Unique Salt-Tolerance-Related QTLs, Evolved in Vigna riukiuensis (Na+ Includer) and V. nakashimae (Na+ Excluder), Shed Light on the Development of Super-Salt-Tolerant Azuki Bean (V. angularis) Cultivars.

Wild relatives of crops have the potential to improve food crops, especially in terms of improving abiotic stress tolerance. Two closely related wild species of the traditional East Asian legume crops, Azuki bean (Vigna angularis), V. riukiuensis "Tojinbaka" and V. nakashimae "Ukushima" were shown to have much higher levels of salt tolerance than azuki beans. To identify the genomic regions responsible for salt tolerance in "Tojinbaka" and "Ukushima", three interspecific hybrids were developed: (A) azuki bean cultivar "Kyoto Dainagon" × "Tojinbaka", (B) "Kyoto Dainagon" × "Ukushima" and (C) "Ukushima" × "Tojinbaka". Linkage maps were developed using SSR or restriction-site-associated DNA markers. There were three QTLs for "percentage of wilt leaves" in populations A, B and C, while populations A and B had three QTLs and population C had two QTLs for "days to wilt". In population C, four QTLs were detected for Na+ concentration in the primary leaf. Among the F2 individuals in population C, 24% showed higher salt tolerance than both wild parents, suggesting that the salt tolerance of azuki beans can be further improved by combining the QTL alleles of the two wild relatives. The marker information would facilitate the transfer of salt tolerance alleles from "Tojinbaka" and "Ukushima" to azuki beans.

Same Locus for Non-shattering Seed Pod in Two Independently Domesticated Legumes, Vigna angularis and Vigna unguiculata.

Loss of pod shattering is one of the most important domestication-related traits in legume crops. The non-shattering phenotypes have been achieved either by disturbed formation of abscission layer between the valves, or by loss of helical tension in sclerenchyma of endocarp, that split open the pods to disperse the seeds. During domestication, azuki bean (Vigna angularis) and yard-long bean (Vigna unguiculata cv-gr. Sesquipedalis) have reduced or lost the sclerenchyma and thus the shattering behavior of seed pods. Here we performed fine-mapping with backcrossed populations and narrowed the candidate genomic region down to 4 kbp in azuki bean and 13 kbp in yard-long bean. Among the genes located in these regions, we found MYB26 genes encoded truncated proteins in azuki bean, yard-long bean, and even cowpea. As such, our findings indicate that independent domestication on the two legumes has selected the same locus for the same traits. We also argue that MYB26 could be a target gene for improving shattering phenotype in other legumes, such as soybean.

Whole genome sequencing data of Vigna nakashimae var. Ukushima and G418.

Vigna nakashimae is one of the closely related species of Vigna angularis (Adzuki bean). Two strain of 'Ukushima' and 'G418' were identified as salt tolerance strains in Vigna nakashimae from gene bank collection. F2 populations from an inter- or intra-specific cross between the sensitive and tolerant strains are useful for the detection of salt tolerance QTL in Vigna nakashimae. Although Vigna angularis reference genome is available and useful for genetic analysis by genotyping-by-sequencing/RADseq in closely related species, it is not enough for isolation of responsible genes. To reveal sequence variation in Vigna nakashimae "Ukushima" and "G418", the whole genome sequencing was performed using Illumina HiSeq X Ten system (411,174,986 and 478,116,282 read). NGS data was deposited in the DNA Data Bank of Japan (DDBJ) under accession number DRA009307.

Construction of an SSR and RAD-Marker Based Molecular Linkage Map of Vigna vexillata (L.) A. Rich.

