the soil may be moist and the temperature high enough for growth throughout the year (Tomooka ). Therefore, strong seed dormancy during the dry season may not be important for this wild accession, and this may suggest why the wild accession possesses a QTL that reduces seed dormancy.

Pod dehiscence

A single QTL for pod dehiscence was detected in rice bean and azuki bean. In rice bean, a pod dehiscence QTL with a relatively high contribution to this trait (42·4 %) was found on LG7. In azuki bean, a QTL with a higher contribution (90·5 %) was detected at a similar map position (Isemura ) and was considered common to the two species (Table 9 and Fig. 4).

Useful QTLs for breeding

The differences in the large-effect domestication QTLs suggest that rice bean and azuki bean harbour specific useful genes and can play important roles as new gene resources for each other. For example, a QTL for twining habit with large effect on LG9 is specific to azuki bean, whereas the 100-seed weight QTL with large effect on LG4 is specific to rice bean (Fig. 4). The seed-dormancy-related QTL with the largest effect was detected on LG4 in rice bean and LG1 in azuki bean. Similarly, the pod-size-related QTL with the largest effect was detected on LG4 in rice bean and LG7 in azuki bean, and no common QTL was found.

Hybrids can be produced between rice bean and azuki bean when rice bean is used as the seed parent (Kaga ). Therefore, it seems that the seed weight QTL on LG4 in rice bean and the QTL for loss of twining habit on LG9 in azuki bean are particularly useful for the breeding of larger-seeded azuki bean cultivars and determinate rice bean cultivars, respectively.

CONCLUSIONS

This is the first report of a genetic linkage map and QTLs for domestication-related traits in rice bean. A genetic linkage map of rice bean was constructed using 223 SSR markers from related legume species and 103 AFLP markers. A total of 326 markers converged into 11 LGs, corresponding to the haploid number of rice bean chromosomes. Using this map, 31 domestication-related traits in rice bean were dissected into 69 QTLs, but the differences between cultivated and wild parents were found to be controlled by only a few major QTLs. Major QTLs for unrelated organs were distributed as clusters on LGs 2, 4 and 7. The inheritance of domestication-related traits was so simple that the few major QTLs explained the phenotypic variation between cultivated and wild rice bean. The high level of genome synteny between rice bean and azuki bean facilitated QTL comparison between the species. Major QTLs in rice bean were found on LG4, whereas major QTLs in azuki bean were found on LG9. The results suggest that the degree of domestication or divergence between wild and domesticated rice bean was lower than in azuki bean. The results provide a genetic foundation for improvement of rice bean. Interchanges of major QTLs between rice bean and azuki bean might be useful to broaden the genetic variation in both species.

SUPPLEMENTARY DATA

Supplementary data are available online at www.aob.oxfordjournals.org/. Table S1: Information for cowpea SSR primer pairs designed in the present study. Table S2: Correlation coefficients between traits in the