Distribution Of Specific Mitochondrial Chimeric Sequences Of Upland Cotton Maintainer Line In Gossypium And Development Of Molecular Markers

Cotton plays an important role in China ’s agriculture.Gossypium plants can be divided into diploid genome(A,B,C,D,E,F,G and K genomes,respectively)and tetraploid genome(AD genome).DNA molecular marker technology has achieved remarkable results in promoting cotton breeding in China.However,there are few studies on mitochondrial genome DNA molecular markers.In the study of cotton,our research group found that there was a specific chimeric sequence with a length of 515 bp in the 3 ’ flanking region of the mitochondrial atp A gene of upland cotton maintainer line P30 B,named N515 sequence.The N515 chimeric sequence was screened horizontally in 34 kinds of cotton by Southern blot and PCR.The results showed that 28 kinds of cotton were amplified in 34 kinds of cotton.The N515 sequences of these 28 kinds of cotton were cloned and sequenced.The genetic polymorphism was analyzed and the following conclusions were drawn :1)The genomic DNA of 34 cotton species from Sanya cotton germplasm nursery in Hainan,China was obtained by improved CTAB method.2)The A genome cotton species G.arboreum and G.africanum were selected;B chromosome group cotton G.anomalum,G.capitis-viridis;D genome cotton species G.aridum,G.raimondii;E genome cotton variety G.aeysianum;F chromosome cotton variety G.longicalyx;according to the results of Southern blot,N515 is a single copy in the above cotton species.3)Primer N515 HEXIN was designed,and 28 of 34 cotton species had N515 sequence.4)The primers were designed again to amplify the N515 region of 28 cotton mitochondrial genomes,and the specific sequence of the N515 region of 28 cotton mitochondrial genomes was determined by cloning and sequencing.5)Through sequence comparison,72 SNP sites were found,and 28 cotton species were divided into 12 different categories:(1)G.herbaceum,G.capitis-viridis;(2)G.africanum;(3)G.sturtianum,G.davidsoni,G.somalese,G.aeysianum,G.incanum and G.nandewarense;(4)G.aridum,G.raymondii,G.gossypioides,G.lobatum,G.laxum,G.schwendimani;(5)G.nelsonii,G.australe;(6)G.bickii;(7)G.mustillinum;(8)G.arboreum,G.anomalum and G.robinsonii;(9)G.stocksii,G.tomentosum and G.darwinii;(10)G.longicalyx;(11)G.hirsutum;(12)G.barbadense.6)According to the specific SNP locus of Gossypium bickii,primers N515-bickii were designed to distinguish Gossypium bickii from other 33 cotton species.7)Primers N515-G were designed according to the specific SNP loci of G.nielsonii and G.australia,which could distinguish G.nielsonii and G.australia from other 32 cotton species.8)The primer pair N515-AD D was designed to distinguish cotton with AD genome group and cotton with D genome group from cotton with other chromosome genomes.N515 marker is of great significance for expanding the germplasm basis of cotton,identifying the polymorphism of cotton mitochondrial genome and understanding the genetic diversity of cotton germplasm resources.By comparing the genetic relationship among 28 cotton species,the biological basis for cotton cross breeding was also provided;To provide a rapid method for cotton mitochondrial genome polymorphism and variety identification,and provide a theoretical basis for breeding new cotton varieties.