Vigna vexillata (L.) A. Rich. (tuber cowpea) is an underutilized crop for consuming its tuber and mature seeds. Wild form of V. vexillata is a pan-tropical perennial herbaceous plant which has been used by local people as a food. Wild V. vexillata has also been considered as useful gene(s) source for V. unguiculata (cowpea), since it was reported to have various resistance gene(s) for insects and diseases of cowpea. To exploit the potential of V. vexillata, an SSR-based linkage map of V. vexillata was developed. A total of 874 SSR markers successfully amplified single DNA fragment in V. vexillata among 1,336 SSR markers developed from Vigna angularis (azuki bean), V. unguiculata and Phaseolus vulgaris (common bean). An F2 population of 300 plants derived from a cross between salt resistant (V1) and susceptible (V5) accessions was used for mapping. A genetic linkage map was constructed using 82 polymorphic SSR markers loci, which could be assigned to 11 linkage groups spanning 511.5 cM in length with a mean distance of 7.2 cM between adjacent markers. To develop higher density molecular linkage map and to confirm SSR markers position in a linkage map, RAD markers were developed and a combined SSR and RAD markers linkage map of V. vexillata was constructed. A total of 559 (84 SSR and 475 RAD) markers loci could be assigned to 11 linkage groups spanning 973.9 cM in length with a mean distance of 1.8 cM between adjacent markers. Linkage and genetic position of all SSR markers in an SSR linkage map were confirmed. When an SSR genetic linkage map of V. vexillata was compared with those of V. radiata and V. unguiculata, it was suggested that the structure of V. vexillata chromosome was considerably differentiated. This map is the first SSR and RAD marker-based V. vexillata linkage map which can be used for the mapping of useful traits.

Reciprocal translocation identified in Vigna angularis dominates the wild population in East Japan.

Using an F2 population derived from cultivated and wild azuki bean, we previously detected a reciprocal translocation and a seed size QTL near the translocation site. To test the hypothesis that the translocation in the cultivated variety contributed to the larger seed size, we performed further linkage analyses with several cross combinations between cultivated and wild azuki beans. In addition, we visually confirmed the translocation by cytogenetic approach using 25 wild and cultivated accessions. As a result, we found the translocation-type chromosomes in none of the cultivated accessions, but in a number of the wild accessions. Interestingly, all the wild accessions with the translocation were originally collected from East Japan, while all the accessions with normal chromosomes were from West Japan or the Sea of Japan-side region. Such biased geographical distribution could be explained by the glacial refugium hypothesis, and supported narrowing down the domestication origin of cultivated azuki bean.

Detection of genome donor species of neglected tetraploid crop Vigna reflexo-pilosa (créole bean), and genetic structure of diploid species based on newly developed EST-SSR markers from azuki bean (Vigna angularis).

Vigna reflexo-pilosa, which includes a neglected crop, is the only one tetraploid species in genus Vigna. The ancestral species that make up this allotetraploid species have not conclusively been identified, although previous studies suggested that a donor genome of V. reflexo-pilosa is V. trinervia. In this study, 1,429 azuki bean EST-SSR markers were developed of which 38 EST-SSR primer pairs that amplified one product in diploid species and two discrete products in tetraploid species were selected to analyze 268 accessions from eight taxa of seven Asian Vigna species including V. reflexo-pilosa var. glabra, V. reflexo-pilosa var. reflexo-pilosa, V. exilis, V. hirtella, V. minima, V. radiata var. sublobata, V. tenuicaulis and V. trinervia to identify genome donor of V. reflexo-pilosa. Since both diploid and tetraploid species were analyzed and each SSR primer pair detected two loci in the tetraploid species, we separated genomes of the tetraploid species into two different diploid types, viz. A and B. In total, 445 alleles were detected by 38 EST-SSR markers. The highest gene diversity was observed in V. hirtella. By assigning the discrete PCR products of V. reflexo-pilosa into two distinguished genomes, we were able to identify the two genome donor parents of créole bean. Phylogenetic and principal coordinate analyses suggested that V. hirtella is a species complex and may be composed of at least three distinct taxa. Both analyses also clearly demonstrated that V. trinervia and one taxon of V. hirtella are the genome donors of V. reflexo-pilosa. Gene diversity indicates that the evolution rate of EST-SSRs on genome B of créole bean might be faster than that on genome A. Species relationship among the Vigna species in relation to genetic data, morphology and geographical distribution are presented.

QTL mapping for salt tolerance and domestication-related traits in Vigna marina subsp. oblonga, a halophytic species.

