Genetic Diversity And Potential Germplasm Selection Of Elymus Sibiricus L. In The Qinghai-Tibetan Plateau

As the type species of the genus Elymus, E. sibiricus L. (Siberian wildrye) is a perennial, high quality forage indigenous to eurasia,. and in China especially in the Qinghai-Tibetan Plateau, there are abundant natural resources of E. sibiricus. E. sibiricus is an important component of native grasslands and has been recently developed as a major cultivated forage in Qinghai-Tibetan Plateau, owing to its good palatability and excellent capability of adaptation to cold and drought condition.Based on the field investigation on ecological distribution and growth habits of E sibiricus populations in the Qinghai-Tibetan Plateau of China, fifty-four wild accessions were collected and tested both in assessment of genetic diversity and germplasm appraisal, according to population ecology, morphological, production performance, gliadin markers and DNA markers, by a comparison with two representative varieties E sibiricus L. cv. Chuancao No. 1 and E sibiricus L. cv. Chuancao No. 2. In addition, employing ear characters diversity, SRAP and SSR markers, we studied the population structure and genetic variation among E. sibiricus populations from the eastern alpine region of Qinghai-Tibetan Plateau. The main results showed as follows:1. According to studying on distribution characteristics, habitat types, community composition and morphological variation of wild E. sibiricus, below results were obtained: (1) E sibiricus were widely distributied in Qinghai-Tibetan Plateau, its habitat types could be classified into three types: alpine and subalpine meadow type, valley grassland type and forest-shrub type; the main community composition including four types: Salix cheilophila var.microstachyoides +E. sibiricus + Deschampsia caespitosa, Hippophae rhamnoides + E. sibiricus + Artemisia; E. sibiricus+ Caragana+ Roegneria, E. sibiricus + E. nutans+ Bromus plurinodes. (2) Natural E. sibiricus produces rich morphological diversity. Among the 30 observed properties, morphological variances most significantly occured in the traits related with forage and seed yield, as for classification index, there are less variation. (3) Based on the phenotypic characteristics, 37 accessions were clustered into three morphological types, and the cluster result don't agree well with geographical distribution but related to altitude. (4) Principlal component analysis results indicated that 17 of the characters measured: Outer and inner glume length, awn length of outer and inner glume, boot leaf width, length of the second leaf from the inflorescence, plant height, lemma length and width, palea length and width, awn length of lemma, floret number in each spikelet, ear length, leaf color, internode diameter, gray grade and spikelet number were the main sources of morphological differentiation of E. sibiricus accessions. 2. Diversities were also detected exist in production performance of different E. sibiricus accessions. Twenty-three accessions could be divided into early maturing type and late maturing type on the basis of phenological observation. In all of the accessions, SAG205119 and SAG205151 showed the shortest growth period (115d), and "E. sibiricus cv Chuancao No. 2" showed the longest growth period (129d). E. sibiricus present the growth peak from beginning of June to the middle of July at the test place. The yield and quality characterizes of the twenty-three accessions also significantly different, the fresh yield was 35.66-87.59g/plant, with an average of 58.16g/plant, the hay yield was 11.09-29.18g/plant, and with an average of 17.96g/plant. The stem-leaf ratio was 1.84-2.71, with an average of 2.17, the crude protein content was 8.27%-14.79%, with an average of 10.96%, most of the accessions have the lower stem-leaf ratio and higher crude protein content than the CK, its means that the E. sibiricus accessions from Qinghai-Tibetan Plateau posses the high quality. Based on the production performance, all of the accessions were clustered into two types, one is high yield and quality, the other is generally showing. Among the former type, six accessions SAG205167, SAG205179, SAG204089, SAG205230, SAG205124 and SAG204451 showed a higher fresh yield and quality than CK, which hold a greater potentiality for further application. As for the "E. sibiricus cv Chuancao No.1" and "E. sibiricus cv Chuancao No.2", the excellent characteristics of them seems were degenerating in this study.3. Acid polyacrylamide gel electrophoresis (A-PAGE) was employed to detect the gliadin genetic diversity among 54 wild accessions of E. sibiricus collected from Qinghai-Tibetan plateau. A total of 42 bands were detected in all accessions, of which 92.86% were polymorphic. The average number of Shannon index to four electrophoretic zones (α,β,γ,ω) was 0.4627. The Nei-Li genetic similarity coefficient of the tested accessions ranged from 0.2424 to 0.9767, and the average was 0.5822. These results suggested that there was a rich genetic polymorphism among the tested wild resources of E. sibirucus. 