| Elymus sibiricus is a perennial forage grass belonging to the genus Elymus of the Triticeae in the Poaceae,which is widely distributed in the high-altitude areas of northwest China.The germplasm resources of wild E.sibiricus are rich with good palatability and feeding quality,which can be used for forage preparation.In addition,it is suitable for the restoration of degraded grasslands because of strong drought and cold resistance,and high grass yield.Flowering time is a vital agronomic trait of herbage,which has important influence on the yield,feeding quality and utilization value of herbage.With the development of molecular biology,the research on the molecular mechanism of flowering and its regulatory network has made major achievements in many crops.However,such research in E.sibiricus is lacking.In this study,we characterized flowering time and related traits of wild E.sibiricus in Gannan and obtained the flowering variation materials.The flowering candidate gene-markers based on the transcriptome sequencing data were developed and validated in the E.sibiricus flowering time extreme populations.And then,the genetic diversity of E.sibiricus with different flowering time were analyzed by developed molecular markers.Finally,the allelic variation analysis of flowering candidate genes in early-,middle-,and late-flowering populations was performed.It is expected to lay a foundation for marker-assisted selection of flowering traits and provide important resources for breeding new early-and late-maturity varieties of E.sibiricus.The main research results are as follows:1.In this study,nineteen agronomic traits of 200 individual plants from 20 wild E.sibiricus populations in Gannan,such as booting stage,heading stage,flowering stage,inflorescence,leaf,stem,seed,and so on,were evaluated for two years.The results showed that there were significant differences in flowering time and related traits among different germplasms.The flowering time between the earliest flowering plant and the last flowering plant was 62 days.The mean phenotypic differentiation coefficient was 82.66%,which indicated that the variation was mainly among populations.The results of the correlate analysis between flowering time and related traits showed that the flowering time was significantly negatively correlated with leaf length,leaf width,number of stem nodes,seed length and TKW(thousand kernel weight)(p<0.01).In addition,the flowering time was significantly negatively correlated with the number of spikelets per reproductive branch(p<0.05).The results of Principal component analysis(PCA)showed that the cumulative of the top five components was85.56%.The phenotypic variation of E.sibiricus was mainly affected by both nutritional and reproductive traits.Clustering analysis results showed that populations with similar flowering times clustered together.A large number of flowering variation materials were obtained in this experiment to provide germplasm resources for breeding early-and late-maturing varieties.2.A total of 155 candidate genes related to flowering traits were screened in 10,591 unigenes that were sequenced from the flowering transcriptome of E.sibiricus in our previous study.A total of 125 EST-SSR markers were developed based on flowering candidate genes.Fifteen polymorphic markers were identified on 20 extreme flowering time E.sibiricus germplasms and the polymorphic information content(PIC)value of the primers ranged from 0.12 to 0.48,with an average value of 0.25.Cluster analysis showed that when the similarity coefficient was 0.71,the 20 E.sibiricus germplasms could be divided into three groups.The germplasms with similar flowering time could be clustered together,and the result of STRUCTURE analysis was the same.The marker 28366 based on gene Hd3 a was able to distinguish early-and late-flowering genotypes with an efficiency of up to 90%.These newly developed EST-SSR markers can be used for molecular marker-assisted selection and germplasm evaluation of flowering traits in E.sibiricus.3.The genetic diversity of wild E.sibiricus with different flowering time in Gannan was evaluated by using 14 pairs of EST-SSR primers.A total of 105 polymorphic bands were amplified and the PIC was 0.13-0.41,with an average value of 0.30.All two hundred samples were divided into two groups at the genetic similarity coefficient of0.66 by UPGMA clustering analysis.The first group mainly included early-flowering populations.The second group mainly included late-flowering populations.The STRUCTURE analysis also divided the 20 wild populations into two groups,which could explain the case that individual plants were classified as other groups in the clustering results.The results provided a basis for accelerating molecular genetic improvement of flowering traits in E.sibiricus.4.Based on the phenotypic and molecular identification,10 early-flowering germplasms,10 mid-flowering germplasms,and 10 late-flowering germplasms were used to analyze flowering candidate genes.A non-synonymous single nucleotide polymorphism(SNP)was found at 320 bp of the ORF region of TRINITY＿DN26735＿c0＿g2,a homologous gene of LIMYB.The mid-and lateflowering genotypes mutated from G(Guanine)to A(Adenine),resulting in the midand late-flowering genotypes changing from R(Arginine)to K(Lysine).A nonsynonymous SNP was found at 146 bp of ORF region of TRINITY＿DN29226＿c0＿g3,a homologous gene of VRN2/ Ghd7.The late-flowering genotype was mutated from G(Guanine)to C(Cytosine),resulting in the mutation of amino acid from E(Glutamate)to D(Aspartic acid).These results provide a basis for further research on the molecular mechanism of flowering and marker-assisted selection in E.sibiricus. |