QTL mapping in F2 population [V. luteola × V. marina subsp. oblonga] revealed that the salt tolerance in V. marina subsp. oblonga is controlled by a single major QTL. The habitats of beach cowpea (Vigna marina) are sandy beaches in tropical and subtropical regions. As a species that grows closest to the sea, it has potential to be a gene source for breeding salt-tolerant crops. We reported here for the first time, quantitative trait loci (QTLs) mapping for salt tolerance in V. marina. A genetic linkage map was constructed from an F2 population of 120 plants derived from an interspecific cross between V. luteola and V. marina subsp. oblonga. The map comprised 150 SSR markers. The markers were clustered into 11 linkage groups spanning 777.6 cM in length with a mean distance between the adjacent markers of 5.59 cM. The F2:3 population was evaluated for salt tolerance under hydroponic conditions at the seedling and developmental stages. Segregation analysis indicated that salt tolerance in V. marina is controlled by a few genes. Multiple interval mapping consistently identified one major QTL which can explain about 50% of phenotypic variance. The flanking markers may facilitate transfer of the salt tolerance allele from V. marina subsp. oblonga into related Vigna crops. The QTL for domestication-related traits from V. marina are also discussed.

Construction of a genetic linkage map and genetic analysis of domestication related traits in mungbean (Vigna radiata).

The genetic differences between mungbean and its presumed wild ancestor were analyzed for domestication related traits by QTL mapping. A genetic linkage map of mungbean was constructed using 430 SSR and EST-SSR markers from mungbean and its related species, and all these markers were mapped onto 11 linkage groups spanning a total of 727.6 cM. The present mungbean map is the first map where the number of linkage groups coincided with the haploid chromosome number of mungbean. In total 105 QTLs and genes for 38 domestication related traits were identified. Compared with the situation in other Vigna crops, many linkage groups have played an important role in the domestication of mungbean. In particular the QTLs with high contribution were distributed on seven out of 11 linkage groups. In addition, a large number of QTLs with small contribution were found. The accumulation of many mutations with large and/or small contribution has contributed to the differentiation between wild and cultivated mungbean. The useful QTLs for seed size, pod dehiscence and pod maturity that have not been found in other Asian Vigna species were identified in mungbean, and these QTLs may play the important role as new gene resources for other Asian Vigna species. The results provide the foundation that will be useful for improvement of mungbean and related legumes.

An SSR-based linkage map of yardlong bean (Vigna unguiculata (L.) Walp. subsp. unguiculata Sesquipedalis Group) and QTL analysis of pod length.

Yardlong bean (Vigna unguiculata (L.) Walp. subsp. unguiculata Sesquipedalis Group) (2n = 2x = 22) is one of the most important vegetable legumes of Asia. The objectives of this study were to develop a genetic linkage map of yardlong bean using SSR makers from related Vigna species and to identify QTLs for pod length. The map was constructed from 226 simple sequence repeat (SSR) markers from cowpea (Vigna unguiculata (L.) Walp. subsp. unguiculata Unguiculata Group), azuki bean (Vigna angularis (Willd.) Ohwi & Ohashi), and mungbean (Vigna radiata (L.) Wilczek) in a BC(1)F(1) ((JP81610 × TVnu457) × JP81610) population derived from the cross between yardlong bean accession JP81610 and wild cowpea (Vigna unguiculata subsp. unguiculata var. spontanea) accession TVnu457. The markers were clustered into 11 linkage groups (LGs) spanning 852.4 cM in total length with a mean distance between adjacent markers of 3.96 cM. All markers on LG11 showed segregation distortion towards the homozygous yardlong bean JP81610 genotype. The markers on LG11 were also distorted in the rice bean (Vigna umbellata (Thunb.) Ohwi & Ohashi) map, suggesting the presence of common segregation distortion factors in Vigna species on this LG. One major and six minor QTLs were identified for pod length variation between yardlong bean and wild cowpea. Using flanking markers, six of the seven QTLs were confirmed in an F(2) population of JP81610 × TVnu457. The molecular linkage map developed and markers linked to pod length QTLs would be potentially useful for yardlong bean and cowpea breeding.

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.

Mapping of quantitative trait loci for a new source of resistance to bruchids in the wild species Vigna nepalensis Tateishi & Maxted (Vigna subgenus Ceratotropis).

