Identification Of Genes Related To Cotton Fiber Development And Analysis Of DIR And GH9 Gene Families

Cotton is an important economic crop,which provides natural fiber materials for textile industry.As the most widely cultivated cotton species,Gossypium hirsutum and Gossypium barbadense are characterized by high yield and superior fiber properties,respectively.It is helpful for clarifying the molecular mechanisms underlying high-quality fiber formation to identify cotton fiber developmentrelated genes.It also provides candidate genes for molecular breeding,promoting the high-efficiency integration of high yield and superior fiber properties.Comparative transcriptome analysis,based on RNA-seq,can identify cotton fiber development-related genes simply and efficiently.At present,the researches are largely concentrated in G.hirsutum,while the progress in G.barbadense has been delayed.It has great development prospects and application values to mine candidate genes for cotton fiber quality improvement by comparing the fiber transcriptomes of G.barbadense with G.hirsutum.In this study,to construct sequencing libraries,ovules of 0 DPA and fibers of 5,10,15,20,25 and 30 DPA were collected from G.barbadense cultivars Pima90-53 and Hai7124.Subsequently,by comparative transcriptome analysis,we identified preferentially expressed genes during fiber elongation and secondary wall thickening in G.barbadense,to explore the molecular mechanisms underlying highquality fiber formation.Furthermore,in comparison with the fiber transcriptomes of G.hirsutum cultivars HY405 and ND13,we identified differentially expressed genes(DEGs)between G.barbadense and G.hirsutum,generating candidate genes for cotton fiber quality improvement.We then veri fied the functions of candidate genes using transgenic Arabidopsis plants and a BC3F5 population.In addition,genome-wide identification and analysis of DIR and GH9 genes were performed to explore their roles in cotton fiber development.The main results are as follows:1.By comparative transcriptome analysis,759 up-regulated DEGs were identified in cotton fibers of 5,10 and 15 DPA as compared to ovules of 0 DPA in G.barbadense.Among them,we picked 346 genes whose gene expression was up-regulated only during fiber elongation and termed them"preferentially expressed genes during fiber elongation in G.barbadense".After GO enrichment analysis,plenty of pathways involved in cotton fiber development were identified,including actin cytoskeleton organization,cell morphogenesis,cell wall modification,fatty acid biosynthetic process,lipid transport,pectin catabolic process,phenylpropanoid biosynthetic process,response to reactive oxygen species,Sadenosylmethionine metabolic process,very long-chain fatty acid metabolic process,and water transport.Among 346 preferentially expressed genes during fiber elongation in G.barbadense,42 up-regulated DEGs were identified in cotton fibers of G.barbadense as compared to G.hirsutum,generating elongation-related candidate genes for fiber quality improvement.The 42 genes showed important application values for molecular breeding,containing some well-known cotton fiber development-related genes.Among the 42 genes,40 genes were cloned from cotton fibers of 10 DPA in G.barbadense cultivar Pima90-53,based on homology-based cloning method.Gbar_A05G037170(auxin signaling transduction),Gbar_D09G016550(short-chain dehydrogenase)and two novel genes Gbar_A05G020690 and Gbar_D12G007360 were transformed independently into A.thaliana plants,and the transgenic plants,compared with WT plants,showed significantly longer leaf trichomes or hypocotyl cells.2.In G.barbadense,2275 up-regulated DEGs were identified in cotton fibers of 20,25 and 30 DPA as compared to ovules of 0 DPA.Among them,we picked 647 genes whose gene expression was upregulated only during secondary wall thickening and termed them "preferentially expressed genes during secondary wall thickening in G.barbadense".After GO enrichment analysis,a lot of cell wall development-related pathways were identified,which were related to the metabolism of cellulose,xylan,glucuronoxylan,arabinan,pectin,lignin,glycoprotein,arabinose,galactose and sucrose.Among 647 preferentially expressed genes during secondary wall thickening in G.barbadense,48 up-regulated DEGs were identified in cotton fibers of G.barbadense as compared to G.hirsutum,generating secondary wall thickening-related candidate genes for fiber quality improvement.Based on the genome sequence differences between G.barbadense and G.hirsutum,SNP molecular markers of 39 genes were successfully developed,which can distinguish the orthologous genes between G.barbadense and G.hirsutum accurately and quickly.Genotyping of a BC3F5 population(donor parent:Pima90-53;recipient parent:CCR18)was performed using the 39 molecular markers.The phenotype data showed that the introgression of secondary wall thickening-related candidate genes into upland cotton could enhance its fiber strength.3.A dirigent(DIR)gene was found among the elongation-related candidate genes,which was rarely reported in the field of cotton fiber development.Hence,genome-wide identification and bioinformatics analysis of DIR gene family were carried out.In this study,107 GbDIRs and 107 GhDIRs were identified in G.barbadense and G.hirsutum,respectively.Phylogenetic analysis showed that cotton DIR genes were classified into four distinct subfamilies(a,b/d,e,and f).Of these groups,DIR-a and DIR-e were evolutionarily conserved,while DIR-b/d expanded considerably and evolved rapidly by recent tandem duplications in cotton.Based on RNA-seq data,we identified candidate DIR genes related to fiber development in G.barbadense and G.hirsutum,and revealed their differential expression by analyzing cis-acting elements in their promoter regions.Overexpression of GbDIR78 in Arabidopsis increased the length of leaf trichomes and dark-grown hypocotyl cells,indicating that GbDIR78 can promote cell elongation.We finally speculate that GbDIR78 participates in the metabolic flux from flavonoids into lignans,which should be a novel alternative way to improve cotton fiber quality.4.An endo-?-1,4-glucanase(GH9)gene was found among the secondary wall thickening-related candidate genes,playing an important role in the biosynthesis and remodeling of cell wall.Hence,genome-wide identification and bioinformatics analysis of GH9 gene family were carried out in this study.A total of 53 GH9 genes were identified in G.barbadense,which were divided into three groups and were distributed on 22 chromosomes.The expansion of GH9 gene family was mainly driven by polyploidization in G.barbadense,and the duplicated genes have been under strong selective constraints.Four members of group A showed high expression levels during cotton fiber development,especially during secondary wall thickening.Six members of group C were highly expressed during cotton fiber initiation and elongation.In addition,the expression patterns of group B were relatively complex.Auxin and ethylene may have played important roles in the transcriptional regulation of GbGH9s.It improved cotton fiber strength by the introgression of GbGH9B6 into G.hirsutum.In summary,we identified a batch of genes that were preferentially expressed during cotton fiber elongation or secondary wall thickening in G.barbadense,and then explored the molecular mechanisms underlying high-quality cotton fiber formation.Furthermore,we identified 42 elongation-related candidate genes for fiber quality improvement,four of which were subsequently confirmed to promote cell elongation.We also identified 48 secondary wall thickening-related candidate genes for fiber quality improvement,and SNP molecular markers of 39 genes were successfully developed.In addition,we carried out genome-wide identification of DIR and GH9 gene families,analyzed their expression patterns during cotton fiber development,and then revealed the transcriptional regulation.The involvement of GbDIR78 in cell elongation was also determined.These findings will contribute to understanding the molecular mechanisms underlying differences in fibre quality between G.barbadense and G.hirsutum,and promote the high-efficiency integration of fiber quality-related genes by molecular marker-assisted selection.