| Object:Cotton is an important cash crop in China and plays a significant role in the national economy.Xinjiang region is a major cotton-producing area in China,accounting for more than 80%and 90%of the total cotton planting area and harvest yield in China.Therefore,it is of great significance to stabilize cotton production in China.However,due to continuous cropping,cotton stalks being returned to the fields,and frequent introduction and cultivation of varieties from other cotton area in recent years,the problem of cotton Verticillium wilt has become increasingly serious in the Xinjiang cotton region,which has become a bottleneck for the stable development of cotton industry in Xinjiang and China.Lignin is the main component of the secondary wall in plants and plays an important role in plant defense against diseases and pests as the first natural physical barrier.In this study,we used 290 natural population of upland cotton Cultivars to investigate lignin traits,conducted genome-wide association analysis（GWAS）,and identified candidate genes that affect lignin biosynthesis and cotton Verticillium wilt resistance.This study provides valuable gene resources for breeding cotton with Verticillium wilt resistance from the role of lignin.The main results are as follows:Methods:In this study,290 re-sequenced natural population of upland cotton Cultivars were used which were planted under controlled temperature and humidity conditions in a greenhouse.The hypocotyls were sampled at the 4th to 5th leaf stage,and their fresh weight was measured.Tissue chemical staining was performed on the cross-sections of the hypocotyls to examine lignin deposition,and the area and thickness of the deposited lignin were extracted using machine learning.The total lignin content,as well as H,G,and S-type lignin monomer contents,were determined using the thioglycolic acid and nitrobenzene oxidation methods,respectively.Additionally,the total soluble phenol content of the hypocotyls was measured.GWAS was performed using various models to identify SNPs related to lignin traits.A MYB-type transcription factor,GhMYB78,was identified on chromosome A09,and its overexpression and RNAi transgenic lines were obtained.The transcriptome of the transgenic strains was sequenced,and the total lignin content and endogenous hormone content were measured.Resistance to Verticillium wilt was investigated by inoculating the transgenic strains with the defoliating Verticillium dahliae strain Vd.991,and their agronomic traits were also investigated.Differential expression genes（DEGs）were screened based on the disease resistance and phenotype of the transgenic lines,and the role of GhMYB78in lignin synthesis and Verticillium wilt resistance was preliminarily elucidated.Results:1.Based on the differences in the deposition site of lignin,the morphology and arrangement of cells,the cross-section was divided into five parts:cortex（including epidermis,Co）,phloem（Ph）,cambium（Ca）,xylem（Xyl）,and pith（Pi）.During this stage,lignin mainly deposited in the xylem and exhibited discontinuous deposition in the phloem.By using machine learning methods,the area and thickness of lignin deposition in different parts of the cross-section were captured to refine and quantify the characteristics of lignin deposition.The results of tissue chemical staining and captured data showed significant differences in the area and thickness of lignin deposition among the population.The relationship between lignin deposition characteristics and lignin content is complex:lignin content traits（G,S,HGS and total lignin）are highly positively correlated with plant fresh weight and main lignin deposition site（phloem,cambium,xylem）,and it’s confused that the lignin content was significantly negatively correlated with FW＿DW and total soluble phenolic content,cortex without lignin deposition.The intricate relationship between lignin deposition sites and lignin content suggests the lignin biosynthesis and regulation is complexity in cotton.The characteristics of lignin deposition and content in natural populations of upland cotton Cultivars were clarified.2.The general linear model（GLM）in Plink software was used for genome-wide association analysis（GWAS）for 61 traits were related lignin after comparing some models,and there were 78 significantly associated SNP loci in 22 traits.Among them,the S and its related traits