| Sorghum is one of the main grain crops in China.It can be divided into grain sorghum,forage sorghum,sweet sorghum and broom sorghum.Forage sorghum has high biomass,rich nutrition and strong stress resistance.It is the representative of new high-quality forage.As a whole plant silage,biomass has always been an important index for forage sorghum breeding,but the research on quality traits is relatively scarce.A genome-wide association study was conducted on the main quality traits of 206 forage sorghum backbone sterile lines and restorer lines from major sorghum producing areas in the world,Taking lignin content as the research focus,we use transcriptome sequencing,gene editing and other biological technologies to mine key genes,so as to provide a theoretical basis for the establishment of molecular assisted breeding system.The main research contents and conclusions of this thesis are as follows:1.206 forage sorghum backbone sterile lines and restorer lines were re-sequenced,and the population structure and genetic diversity were analyzed(1)All materials can be roughly divided into two groups.The first group includes 60 American materials and 1 Asian material × American materials,The second group includes Europe,America,Asia and Asia ×America and Asia × Most varieties in Australia are not completely divided into several subgroups or subgroups,indicating that the genetic background of varieties in these regions is richer and the genetic improvement among varieties is more frequent.(2)The genetic diversity of the materials was analyzed.The results showed that Asia,America and Asia× America,Asia × More complex genetic backgrounds can be found in Australian varieties,while the genetic diversity of germplasm resources from Europe is relatively narrow.Linkage disequilibrium analysis showed that the population of European materials was not only small in number,but also low in genetic improvement rate.2.Genome wide association study was conducted on the main quality traits of 206 forage sorghum(1)From 2017 to 2019,the contents of lignin,acid detergent fiber,ethanol soluble carbohydrate and NH3-N of all materials were detected by near infrared spectroscopy.(2)GWAS analysis results show that: there are 9 QTLs related to lignin content,distributed on chromosomes 1,4,6,7 and 9 of sorghum,including 184 candidate genes,of which 13 have high homology with the genes found in corn,rice and other crops.Eight QTLs related to acid detergent fiber content were distributed on chromosomes 2,5,6,7,8 and 9 of sorghum,including 91 candidate genes,of which 44 had high homology with the genes found in rice and maize.A total of 16 QTLs related to alcohol soluble sugar content were distributed on chromosomes 1,3,5,6,7,9 and 10 of sorghum,including 302 candidate genes,of which 25 had high homology with the genes found in rice.Eight QTLs related to ammonia nitrogen content were distributed on chromosomes 1,2,4 and 6 of sorghum,including 35 candidate genes,of which 21 had high homology with the genes found in rice and alfalfa.The results of allelic haplotype analysis of Forage Sorghum in different regions showed that European materials did not contain haplotypes that reduced lignin content;The contents of acid detergent fiber,ethanol soluble carbohydrate and NH3-N in Forage Sorghum materials containing AA,CC,GG and TT were significantly higher than those of other haplotypes.3.At jointing stage,select 3 pairs(6,18 comparison groups)of materials with extreme differences in lignin content,and take their stems and leaves for transcriptome sequencing and metabolic pathway analysis respectively(1)Transcriptome sequencing showed that there were 76420 differentially expressed genes in lignin content,including 18175 up-regulated genes and 17393 down regulated genes in leaf group,24285 upregulated genes and 14794 down regulated genes in stem group.Key transcription factors mainly come from C2H2,WRKY,MYB,b HLH and other families,with a total of 2680.(2)GO enrichment analysis showed that the differential genes were mainly enriched in the biological process of plant cell wall and secondary cell wall.KEGG pathway analysis showed that the most significant metabolic pathway of lignin synthesis was phenylpropane biosynthesis pathway.KO analysis results show that the lignin type of materials with low lignin content is mainly guaiacyl lignin,and the materials with high lignin content are p-hydroxyphenyl lignin or syringyl lignin.Transcriptional sequencing analysis showed that there were 31 genes involved in phenylpropane biological metabolic pathway(lignin metabolism),of which17 were up-regulated and 14 were down regulated.4.Functional verification of candidate genes based on gene heterologous expression and CRISPR-Cas9technologyThe genes LOC8067848 and LOC8074615 in GWAS and RNA-Seq analysis results were heterologously expressed and knocked out in Rice Variety Zhonghua 11,The results showed that the overexpression of gene LOC8067848 could reduce the lignin content of rice leaves and stems by 5.91% and 15.51%,and the overexpression of gene LOC8074615 could reduce the lignin content of rice leaves and stems by 13.36% and23.69%.When the homologous genes of rice were knocked out at the same time,the wood content of stem and leaf was 44.65% and 22.91% higher than that of the control.In summary,after genome-wide association analysis,transcriptome sequencing,gene editing and other biological technology analysis of the lignin content of 206 forage sorghum from major sorghum producing areas in the world,the gene LOC8067848 on chromosome 7 and the gene LOC8074615 on chromosome 9of sorghum are important genes to reduce the lignin content of forage sorghum.It can be reasonably used in the breeding of new varieties with lodging resistance and easy digestion... |
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