Genetic Divergence and Relationship Among Opisthopappus Species Identified by Development of EST-SSR Markers

Abstract

Opisthopappus Shih is an endemic and endangered genus restricted to the Taihang Mountains that has important ornamental and economic value. According to the Flora Reipublicae Popularis Sinicae (FRPS, Chinese version), this genus contains two species (Opisthopappus longilobus and Opisthopappus taihangensis), whereas in the Flora of China (English version) only one species O. taihangensis is present. The interspecific phylogenetic relationship remains unclear and undefined, which might primarily be due to the lack of specific molecular markers for phylogenetic analysis. For this study, 2644 expressed sequence tag-simple sequence repeats (EST-SSRs) from 33,974 unigenes using a de novo transcript assembly of Opisthopappus were identified with a distribution frequency of 7.78% total unigenes. Thereinto, mononucleotides (1200, 45.39%) were the dominant repeat motif, followed by trinucleotides (992, 37.52%), and dinucleotides (410, 15.51%). The most dominant trinucleotide repeat motif was ACC/GGT (207, 20.87%). Based on the identified EST-SSRs, 245 among 1444 designed EST-SSR primers were selected for the development of potential molecular markers. Among these markers, 63 pairs of primers (25.71%) generated clear and reproducible bands with expected sizes. Eventually, 11 primer pairs successfully amplified all individuals from the studied populations. Through the EST-SSR markers, a high level of genetic diversity was detected between Opisthopappus populations. A significant genetic differentiation between the O. longilobus and O. taihangensis populations was found. All studied populations were divided into two clusters by UPGMA, NJ, STRUCTURE, and PCoA. These results fully supported the view of the FRPS, namely, that O. longilobus and O. taihangensis should be regarded as two distinct species. Our study demonstrated that transcriptome sequences, as a valuable tool for the quick and cost-effective development of molecular markers, was helpful toward obtaining comprehensive EST-SSR markers that could contribute to an in-depth assessment of the genetic and phylogenetic relationships between Opisthopappus species.

Keywords: EST-SSRs development; Opisthopappus; phylogenetic relationship; population genetics; transcriptome sequencing.

FIGURE 1

Geographic distribution of the 19 sampled populations of Opisthopappus in China. For population abbreviations, see Table 1 for details.

FIGURE 2

Dendrogram of Opisthopappus populations, generated using UPGMA (A) and NJ (B) cluster analysis. Bootstrap analysis was applied using 1000 replicates and only bootstrap values (%) >50 were given. Nineteen populations were clustered into two groups corresponding to O. longilobus and O. taihangensis in both UPGMA and NJ trees.

FIGURE 3

The principal coordinates analysis (PCoA) for Opisthopappus. PC1 explained 43.04% variations of genetic components and PC2 explained 21.10% variations of genetic components. The O. longilobus and the O. taihangensis could be divided into two different groups corresponding to their distribution on axis1 and axis2.

FIGURE 4

Results of the Bayesian clustering analysis conducted using STRUCTURE. (A) The ΔK plot shows that K = 2 got the highest ΔK-value and K = 3 got the second-best ΔK-value, meaning that the most probable grouping number could be two or three. (B) Estimated genetic structure for K = 2 obtained for Opisthopappus. (C) Estimated genetic structure for K = 3 for Opisthopappus.

FIGURE 5

Geographic distance was positively correlated with genetic distance. The generalized linear regression model (GLRM): y = 0.0011x + 0.2117, R² = 0.2082. Mantel test: r = 0.4565, P = 0.001.