HS（the sum of H and S）,GS（the sum of G and S）,and HGS（the sum of H,G,and S）had good co-localization on chromosomes A01,A07,A08,and D12.Excellent allele analysis found that 11 non-synonymous mutations in the coding region of genes were significantly associated with phenotypes.The haplotype of Gh GNA1（Gh＿A08G0953）significantly affected the content of G,S,HGS,S/H and was also significantly correlated with the disease index of Verticillium wilt,suggesting that Gh GNA1 affect the resistance to Verticillium wilt by mediating lignin biosynthesis,it a candidate gene for coordinating the synthesis of lignin and improving cotton’s resistance to Verticillium wilt.Gh NGA1 could be a candidate gene for coordinating the synthesis of lignin and improving resistance to Verticillium wilt in cotton.After silencing the genes Gh LCBK1（Gh＿D12G0104）,Gh ERG2（Gh＿A01G1566）,and Gh＿D13G1741 by virus-induced gene silencing（VIGS）experiments,compared to TRV:00,the lignin deposition in the first internode decreased,and the development of phloem and xylem slowed slow down.The lignin biosynthesis genes in the first internode of TRV:Gh LCBK1 and TRV:Gh＿D13G1741 were down-regulated.most genes in the phenylpropane metabolism pathway were up-regulated in TRV:Gh ERG2 except for a few laccase genes.This indicates that these genes are involved in lignin/phenylpropane metabolic processes.Infection of the silenced strains with Vd.991 revealed that TRV:Gh LCBK1-silenced were more sensitive to Vd.991,while silencing of TRV:Gh＿D13G1741,TRV:Gh ERG2,and TRV:Gh NGA1 enhanced resistance to Verticillium wilt compared to TRV:00.Therefore,Gh LCBK1,Gh＿D13G1741,Gh ERG2,and Gh NGA1 were identified as candidate genes that affect lignin synthesis and mediate resistance to Verticillium wilt.3.Using the 3VMr MLM model for GWAS analysis of of H-type lignin,a significant SNP site A09＿49533397 was found within the LD-Block of 142 kb,where only two SNPs causing non-synonymous mutations in the CDS region of the gene Gh＿A09G0645.1.The haplotype of Gh＿A09G0645.1 significantly affects lignin content traits.Sequence analysis showed that Gh＿A09G0645.1 belongs to the typical R2R3 of MYB transcription factors,which is in the same branch as At MYB78 and is named GhMYB78.Experimental results showed that the expression of GhMYB78 is induced by JA,H2O2,and Vd.991,and GhMYB78 located in the nucleus,and has self-activation activity.RNA＿seq data from transgenic lines and WT indicated that the lignin biosynthesis genes was up-regulated in GhMYB78 overexpression lines,suggesting that GhMYB78activated the lignin synthesis pathway.Compared to WT,the lignin content in the hypocotyls of the GhMYB78 overexpression lines increased before and after infected with Vd.991.DEGs involved in the lignin synthesis pathway were screened from the transcriptome,and their promoters were cloned.Compared to WT,GhMYB78 overexpression lines upregulated expression of lignin biosynthesis genes（according to transcriptomic data）and increased lignin content（before and after inoculation with Vd.991）.Additionally,GhMYB78 overexpression line exhibits shortened internode length in the main stem and fruit branches,dwarfed,and a compact plant architecture.Dual Luciferase Reporter Gene Assay（DLR）experiment showed that GhMYB78 could activate the promoters of lignin biosynthesis related genes Gh C4H-1,Gh C4H-2,Gh4CL-1,Gh4CL-3,Gh4CL-4,Gh CCo AOMT,Gh HCT,and Gh LAC1 in tobacco leaves,indicating that GhMYB78 could bind the promoters of genes of lignin biosynthesis and activate lignin synthesis.Meanwhile,the content of JA was promoted and the IAA content was inhibited in the GhMYB78 overexpression lines,leading to a decrease in the expression of genes involved in lateral organ formation and a reduction in lateral root quantity.Treatment with low concentration of IAA(1×10-9M)can partially restore the lateral root reduced phenotype in the overexpressing strains.GhMYB78 may be involved in inducing lignin and JA synthesis,thereby weakening IAA in mediating changes in cotton plant architecture.These factors may contribute to the internode shortening and dwarfing of GhMYB78 overexpressing lines.After inoculation with Vd.991,the overexpressing lines exhibit a significant increase in lignin content and activate gene expression related to JA and SA synthesis and signaling transduction pathways,and enhanced the resistance to Verticillium wilt.. |
