Genetic diversity and population structure of Indigofera szechuensis complex (Fabaceae) based on EST-SSR markers

Indigofera, the third largest genus of Fabaceae, comprises approximately 750 species worldwide with a pantropical distribution. Eight Indigofera species, namely I. calcicola, I. delavayi, I. franchetii, I. hancockii, I. lenticellata, I. pendula, I. rigioclada, and I. szechuensis, are considered a species complex because of their morphological similarities and the phylogenetic analysis based on Internal Transcribed Spacer (ITS). Small populations of these species are allopatrically distributed in Hengduan Mountains in China. Although considerable EST-SSR markers have been developed from the transcriptome of I. szechuensis, no codominant markers have been applied to study population genetic structure of the complex. In this study, we selected 66 EST-SSR markers which were transferable in Indigofera szechuensis complex for estimating polymorphism, of which 44 EST-SSRs (66.67%) were polymorphic. Amplification with selected 23 polymorphic EST-SSRs revealed a moderately high genetic diversity in this complex. The mean value of Observed number of alleles, Expected heterozygosity, Polymorphism information content and Shannon's information index was 10.4783, 0.4335, 0.6228 and 1.4369 respectively. A total of 758 genotypes were detected, with an average of 32.9565 genotypes per locus. The Mantel test showed a significant correlation between genetic and geographic distance (r=0.0748, P=0.0100). High differentiation and limited gene flow were detected among all populations (F_st=0.3589, Nm=0.5168). The PCoA and structure analysis grouped 31 populations of Indigofera szechuensis complex into five main species. I. delavayi was obviously separated from other species, and the result was in accordance with that of morphology and phylogeny. I. pendula was a separate species, and had two distinct phenotypes. Four I. szechuensis populations (Pop23, Pop24, Pop27 and Pop30) were the same species. I. calcicola, I. hancockii, I. rigioclada, I. franchetii, I. lenticellata, and I. szechuensis, distributed in southwest of Jinshajiang, could be considered as the same species; the species of I. franchetii, I. lenticellata, and I. szechuensis distributed in northeast of Jinshajiang could be considered as another separate species. Given the above information, the morphological classification of I. calcicola, I. franchetii, I. hancockii, I. lenticellata, I. rigioclada, and I. szechuensis was incredible.