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.

糙毛豇豆（Vigna reflexo-pilosa）包含一种被忽视的栽培作物，是豇豆属（Vigna）中唯一的四倍体（tetraploid）物种。尽管此前研究推测糙毛豇豆的一个基因组供体为三出豇豆（V. trinervia），但构成该异源四倍体（allotetraploid）物种的祖先类群尚未被确凿鉴定。
本研究开发了1429个小豆表达序列标签-简单序列重复（EST-SSR）标记，从中筛选出38对EST-SSR引物：此类引物在二倍体物种中可扩增得到单一条带，在四倍体物种中则扩增得到两条清晰分离的条带。利用这些引物，我们对7个亚洲豇豆属物种的8个类群共268份种质资源开展了分析，所涉类群包括糙毛豇豆光滑变种（V. reflexo-pilosa var. glabra）、糙毛豇豆原变种（V. reflexo-pilosa var. reflexo-pilosa）、细茎豇豆（V. exilis）、硬毛豇豆（V. hirtella）、最小豇豆（V. minima）、野绿豆（V. radiata var. sublobata）、细茎豇豆（V. tenuicaulis）与三出豇豆（V. trinervia），旨在明确糙毛豇豆的基因组供体。
由于本研究同时分析了二倍体与四倍体物种，且每对SSR引物均可在四倍体物种中检测到两个位点，因此我们将该四倍体物种的基因组划分为两种不同的二倍体类型，即A基因组与B基因组。38对EST-SSR标记共检测到445个等位基因（allele）。硬毛豇豆（V. hirtella）的基因多样性最高。
通过将糙毛豇豆的特异性PCR扩增产物分配至两个不同基因组，我们成功鉴定出糙毛豇豆的两个基因组供体亲本。系统发育分析与主坐标分析（principal coordinate analysis）结果显示，硬毛豇豆为一个物种复合群，至少包含3个独立的类群。两项分析均明确证实，三出豇豆（V. trinervia）与硬毛豇豆的一个类群为糙毛豇豆的基因组供体。
基因多样性分析结果表明，糙毛豇豆B基因组上的EST-SSR进化速率可能快于A基因组。本研究还结合遗传数据、形态学特征与地理分布，阐明了豇豆属物种种间的演化关系。