Genetic Characteristics And Gene Mapping Of A Thermal Sensitive Virescent Mutant Tsv In Gossypium Hirsutum L.

Leaf-color mutants are widely found in nature and can be found in many plants,such as Arabidopsis thaliana,rice,maize,cotton,etc.The types of leaf color mutations are also varied,including yellowing,albinism,speckled,stripe,etc.Leaf color mutants are also widely studied as ideal materials for studying chloroplast development,chlorophyll synthesis and photosynthesis.In this study,the thermal sensitive virescent mutants were produced during tissue culture of cotton,tsv mutants were isolated and purified,and their physiological and biochemical characteristics and genetic characteristics were studied respectively.The target genes were mapped by highthroughput sequencing technology and map cloning,and candidate genes were screened for preliminary verification.The main research results are as follows:1.The thermal sensitive virescent mutant tsv has a virscent phenotype at 23℃environments,while tsv mutants at 28℃ environments is similar to the normal plants.The main reason is the temperature difference between two environments.tsv mutant paraffin section showed that palisade tissue dysplasia,transmission electron microscope found seriously disrupted chloroplast development,the chlorophyll and carotenoid content decreased significantly,and shows a leaf etiolation.The above results led to the net photosynthetic rate decreased greatly.Transmission electron microscope observation and growth parameter measurement showed that there was no obvious starch grain accumulation in the chloroplast,and its dry weight was significantly lower than that of the control.2.We studied the genetic characteristics of tsv mutants by constructing the F2 generation population with tsv mutants,and speculated that the character was controlled by a pair of recessive gene.The extreme phenotype cottons of F2 generation population were constructed the BSA-seq pool,and the SNP and In Del outcomes of BSA-seq was analyzed by different ways,including Euclidean Distance(ED)and SNP index algorithm.The results showed that a 3.84 Mb interval was obtained from the intersection of SNP and In Del,which was located in 0-3.84 Mb of A12 chromosome.3.Transcriptome sequencing of tsv mutants and CCRI 24 at 23℃ and 28℃showed that 1758 genes were significantly different between tsv mutants and CCRI 24 at 23℃;862 genes were significantly different between the tsv mutants and the control CCRI24 at 28℃,while the number of genes the tsv mutants between 23℃ and 28℃reached 9863.Go enrichment analysis showed that the three groups of data were enriched to the pathway that might be related to the virscent,such as r RNA metabolism,ribosome biosynthesis,heme binding pathway,tetrapyrrole binding pathway,ribosome and ribosomal protein complex were enriched.KEGG is also enriched in photosynthesis pathway,photosynthetic antenna protein,ribosome biosynthesis,protein processing of endoplasmic network,RNA transport,RNA degradation,flavonoids biosynthesis,etc.,which proves that transcriptional data are reliable and can provide some guidance significance.4.The SSR marker of whole Gossypium hirstum genome was obtained from the two generations database of Nanjing Agricultural University.The marker was selected from the BSA-seq related region and verified by polyacrylamide gel.It was found that the trait was linked with the number of SSR markers on chromosome A12.Finally,8pairs of markers were used to draw a genetic map with a genetic distance of 16.5c M.The candidate gene was located in SSR marker A12＿339 and A12＿298,The genetic distances to the mark A12＿339 and A12＿298 were 0.6c M and 2.1c M,respectively.The physical distance between the two markers is 1.66 Mb.5.Based on the transcriptome data and functional annotation on cotton genome website,six candidate genes for virus induced gene silence experiment(VIGS).In the experiment of VIGS,the plants treated with two different genes showed different phenotypes compared with the blank control.Two genes,Gh＿A12G0013 and Gh＿A12G0158,encoding ribosomal biosynthetic protein BOP1 and 4-hydroxy-3-methylbuten-2-enyl diphosphate reductase respectively,may be related to the virescent trait.The cotton seedling was infected with those genes had different phenotypes,including leaf shrinkage,leaf vein yellowing and leaf reticulate yellowing.The efficiency of VIGS was detected by q RT-PCR,and with a more than 60% and 70%efficiency of VIGS.it can infer the phenotype was caused by these two gene silencing.