Deletion Analysis Of 5’UTR Sequence Of Cotton Mitochondrial Atp9 Gene And Development Of Molecular Markers

Cotton has attracted wide attention due to its great economic and scientific research value.Among the 54 known cotton species,47 are diploid cotton species,including 8karyotypes such as A,B,C,D,E,F,G and K,and 5 are allotetraploid cotton species(AD genome).These cotton species are widely distributed and have different living environments,resulting in very rich germplasm resources.At the same time,they also have many excellent characteristics that cultivated cotton lacks,such as disease resistance,stress resistance,cytoplasmic male sterility,etc.However,there are few methods for molecular identification.It was found that there was a repetitive sequence deletion site in the 5’UTR of cotton mitochondrial atp9 gene.Previous studies showed that this site was different between cotton CMS line and maintainer line,and the deletion of this site was also found in wild type cotton,indicating that this site may be related to the evolution of cotton mitochondrial gene.This site may provide us with rich genetic information and can be used to identify different cotton varieties.For this reason,we compared 33 kinds of cotton horizontally.The specific results are as follows :(1)We used CTAB method to extract 33 kinds of cotton genomic DNA.The A260 /A280 ratio is close to 1.8,and the sample purity is good.(2)Using the extracted genomic DNA as a template,and using atp9meiqie-F and atp9meiqie-R as upstream and downstream primers,we carried out PCR amplification.Finally,the 5’UTR target sequence of atp9 gene in 33 cotton species was successfully amplified,and the fragment length was about 750 bp.(3)The PCR products of Gossypium mitochondrial atp9 gene and 5’UTR sequence were digested by Eco R I restriction site to determine whether there was a deletion of site(AATTT).The results of enzyme digestion experiments showed that 27 of 33 cotton species were missing;six cotton species were not missing : G.longicalyx、G.hirsutum、G.barbadense、G.tomentosum、G.mustillinum、G.darwinii.(4)According to SSR loci and SNP loci,the phylogenetic tree drawn by Clustalx and Phylip software divided 33 cotton species into 6 different groups.Combining the electrophoresis map with the world cotton origin map,it was found that the closer to the origin of cotton seeds,the higher the similarity of the 5’UTR sequence of atp9 gene,which proved that the sequence was related to the origin of cotton seeds.(5)Primer pair atp9-SDKS was designed according to the specific sequence of G.stocksii,which could distinguish G.stocksii from other cotton species.(6)According to the specific SNP of E genome,a primer pair atp9-E was designed to distinguish G.stocksii,G.somalense,G.aeysianum and G.incanum and other cotton species.(7)According to the specific sequence of G.bickii,the primer pair atp9-BKS was designed to distinguish G.bickii from other cotton species.(8)According to the specific SNP of D genome,a primer pair atp9-D was designed,which could distinguish D genome cotton such as G.thurberi,G.harknessii,G.klotzschianum,G.davidsonii,G.aridum,G.raimondii,G.gossypioides,G.laxum,G.lobatum,G.schwendimanii,G.armourianum,G.turneri and G.trilobum and other cotton species.(9)Primer pair atp9-AD was designed according to the cotton specific sequence of AD genome,which could distinguish G.hirsutum,G.barbadense,G.tomentosum,G.mustillinum and G.darwinii from other cotton species.The 5’UTR locus of atp9 gene provides us with abundant genetic information,which can be used to identify different cotton varieties and establish the techniques and methods to identify different cotton varieties at the molecular level.It provides theoretical basis and technical reference for molecular identification and molecular marker-assisted breeding of cotton varieties.