Construction Of High Idensity Genetic Map For Tetraploid Cultivated Cotton Species And Fine Mapping Of Red Plant R₁and Trichomes T₁in Cotton

Cotton is one of the important cash crops in the world, and the quality and color of the cotton fibers can greatly enhance its economic value. The high-density genetic map is the basement of genome research in plant. It can not only shed light on the structural features of the genome, but also lay the foundation for the fine mapping of genes related to qualitative and quantitative traits. Trichomes and red plant traits were controled by a single dominant gene in cotton. There were similarities between the development of leaf hairs and the development of the cotton fiber, and the leaf hairs had insect-resistant characteristics. Pigmentation gene of the red plant can be effectively used to cultivate natural colored cotton. To elucidate the mechanism and provide important genetic resouces for fiber quality improvement, insect resistance enhancement and natural colored cotton cultivation, we mapped the two key genes and tagged preliminary the candidate genes. In this paper, we updated our backbone genetic map and analyzed the chromosome structural features in allotetraploid cotton. Based on the high-density genetic map, two genes related to quality trait, T1and R1, were fine mapped and candidate genes were further mined. Using different cotton EST database and genome sequene of G. raimondii, the development, identification, classification of genome-wide WRKY transcription factors family in cotton were carried out and preliminary expressional analysis was performed. The main findings are as follows.1The construction of high-density genetic map and analysis of genomic structureTotal1431primer pairs were used, including newly developed gSSR, publicly released CER, CGR, COT, DC, DPL, SHIN and HAU primers, to screen poplymorphisms between TM-1and Hai7124in order to enhance our backbone genetic map in allotetraploid cotton. The updated map consists of3,414loci in26linkage groups covering3,667.62cM with an average inter-locus distance of1.08cM. By the analysis of loci, we draw the following results: Among2898primer pairs produced3414loci in TM-1and Hai7124,425primer pairs amplified two or more loci, resulting in total of941duplicate loci. Four marker types, morphological marker, CAPs, SNP and BAC-end, did not find duplicate loci.693duplicated loci were identified by326SSR primer pairs, with574duplicated,111triplicated, and8tetraplicated loci. Some loci were present on the homeologous chromosomes, some loci found to involve in the same chromosomes, and other loci spanned the different chromosomes, implying duplication of multiple round and genome rearrangement of both intrachromosome and interchromosome in the process of evolution.The cluster of loci were also observed in26linkage group,86clusters involved in617loci (≥5loci/cM) were discovered on the25linkage group besides A1(Chr.01). Of them,31clusters contained229loci from At subgenome, and55clusters contained388loci from Dt subgenome.19candidate genes islands (≥5EST-SSR loci/cM) and one retrotransposon-rich region were discovered by clusters distribution of marker loci.300loci showed non-mendelian segregation (P<0.05). A total of12segregation distortion regions (SDRs) were detected on11linkage groups. There were two SDRs in D10linkage group. Among12SDRs, six were on the At subgenome and six on the Dt subgenome, with8SDRs skewed toward G. hirutum TM-1and four SDRs skewed toward the heterozygote.2The fine mapping of two genes related to qualitative traits and screening of candidate genesBased on the high-density genetic map and two populations, Subl6×T586F2and [(Hai7124xT586)xHai7124] BC1populations, the red plant R1gene and the trichomes T1gene were fine mapped. As a result, R1was mapped between NAU4956and NAU6752, with only0.49cM to the nearest maker loci (NAU6752) by the Sub16×T586F2population contained1259individual plants;the T1was located between NAU5434and NAU1277, with only0.6cM to the nearest marker loci (NAU5434) by the [(Hai7124xT586)xHai7124] BC1population contained835individual plants.To find their candidate genes, we used the sequence of markers as probes to anchor the G. raimondii genome scaffolds. The ORF were searched by Fgenesh program and functional annotation by BLAST2Go. Based on pigment synthesis pathway, we selected six candidate genes, named for RG1-RG6, for cloning R1gene; According to Arabidopsis trichomes development research, we selected six candidate genes for cloning T1gene, named for TG1-TG6. Morphological and electron microscope scanning analysis show that the pigment accumulates is more and more in T586leaves as development process, and the density of leaf hairs of T586is significantly higher than Hai7124in each development stage, though the hairs of the leaves become sparser with the leaves development. The leaf hairs can only be observed around the nervation and the edges of the leaves of Hai7124on the mature leaves, while the leaves of T586are covered with leaf hairs of high density. Based on the study, cotton leaves of different development stage (leaf buds; young leaf; mature leaf) of T586and Hai7124were collected for further expression analysis of candidate genes. Q-PCR results indicated that among the6candidate genes, RG4gene had a significantly higher expression level in the mature leaves that accumulated the most pigment of T586than in the leaves of Hai7124of