Azuki bean breeders have long been interested in producing azuki bean [Vigna angularis (Willd.) Ohwi & Ohashi] varieties with bruchid resistance. A new bruchid (Callosobruchus spp.) resistance source was found in V. nepalensis Tateishi & Maxted, a species that is cross compatible with azuki bean. Quantitative trait loci (QTLs) analysis for resistance to C. chinensis (L.) and C. maculatus (F.) was conducted using F2 (V. nepalensisxV. angularis) and BC1F1 [(V. nepalensisxV. angularis)xV. angularis] populations derived from crosses between the bruchid resistant species V. nepalensis and bruchid susceptible species V. angularis. Resistance was measured using two traits, percentage of seeds damaged by bruchids and the time taken for adult bruchids to emerge from seeds. Based on the results from both populations seven QTLs were detected for bruchid resistance; five QTLs for resistance to C. chinensis and two QTLs for resistance to C. maculatus. The different locations found for some resistance QTL to the two bruchid species suggests different resistance mechanisms. QTLs on linkage group (LG) 1 and LG2 for bruchid resistance to C. chinensis co-localized with seed size QTLs suggesting that incremental increase in seed size accompanied susceptibility to C. chinensis. Based on linked markers the QTL on these two linkage groups appear to be the same as previously reported in other Asian Vigna. However, several other QTLs were newly detected including one on LG4 that appears unrelated to seed size. Transfer of these new sources of bruchid resistance from V. nepalensis to azuki bean will be aided by the progress being made in azuki genome mapping.

The genetics of domestication of the azuki bean (Vigna angularis).

Genetic differences between azuki bean (Vigna angularis var. angularis) and its presumed wild ancestor (V. angularis var. nipponensis) were resolved into QTL for traits associated with adaptation to their respective distinct habits. A genetic linkage map constructed using progenies from a cross between Japanese cultivated and wild azuki beans covers 92.8% of the standard azuki bean linkage map. A reciprocal translocation between cultivated and wild azuki bean parents was identified on the basis of the linkage map having a pseudolinkage group and clustering of seed productivity-related QTL with large effect near the presumed breakpoints. In total, 162 QTL were identified for 46 domestication-related traits. Domestication of azuki bean has involved a trade-off between seed number and seed size: fewer but longer pods and fewer but larger seeds on plants with shorter stature in cultivated azuki bean being at the expense of overall seed yield. Genes found related to germination and flowering time in cultivated azuki bean may confer a selective advantage to the hybrid derivatives under some ecological conditions and may explain why azuki bean has evolved as a crop complex in Japan.

Genome dissection of traits related to domestication in azuki bean (Vigna angularis) and comparison with other warm-season legumes.

BACKGROUND: The objective of this study was to dissect into quantitative trait loci (QTLs) the large morphological and physiological differences between cultivated azuki bean (Vigna angularis) and a wild relative and to infer the commonalities of the QTLs for domestication-related traits across the Asian Vigna and with other warm-season legumes. METHODS: Two linkage maps, for the BC(1)F(1) and F(2) populations, respectively, from the same cross between azuki bean and V. nepalensis were developed. Using these linkage maps QTLs for 33 domestication-related traits were analysed and mapped. The location of mapped QTLs was compared with locations of similar QTLs in other warm-season legumes. KEY RESULTS: QTLs were detected for seed-, pod-, stem- and leaf-related traits. Most traits were controlled by between two and nine QTLs but several traits, such as pod dehiscence, were controlled by single genes. QTLs for domestication-related traits were restricted to particular regions of the azuki bean genome, especially linkage groups 1, 2, 4, 7 and 9. Linkage groups 1 and 2 had QTLs for a suite of traits including pod size, germination, seed size and lower stem length. QTLs on linkage groups 7 and 9 were associated with upper stem length, maximum leaf size and pod and seed size. Pleiotropy or close linkage of genes for domestication-related traits is suggested in these regions. While some QTLs are common to azuki bean and other warm-season legumes, many are recorded for the first time in azuki bean. CONCLUSIONS: QTLs for a large number of domestication-related traits have been mapped for the first time in azuki bean. QTLs with unexpected effect and new QTLs for traits such as seed size have been found. The results provide a foundation that will be useful for improvement of azuki bean and related legumes.