Screening And Application Of The Chromosome-specific BAC Clone In D-genome Diploid Cotton

Cotton is the most important fiber cash crop in the world.At present,The genome sequencing work has entered the agenda,and the construction of cotton high-resolution cytogenetic map by fluorescence in situ hybridization(FISH) is an important basic work in genome research.Molecular markers come from different chromosomes in genetic maps were as a media use to screening chromosome-specific BAC clones in the BAC library,then to them as a probe for fluorescence in situ hybridization to fully identify chromosomes or integrate the maps between cytology and genetic maps. This strategies was a large number of applications received by other crops.To speed up constructed high-resolution cytogenetic map of cotton.In this study,we firstly investigated on the work of chromosome-specific BAC clones for screening and identificating,and after the experimental demonstration has been to identify a set of diploid D-genome chromosome-specific BAC probe.On the basis of obtain two types of rDNA BAC clones,Groups of 7 diploid species distribution of rDNA loci are discussed.Application of 45S rDNA,5S rDNA and the D-genome BAC centromere region probes,build a high-resolution cytology map on the G.thurberi (D-genome) pachytene chromosomes,For the D-genome chromosome-specific BAC probe in pachytene on the final application and construction of D-genome high-resolution cytogenetic cotton laid the basis for the work.The results are as follows:●Make use of the chromosome-specific SSR marker NAU2440(Chr.3),NAU2353(Chr.10),BNL3103(Chr.25) come from the genetic linkage map(Han et al.2006),from Pima90 BAC library screening and obtain,respectively,425-D-2,429-J-7,189-I-4 BAC clone.The above BAC clones can produce chromosome-specific hybridization signals by FISH,therefore,BAC clones 425-D-2,429-J-7,189-I-4 can be used as the FISH probes to identify Chr.3,Chr.10,Chr.25 of the tetraploid cotton.●On the basis of access to a set of chromosome-specific BAC clones of the tetraploid cotton, Apply dual-color FISH,by the D-genome centromere region probe(BAC 150D24) to distinguish chromosome arm length,D-subgenome 13 chromosome-specific BAC clones were targeted in the diploid D-genome(G.thurberi)metaphase chromosome arm successfully,FISH hybridization results showed:D-subgenome 13 BAC probes in the corresponding chromosomes of D-genome chromosomes can have specific fluorescent signals.The D -genome chromosomes with FISH signals derived from the D-subgenome chromosome-specific BACs were proposed a new chromosome nomenclature,according to the homeologous relationship between D- subgenome and D -genome chromosomes.●13 D-subgenome of chromosome-specific clones have been analysed through the physical map and genetic map of the comparative mapping,Although some markers located in the middle of linkage group,however,cloning of the corresponding in situ hybridization showed that it located in end or near the end of the chromosomes.Suggest that the cotton genome distal of chromosomes are recombinant active section;Some markers located in end or near the end of linkage group,But FISH showed that it located in the middle of chromosomes;This situation may be caused by relatively low coverage rate of linkage group.13 probes of distribution physical have better consistency in the D-subgenome and D-genome chromosomes.It proves that does exist the majority of chromosome segment homology between D-subgenome and D-genome chromosomes.●Because rDNA is a highly repetitive sequence,it could be a marker for FISH to identify chromosome.We used 45S and 5S rDNA primers,screened four 45S rDNA BAC clones: 181-C-3,181-J-11,183-C-22,183-G-12;two 5S rDNA BAC clones:182-F-9,188-H-17,from the Pimag0 BAC library and verified through FISH.When sequencing the 45S rDNA BAC clones,obtain a 533bp DNA fragment.It compared with known sequences in the NCBI database. The results showed that:it reached 98%homology with the 26S sequence of tea tree,kapok tree,magnolia,lotus and some animals.●On the basis of obtain to cotton rDNA BAC clones,Selected representatives of their species for the study from A,B,C,D,E,F,G,7 genome of diploid cotton species,Explore the rDNA locis and the 45S and 5S of seven groups linear relationship.It is found that 45S signal have 3-4 pairs in seven groups of diploid cotton.But,signals' strength and size are different;G.incanum of E genome has two pairs of 5S signals,but the others only have a pair of 5S sigal.45S and 5S linear relationship:45S and 5S are collinearity in the A,C,D,F,G -genome.However,45S and 5S are non-collinearity in the two cotton species of B-genome and G.somalense/G,areysianum of E-genome.G.incanum of E genome has two pairs of 5S locis,the existence of a pair was separate and the other with 45S locis is collinearity.●The special rDNA loci in B,E,F genome we found could be play an important role in classification for cotton.Acquisition four types distribution pattern,according to evolution of rDNA speculated:In the diploid cotton species,distribution of non-collinear--- the two cotton species of B-genome and G somalense/G.areysianum of E-genome were the most primitive species,It towards the direction of the evolution of collinearity of 45S and 5S,During the Egenome -G.incanum,F-genome-G.longicalyx are a "turbulent" period in the entire diploid cotton species,may be 45S and 5S chromosomes from non-collinear to collinear process of the transition phase,eventually reach collinearity of 45S and 5S chromosomes---A,C,D,G genome. ●Further optimization of the cotton pachytene-FISH system.The first time in the D-genome pachytene chromosomes,make use of 45S,5S and centromere region repeats of BAC clones as probes,a high-resolution cytology map of D-genome was built initially.