| Bromus L.is a genus of approximately 150 C3 grass species distributed widely in Asia,Europe,Africa,and the Americas,and today,introductions are widespread in the temperate world.The genus is taxonomically difficult with several unresolved species complexes,especially in section Ceratochloa,which is also one of the most important section contains the major agricultural species.Prairie grass(Bromus catharticus Vahl.)is a typical excellent cool season forage grass species in Ceratochloa with both annual and biennial growth forms.However,its process of taxonomy research and molecular breeding is stagnating due to very limited genomic resources.So development of genomic resource and valuable trait related genes is necessary to break the bottleneck and speed up the breeding process.The present research first developed transcriptomic resource of prairie grass,and identified tissues development related genes and metabolic pathways.Based on large-scale transcripts,we developed lots of molecular makers and applied in genetic diversity and population structure research.Finally,we combined traits and EST-SSRs to employ association analysis.The paper was to provides a valuable resource for ongoing research into this agronomical and forage important species.1.De nove assembly and annotationEleven experimental tissues of BCS1103 were collected at different development stages,including seed(marinating 24h),whole seeding(5d and 21 d after germination),tufted leaves at tillering stage,flag leaves and top second leaves at early filling stage,stems(tillering stage,early filling stage and late maturing stage),seeds(8 days and 20 days after fertilization).Average 48945309 raw reads were and total 257773 unigenes were obtained.The average length of unigenes were 1629 bp,and 193082(74.9%)genes were matched to at least one data base.121098 unigenes were assigned into 56 GO classes,including 25 biological processes,21 cellular component ontologies and 10 molecular functions.Out of160662 unigenes with perfect hits in NR database,44775 sequences were assigned to the COG function classification with 25 clusters.KEGG pathways analysis showed 60321 significantly matched unigenes were grouped into five categories including 130 KEGG pathways.2.Identification of development related genes and pathwaysThrough the comparisons analysis on seed filling,seed germination,flag and top second leaf development,stem development process,we found 1316 up-regulated genes and 673 down-regulated genes from 8 DAP to 20 DAP,and 2463 up-regulated genes and2173 down-regulated genes from 1 d to 5d germination.609 genes changed during the two processes,and most of them showed opposite trends.Leaves showed relative small number of DEGs.When compare to leaves in tilling stage,209 and 377 genes were up-regulated,446 and 561 genes were down regulated in flag and top second leaves respectively.434 genes were differently expressed in both leaf types.The DEG analysis showed more complex development and growth process of stems.Compare to stems in tilling stage,stems at 8 DAP showed 1989 genes up-regulated and 3323 genes down-regulated.While3025 genes were up-regulated in 20 DAP,and 1804 genes were down-regulated,when compared to stems at 8 DAP.We also analysed the pathways significantly enriched by up and down regulated genes,and found these genes were significantly related to the tissues development and functions.3.Maker development,genetic diversity and phenotypic diversityA total of 37288 SSRs were successfully generated and used to design primers,among these SSRs,tri-nucleotide,mono-nucleotides and di-nucleotides were most abundant.From 420 randomly selected and synthesized primers,we found 52 polymorphic primers to perform genetic diversity and population structure of 40 B.catharticus accessions.The result showed the average genetic distance of tested accessions were 0.298 indicating relatively low diversity.UPGMA and STRUCTURE analysis gathered all the accession into 6 subpopulations.Diversity analysis and AMOVA analysis based on development states,geographic distribution and population structure showed South America accessions and wild accessions showed high diversity.The genetic differentiation of population based on development states(Fst=0.057)and geographic distribution(Fst=0.126)were low,while population structure based subpopulations showed high genetic differentiation(Fst=0.593).On the other hand,the phenotypic estimation showed the average Euclidean distance was 25.92.UPGMA and PCA analysis both divided all accessions into 4 groups,and each groups had clear characteristics.In addition,genetic distance and phenotypic distance of the accessions showed high relationship(r=0.667,P<0.001),and both genetic and phenotypic data showed a certain level of correlations with geographic information.4.Maker-traits associationTo identify the QTLs,we performed LD analysis,and then association analysis based on population structure with GLM model.The result showed LD was existed between 152EST-SSRs,and 108 locus were associated with 9 traits.Besides,some maker locus were associated with two or more traits,while most traits were associated with multiple maker locus. |
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