Study On Whole Genome Sequencing And Functional Genes Of Key Traits In Cleistogenes Songorica And Melilotus Albus

Genome sequence is biology genetic resource and foundation for study of gene function and molecular mechanisms.The candidate genes of key traits can be identified based on the whole genome DNA makers(SNP),which can be applied in the molecular breeding and shorten the breeding time.C.songorica is perennial C₄ grass with super drought stress resistance and dimorphic flower.The dimorphic flowering mechanism benefit for surviving and reproducing of C.songorica under extreme conditions.Melilotus is widely used as a traditional medicine,forage,and green manure.However,as a kind of forage,the high content of coumarin limits Melilotus application.It is little known about functional gene of dimorphic flower of C.songorica,and coumarin biosynthesis key enzyme of Melilotus.Here,we conducted whole genome sequencing of C.songorica cv‘Tenggeli'and M.albus,combining the transcriptome,metabolome,BSA and re-sequencing to study functional gene of key traits.The main results are as follows:1.A chromosome level genome sequence of C.songorica cv‘Tenggeli'were assembled.Contig-level assembly comprised the genome of 540.12 Mb,with a contig N50 of 21.28 Mb.528.52 Mb contigs were anchored to 20 pseudochromosomes,and telomeres of all 20 chromosomes were assembled.A total of 54,383 protein-coding genes were predicted in C.songorica.Whole genome duplication occurred around 19.2million year ago(Mya).Based on relationship between C.songorica and Oryza sativa,the A and B sub-genome of C.songorica were separated,and chromosomal rearrangements had been occurred between B2 and B5,B1 and B8.Phylogenomic analysis revealed that C.songorica is a member of Chloridoideae,has a closer relationship with Oropetium thomaeum.Expanded and conserved family genes were significantly enriched in metabolic pathways implicated in stress responses,including fatty acid elongation,phenylpropanoid biosynthesis,and starch and sucrose metabolism.2.A flowering gene network of C.songorica were constructed including 302members of 83 gene families.Twelve transcription factor families related flowering were identified,and 168 members of them correlated with ABCDE model genes(AMGs)based on WGCNA analysis.Based on target sites prediction and expression analysis,we speculated that the interactions of mi R156ab-Cs SPL17-AMG may be involved in dimorphic flower differentiation,Cs MYB219 may regulate flower development under drought stress,mi R159-Cs MYB123-B-class gene module may be an important regulator for divergence of dimorphic floral structure.mi R172l is specifically targeted to Cs AP2?9,the interaction between them may regulate cleistogamy.Cs AP2?9 overexpression rice lines showing abnormal palea,and smaller and thinner lodicules;and mi R172l overexpression rice lines showing longer filaments,and reduced anther numbers.These results validated the functions of Cs AP2?9 and mi R172l in regulating lodicule,filament and anther development,respectively.3.M.albus(2n=16)genome were assembled to 1.05 Gb,with contig N50 of 7.49Mb.1.04 Gb(99.40%)of the assembled genome were anchored into 8pseudochromosomes.A total of 41,910 genes were predicted in the M.albus genome.Phylogeny analysis showed that M.albus is close with Medicago truncatula,and the divergence between them occurred?14.16 Mya.71.42%of the M.albus genome was annotated as repeated sequences,long terminal repeat(LTR)occupying 54.99%of the genome.Burst insert(0.05?0.85 Mya)of intact LTR was notably less than the divergence time(14.16 Mya)of M.albus and M.truncatula.The intact LTRs burst insertion patterns of M.albus was different from other three legume species,and the intact LTRs of M.albus were relatively young and highly dynamic.4.The 94 Melilotus accessions from various geographic regions were conducted whole genome re-sequencing.A total of 4,999,288 SNPs and 606,387 In Dels were discovered.The introgression existed between M.officinalis group and M.albus group,and gene flows was from M.albus to M.officinalis.Linkage disequilibrium analysis indicated that M.officinalis has relatively larger genetic diversity than that of M.albus.We inferred that M.albus and M.officinalis experienced bottlenecks mirroring by the continual Ne declines during the recent 0.2 Mya,coinciding with the period of the quaternary glaciation(0.01-2 Mya).The LTR burst insert may benefit for surviving of Melilotus populations.We identified two genes associated with flower color,five genes associated with coumarin biosynthesis by selective sweep analysis.Based on GWAS analysis,two MAH1 genes,related to wax biosynthesis,and XGT and XXT1 genes,related to cell walls development,were identified.5.Pathway of coumarin biosynthesis was constructed and thirteen gene families involved in it were identified in M.albus.We identified 8 candidate genes of coumarin biosynthesis based on WGCNA and BSA analysis of the near isogenic lines(NILs).Three differential metabolites,coumaric acid,coumaroyl glucoside and coumarin,were identified by metabolomics analysis of NILs.We predicted that BGLU(?-glucosidase)may play important role in coumarin biosynthesis.A cluster of six BGLU genes was identified which could regulate coumarin biosynthesis.The function of MaBGLU1 was verified by over-expression in Melilotus and heterologous expression in vitro,which demonstrated that BGLU contributes to the production of the coumarin derivative scopoletin and that its nonsynonymous variation may drive the divergence of BGLU enzyme activity.q RT-PCR analyses showed an obvious increase in relative expression levels in the positive MaBGLU1 overexpressing lines compared with control M.albus.