54 wild accessions can be clustered into 4 groups at GS = 0.562 level on dendrogram. The principal coordinates (PCA) reflected almost the same relationships among the studied materials as showed in cluster analysis. Moreover, the accessions from the same origin frequently clustered into one group. Genetic differentiation of between and within five eco-geographical groups of E. sibiricus is estimated by Shannon's diversity index, which shown that 68.17% genetic variance existed within group, and 31.83% genetic variance was among groups. The unweighted pairwise groups method using arithmetic average (UPGMA) cluster analysis based on Nei's unbiased measures of genetic identity was assayed for five geographical groups of E. sibiricus, which indicated that there was a significantly positive correlation between genetic differentiation and geographical habits among the five groups.4. Sequence-related amplified polymorphism (SRAP) molecular markers were used to detect the genetic diversity of 52 wild accessions of Elymus sibiricus L. collected from Qinghai-Tibetan plateau. A total of 318 fragments were identified with 16 SRAP primers sets, of which 86.48% were polymorphic. For each primer set, there were 14-27 fragments were detected, with an average of 19.88, the polymorphism information content (PIC) was 0.122-0.326, with an average of 0.24, and the marker index (MI) of SRAP was 4.26. The genetic similarity (GS) coefficient of the tested accessions ranged from 0.5064 to 0.9586, with an average of 0.7921. The Nei's index of diversity (He) at the species level was 0.2270, and the Shannon's index (Ho) was 0.3472. These results suggested that there was rich genetic diversity among the tested wild resources of E. sibiricus. The results demonstrated a strong geographic effect on molecular variation of the local E. sibiricus as indicated by unweighted pairwise groups method using arithmetic average (UPGMA), and 52 wild accessions were clustered into five group at GS=0.80 level on dendrogram. Genetic differentiation between and within five eco-geographical groups of E. sibiricus was estimated by Shannon's diversity index, which showed that 65.29% genetic variance existed within group, and 34.71% genetic variance was among groups. Based on Nei's unbiased measures of genetic identity, UPGMA cluster analysis measures of five eco-geographical groups of E. sibiricus, indicated that there was a correlation between genetic differentiation and eco-geographical habits among the groups.5. The genetic diversity of 52 E. sibiricus accessions were evaluated by SSR markers A total of 318 fragments were identified with 18 SSR primers sets, of which 204 (86.48%) were polymorphic, and each primer generated 13.1 fragments, the polymorphism information content (PIC) per primer was 0.267-0.471, with an average of 0.35, and the marker index (MI) of SSR was 3.98. The genetic similarity (GS) coefficient of the tested accessions ranged from 0.622 to 0.895, with an average of 0.766. The Nei's index of diversity (He) of the 52 accessions was 0.3286, and the Shannon's index (Ho) was 0.4851. These results suggested that there was rich genetic diversity among the tested wild resources of E. sibiricus. 52 accessions could be divided into five main groups by cluster and principal component analysis, the accessions from the same region or with the same habitat type tends to were classified into the same group, indicating the geographical distribution of genetic diversity of E. sibiricus.6. The variation in 15 ear characters of 13 populations of Elymus sibiricus L. were researched in the present study. Results from the Shannon-weaver index analysis showed that there was an abundant genetic diversity (H'=1.7937) among these populations in ear characters, the genetic variation within populations (69.31%) was greater than that among populations (30.69%). Cluster analysis showed that 13 populations could be categorized into 3 groups. Principlal component analysis results indicated that 7 of the characters measured: Ear length, Ear width, Weight of single ear, Spikelet length, Palea length, Lemma length and Spikelet per rachis were the main sources of ear characters variation of 13 E.sibiricus populations. Results from the correlation analysis showed that altitude, latitude, longtitude and precipitation have remarkable influence on the ear characters variation of E. sibiricus, but mean annual temperature has little influence on it. Based on the genetic information available for E. sibiricus , some conservation strategies were proposed.7. In present study, the genetic diversity and population structure in eight natural populations of E. sibiricus from Qinghai-Tibetan Plateau of China was analyzed by means of sequence-related amplified polymorphism (SRAP) and Microcatellite markers (SSR). A total of 384 fragments were identified with 16 SRAP primers sets, of which 86.98% were polymorphic. Meanwhile, a total of 221 alleles were detected at 16 loci, with 192 (86.88%) being polymorphic, indicating considerable genetic variation at the species level. The mean gene diversity (He) was estimated to be 0.1092 and 0.1296 within populations detected by SRAP and SSR markers respectively, and 0.2434 and 0.3732 at the species level. A high level of genetic differentiation among populations was detected based on Nei's genetic diversity analysis both in SRAP (Gst=0.5525) and SSR (Gst=0.5158) markers, and an indirect estimate of the number of migrants per generation (0.4050 by SRAP markers, 0.4694 by SSR markers) showed that gene flow was low among populations. Shannon's index analysis and AMOVA analysis displayed the same result that mainly genetic variation of Elymus sibiricus existed among the populations. In addition, a geographical pattern of population differentiation, where the populations from south, north and middle area of sampling sites were clearly separated from each other, was revealed by cluster analysis. Based on the genetic information available for the native E. sibiricus, we proposed that it should be advisable to collect and preserve the native Elymus sibiricus germplasm pool in a larger extent.