Identification of Diploid Stylosanthes seabrana Accessions from Existing Germplasm of S. scabra Utilizing Genome-Specific STS Markers and Flow Cytometry, and Their Molecular Characterization

Abstract

Stylosanthes seabrana (Maass and 't Mannetje) (2n = 2x = 20), commonly known as Caatinga stylo, is an important tropical perennial forage legume. In nature, it largely co-exist with S. scabra, an allotetraploid (2n = 4x = 40) species, sharing a very high similarity for morphological traits like growth habit, perenniality, fruit shape and presence of small appendage at the base of the pod or loment. This makes the two species difficult to distinguish morphologically, leading to chances of contamination in respective germplasm collections. In present study, 10 S. seabrana accessions were discovered from the existing global germplasm stock of S. scabra represented by 48 diverse collections, utilizing sequence-tagged-sites (STS) genome-specific markers. All the newly identified S. seabrana accessions displayed STS phenotypes of typical diploid species. Earlier reports have conclusively indicated S. seabrana and S. viscosa as two diploid progenitors of allotetraploid S. scabra. With primer pairs SHST3F3/R3, all putative S. seabrana yielded single band of approximately 550 bp and S. viscosa of approximately 870 bp whereas both of these bands were observed in allotetraploid S. scabra. Since SHST3F3/R3 primer pairs are known to amplify single or no band with diploid and two bands with tetraploid species, the amplification patterns corroborated that all newly identified S. seabrana lines were diploid in nature. Flow cytometric measurement of DNA content of the species, along with distinguishing morphological traits such as flowering time and seedling vigour, which significantly differ from S. scabra, confirmed all identified lines as S. seabrana. These newly identified lines exhibited high level of similarity among themselves as revealed by RAPD and STS markers (>92% and 80% respectively). Along with the enrichment in genetic resources of Stylosanthes, these newly identified and characterized accessions of S. seabrana can be better exploited in breeding programs targeted to quality.

Low cytomolecular diversification in the genus Stylosanthes Sw. (Papilionoideae, Leguminosae)

“…The small variation of DNA content (1.4 fold) in the Stylosanthes species analyzed here corroborates a scenario of karyotypic stability. Although the average of DNA estimation is in agreement with other estimations for the genus, we observed different C-values for two diploid species (S. seabrana B.L.Maass & 't Mannetje and S. viscosa) for which 5.45 pg were reported in the literature (Chandra and Kaushal, 2009). Variation of DNA content within a genus may be due to different factors such as recombination, deletion and retrotransposition (Piegu et al, 2006;Baziz et al, 2014).…”

“…DNA contents are known for 23 species of Arachis, with an average of 2C = 2.83 pg (http://data.kew.org/cvalues). On the other hand, Stylosanthes karyotype data are extremely scarce, with most data coming from chromosome counting, and few species with genome size and molecular cytogenetics were investigated (Vieira et al, 1993;Chandra and Kaushal, 2009;Lira, 2015;Marques et al, 2018). Most of the species of the genus are diploids (2n = 20) but few polyploid species (2n = 40, 60) were reported (Cameron, 1967;Karia, 2008).…”

Radiation of the Nod‐independent Aeschynomene relies on multiple allopolyploid speciation events

“…This can explain why these species share a high similarity for morphological traits and are then sometimes misidentified (Rudd, ; http://www.fao.org). The difficulty in distinguishing species in polyploid complexes due to little or overlooked morphological differentiation is a recurrent problem as already reported notably for Stylosanthes scabra (Chandra & Kaushal, ), Hordeum murinum (Ourari et al ., ) and Mercurialis annua (Obbard et al ., ).…”

“…For polyploid species the use of genetic markers as well as molecular phylogenies based on the sequence of nuclear single‐copy genes have been valuable tools to investigate their genomic structure and to identify among diploid species those representing potential progenitors ( Coffea arabica : Lashermes et al ., ; Arachis hypogaea : Jung et al ., ; de Carvalho Moretzsohn et al ., ; Ma et al ., ; Stylosanthes scabra : Chandra & Kaushal, ; Hordeum murinum : Jacob & Blattner, 2010; Mercurialis annua : Korbecka et al ., ). In the case of the polyploid A. indica forms, these two approaches unambiguously indicated that in addition to A. evenia ssp.…”

Molecular mapping of test mapping strain for 18th linkage group recessive genes elp, ch-2 and mln in silkworm (Bombyx mori)

“…已达 5 000 余年, 是由仍在桑园中生存的野桑蚕 (Bombyx mandarina)驯化而来 [1] , 自 20 世纪初以来 一直是遗传学研究的重要对象。在家蚕性状遗传研 究中, 在第 18 染色体先后发现了第二隐性赤蚁(The second recessive gene of chocolate larvae, ch-2) [2,3] (蚁 蚕全身暗赤褐色, 食桑后幼虫体色逐渐正常)、显性 短节油蚕(Dominant obese translucent, Obs)(蚕体短 而肥大的显性油蚕) [4] 、长形卵(Ellipsoid egg, elp) [5,6] (突变体的卵长轴方向较正常卵显著增长, 呈长椭 圆形)、 暗化型(melanism, mln) [4,7] (主要特征为蚕蛾身 体 和 翅 灰 黑 色 ) 和 白 蚁 (Undevelopped gonads, gon)(蚁蚕体白色) [4] 。典型遗传分析结果表明这 5 个 突变基因均位于第 18 染色体, 除gon基因尚未定位 外, 其余 4 个突变基因在其遗传连锁图谱上的位点 分别为 0.0、6.2、16.1 和 41.5 [4] 。本研究所育成了家 蚕第 18 连锁群包含ch-2、elp、mln三隐性突变的测 交系统W18 [8] 。 随着分子生物学技术在家蚕中的应用与发展, 先后建立了家蚕AFLP、RAPD、RFLP、SSR、SNP 等分子遗传图 [9~15] , 为家蚕突变基因的定位与克隆 奠定了基础。自序列标签位点(Sequence-tagged sites, STS) [16] 应用于人类基因组作图以来, 由于其在基因 组位点中的唯一性, 已被广泛应用于遗传连锁图构 建、功能基因定位克隆和种质资源鉴定。简单重复 序列(Simple sequence repeat, SSR)又称微卫星 (Microsatellite)序列, 由于其具有在真核生物基因组 中分布广、多态性丰富、呈共显性遗传、实验重复 性和稳定性好以及能够高通量操作等优点, 已经成 为功能基因定位克隆、数量性状分析、指纹图谱构 建及资源多样性、分子标记辅助选择育种等的有力 工具 [17,18] , Miao等 [13] 构建了家蚕SSR分子连锁图, 并 将其与家蚕突变基因连锁图进行了对应。家蚕基 因组精细图谱已经于 2008 年公布 [19] , 测序的结果覆 盖了家蚕基因组 432 Mb的 8.5 倍, 将 87%的scaffold 分配到家蚕的 28 条染色体, 预测基因 14 623 个。这 项工作为家蚕功能基因的定位和克隆提供了强有力 的基础。 在此基础上, 2008 年, 相辉等 [20] 利用定位克 隆的方法对E Kp 位点进行了分离。 同年, 日本Ito等 [21] 研 究了决定家蚕浓核病毒…”