the same development stage, while others did not show significant differences. TG5gene showed significant expression difference between the leaves of T586and Hai7124at every development stage compared with other five candidate genes, and the expression level in T586and Hai7124decreased with the development of the leaves, and the results in consistant with the distribution of the density of leaf hairs, while other candidate genes did not show regularities in the expression mode. Taking all the above results into consideration, RG4and TG5are considered to be the two candidate genes that controls two important qualitative traits, namely Red plant R1and Leaf Hairs T1, respectively. Blast2Go results indicated that RG4encoded a phytoene synthase, while TG5acted as a bHLH transcript factor.3The genome-wide analysis of WRKY transcription factor family in cottonIt has been reported WRKY transcription factors family involved in plant growth, development, senescence and response to various biotic and abiotic stress. AtWRKY44involved in trichomes development in Arabidopsis.To clarify the possible role of WRKY transcription factors family in cotton growth and development, the development, identification, classification of genome-wide WRKY transcription factors family in cotton were carried out and preliminary expressional analysis was performed.In recent years, a large number of EST sequences of cotton have been releasd in NCBI. Using the Pfam database provides WRKY transcription factor seed file, total2450seed sequences compared to the EST database of cotton, further we got535EST sequences related to WRKY transcription factor. By the535EST sequences as probes and electronic cloning technology, we got the95contigs contained full or partial WRKY domain. Of them,21Contig lacked the zinc finger structure, and remaining74Contigs were able to find a complete WRKY domain. These74Contigs were named Contigl to Contig74. Based on the WRKY domain characteristics and evolutionary analysis,74Contigs were divided into three groups and five subgroups, Group I contained16Contigs, Group Ⅱ49and Group Ⅲ9; the Group Ⅱ was divided into five subgroup, Group Ⅱ a, Group Ⅱb, Group Ⅱc, Group Ⅱd and Group Ⅱe. The each subgroup member was5,2,25,12and5Contigs, respectively.Due to cotton EST sequences could not cover the entire cotton transcriptome in NCBI. We scanned the G. raimondii genome scaffolds released publicly by the WRKY transcription domain. This family was divided into three categories, with included109members. Group Ⅰ contained19members, Group Ⅱ78, GroupⅢ12. By constructing the phylogenetic tree using G raimondii and arabidopsis WRKY domain, the Group Ⅱ was further divided into five subgroups, Group Ⅱa, Group Ⅱb, Group Ⅱ c, Group Ⅱd and Group Ⅱe. The each subgroup member was7,15,29,15and12, respectively.By the alignment of the WRKY domain from G. Raimondii WRKY transcription factors, the variations of the WRKYGQK conserved domain was found. The domain of WRKYGQK become the WRKYGKK and WRKYGHK in the Group Ⅱc subgroup. The members from Group Ⅱc, including GrWRKY31, GrWRKY39, GrWRKY46, GrWRKY35and GrWRKY78, contained the variation of the WRKYGKK type, and the variation of the WRKYGHK type from GrWRKY8. The WRKY transcription factors not only included the variation of WRKYGQK domain, the zinc finger structure also had variation. The genes of zinc finger structure variation from Group Ⅰ N、Group Ⅰ C and GroupⅢ.Integrated WRKY transcription factor from cotton EST database and the genome of G raimondii, we found that there were70Contigs are able to find highly homologous sequence in the D genome, Contig69,70,71and73did not find similar homologous sequences in the D genome. They might be new genes in the tetraploid cotton evolutionary process or they were proprietary genes of At subgenome, so they can not find homologous sequence in D genome.Based on in silico cloning technology, we cloned several WRKY genes in G. hirsutum cv TM-1. Total the full-length sequence of five genes were obtained, GhWRKY19, GhWRKY23, GhWRKY60, GhWRKY87, and GhWRKY95. GhWRKY19ORF length is1401bp, encoding466amino acids, containing four introns; GhWRKY23ORF length is942bp, encoding313amino acids, containing four introns; GhWRKY60ORF is1068bp, encoding355amino acids, contain two introns; GhWRKY87ORF length is807bp, encoding268amino acids, containing two introns; GhWRKY95ORF is966bp, encoding321amino acid residues, containing two introns. The Q-PCR analysis in the TM-1showed that GhWRKY38highly expressed in the root; GhWRKY87and GhWRKY43in the leaves; GhWRKY74in roots and stems; GhWRKY60in the roots and leaves in high. Three WRKY members.(GhWRKY19, GhWRKY21and GhWRKY23) were highly expressed in the early fiber development. But GhWRKY19highly expressed in leaves and GhWRKY21in the roots and leaves.Using the three-leaf stage seedlings of G hirsutum cv Jinmian19as test material, some abiotic stress treatment, such as salicylic acid (2mM SA), methyl jasmonate (100μM MeJA), abscisic acid (200μM ABA), gibberellin (of500μM GA), salt (200mM NaCl), drought (20%PEG6000), ethylene (5mM ET), were induced. We analyzed expression levels of WRKY genes in abiotic stress-induced Oh,2h,4h,6h,8h,10h,12h,24h. The results show that the expression of GhWRKY23, GhWRKY43, GhWRKY60, GhWRKY74; and GhWRKY87after i.nduced by the seven kinds stress significantly increased GhWRKY19was not induced by the PEG and ET, GhWRKY21and GhWRKY38was not induced by salt stress.