| Elymus sibiricus L.is one of the important perennial forges in the Qinghai-Tibet Plateau.Given its high feed value and its excellent stress resistance,E.sibiricus has become the main variety of ecological restoration of alpine grassland and an important carrier for the construction of green organic product export places.However,with the increase of growth years,the degradation of spike traits directly resulted in the decrease of seed yield,which became the bottleneck of sustainable utilization of E.sibiricus resources.Screening the E.sibiricus germplasm with good panicle phenotypic traits and clarifying the panicle development mechanism can provide a theoretical basis for solving the degradation of the production function of the E.sibiricus population and improving seed yield.In this study,53 E.sibiricus germplasms from different ecological regions of the Qinghai-Tibet Plateau were selected as experimental materials,and molecular markers and spike phenotypic diversity were used to compare the genetic background and spike morphological variation of the tested germplasms,to establish a new spike type classification standard based on the difference of key spike traits.At the same time,the differences of key enzyme activities,hormone and carbohydrate contents between dense spike and loose spike type E.sibiricus were systematically compared,and the key physiological processes of seed formation of different spike types E.sibiricus were determined.In addition,transcriptome technology was used to explore key genes and regulatory pathways of spike traits development in E.sibiricus,aiming to provide a theoretical basis for spike type classification,spike traits improvement,and seed yield improvement of E.sibiricus.The main results are as follows:1.The genetic variation and genetic structure of 53 E.sibiricus germplasm were evaluated based on SSR molecular markers,and the results showed that six of the seven primer pairs had high polymorphism,and the variation in the polymorphic information content of individual site among 53 E.sibiricus samples ranged from 0.055 to 0.862,with an average of 0.566.The mean value of Shannon’s information index(I)was 1.348,and the observed heterozygosity and expected heterozygosity ranged from 0.019 to0.956 and 0.055 to 0.873,respectively.The 53 E.sibiricus germplasm showed rich genetic diversity.Population genetic analysis showed that the population genetic distance variation ranged from 0.029 to 0.479 and Nei’s genetic concordance ranged from 0.796 to 0.972.The mean population fixed coefficient was 0.288 and the mean gene flow was 1.085.The percentages of inter-and intra-population variation were1.575% and 98.425%,respectively.It indicated that the 53 E.sibiricus variation with different altitude gradients occurred mainly within the populations,with the two populations between 2400-2800 m and 2800-3150 m in altitude being relatively more closely related.2.The analysis of morphological diversity of spikes showed that the traits differed significantly among the 53 E.sibiricus materials,and the results of the two years of experimental data were consistent.The coefficients of variation of 13 spike traits and seed yield ranged from 8.23% to 27.54%,and those with coefficients of variation >20.00% were: grain number per spike,grain weight per spike,grain density,spike mass index,dry weight per spike and seed yield per plant.Correlation analysis showed that spike length,spikelet number,spikelet length,seed length to width ratio,grain number per spike,grain weight per spike and spike mass index were significantly and positively correlated with seed yield per plant.Path analysis showed that the 13 traits were divided into four levels according to their effects on seed yield,and Sd L/W,SL,and DWS had positive direct effects on seed yield as first-order factors,while they were directly or indirectly influenced by the traits in other levels.The combined path analysis,principal component and gray correlation analysis concluded that dry weight per spike,grain weight per spike,spike mass index,grain number per spike,and grain density were the key traits affecting seed yield of E.sibiricus.3.Based on the above study and the field performance of E.sibiricus,dry weight per spike,grain density and spike mass index were selected as the basis for the classification of E.sibiricus spike type,and the K-means clustering method and the field performance of E.sibiricus germplasm were combined to establish the spike type classification criteria suitable for E.sibiricus on the Qinghai-Tibet Plateau.Using dry weight per spike as the classification index,the test materials could be divided into different spike types,namely,thin,medium,and heavy spike type.According to density,the test materials could be divided into loose,semi-dense,and dense spike types.According to the spike mass index,the following spike types were identified: slender,well-proportioned,and large spike types.The seed yield of heavy,dense,and large spike types was significantly higher than that of thin,loose,and slender spike types.The germplasm selection of E.sibiricus in the Qinghai-Tibet Plateau region should be based on heavy,dense and large spike type germplasm.4.The spikelet length,spikelet width,grain number per spike,grain density,grain weight per spike,dry weight per spike,thousand kernel weight and seed yield per plant of the dense spike type E.sibiricus germplasm ‘16-118’ were better than those of the loose spike type germplasm ‘10-005’.Both flag leaf and spike photosynthetic capacity were higher and starch accumulation was higher in the dense spike type than in the loose spike type E.sibiricus;moreover,the effect of photosynthetic rate per organ on seed formation was more studied than the photosynthetic rate per unit area.During the process of seed filling,the content of zeatin in the seeds of both materials showed an increasing and then decreasing trend,while growth hormone and abscisic acid showed an increasing trend and gibberellin gradually decreased.5.Transcriptome sequencing was used to analyze the differentially expressed genes in young spikes of dense and loose spike type E.sibiricus at key stages of young spikes development(double ridge stage,spikelet differentiation stage and floret differentiation stage).The results showed that: compared with the double ridge stage,there were 2486 up-regulated and 3354 down-regulated genes in the spikelet differentiation stage of the dense spike type E.sibiricus,and a total of 2407 upregulated and 3119 down-regulated genes were identified in the spikelet differentiation stage of the loose spike type E.sibiricus;there were 4193 and 4989 up-regulated and down-regulated genes in the spikelet differentiation stage of the dense spike type E.sibiricus,and 7869 and 7743 up-regulated and down-regulated genes in the spikelet differentiation stage of the loose spike type E.sibiricus,respectively.KEGG annotation indicated that the above differentially expressed genes were mainly enriched in galactose metabolism,starch and sucrose metabolism,and plant hormone signal transduction pathways.Further analysis showed that several key TFs,such as Dof,ERF,C2H2,BBR＿BPC,BSE1,MYB＿related,LBD and MYB,play important roles in regulating young spike differentiation by directly or indirectly regulating the expression of downstream genes and activating carbohydrate and hormone signaling pathways,thereby regulating young spike development. |
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