Study On The Application Of Chromosome Microdissection And DNA Fiber-FISH In Cotton

Cotton is one of the main natural fiber crops in the world.Cotton genome sequencing has entered the substantive procedures,so it is urgent to accumulate basic data of cotton gene sequencing and to resovle critical techniques.There are rich secondary metabolites in cotton,such as tannins and gossypol,so it is difficult to make good chromosome slides.What's more,the cotton chromosomes are small and similar.The aim of this study was to explore and establish the system of DNA FISH,which may resolve the cotton genome sequencing completely in the end.At the same time,we explored the single chromosome microdissection,constructed a single chromosome library and accumulated basic data for the cotton genome sequencing,gene cloning,chromosome painting and even the evolution of the Gossypium.The followings are main results:1.Establishment of chromosome microdissection and microcloning system in cotton.After pre-hypotonicity, enzymolysis,post-hypotonicity and squashed with cover slide,the mitosis metaphase chromosomes slides suitable for cutting was obtained.Based on these,we established an efficient procedure of micro-dissecting a single chromosome.Amplified production of single chromosome were verified by Southern blotting,SSR primer amplification and fluorescence in situ hybridization,and a complete system of verification and analysis was formed.We established a highly efficient,fast,easy-to-use system of chromosome microdissection and microcloning in cotton for the first time.2.Studying on cotton chromosome painting.After carrying out chromosome painting between G.arboreura with six other cotton species,it was found that the diploid cotton G.arboreum has high homology with diploid cotton G.herbaceum.Compared with five other tetraploid cotton,it was found that G.arboreum had higher homology with G.hirsutum and G.barbadense,but had lower homology with G.tomentosum,G. darwinii,and G.mustelinum,the lowest is G.mustelinura,which illustrated that the theory of tetraploid cotton polyphyletic origin may be more believable.3.Constructing a single cotton chromosome library.We constructed a single chromosome library of the 5th chromosome with large satellite of G.arboreum Shixiya 1.The library consisted of approximately 37,000 recombinant clones.The size of the inserts varied from 500～1800bp with an average of 750bp.The construction of the 5th chromosome library of G.arboreum Shixiya 1 would facilitate the specific probe screening,genetic map construction,important gene cloning and accumulate of basic data for sequencing on the chromosome.4.Cloning RGA from a single cotton chromosome.Four nucleotide sequence P7,P12,P19 and P23 were obtained after amplified in second round LA-PCR pool of the 5th chromosome in G.arboreum Shixiya 1 with disease-resistant homologous primers P1 and P2 of rice.The blast results showed that the four sequences were the NBS-LRR-type resistant gene analog(RGA) in cotton.Clustering analysis indicated that the sequences of P7,P12,P19 were high homologous and in the same cluster,so we speculated they originated from the same gene cluster,but the P23 was in other cluster and homologous with RPS2 gene of Brassica nigra and RGA30 gene of Brassica napus.5.Establishing cotton FISH system.The cotton DNA-fiber was well extracted in cotton for the first time. An integrated cotton FISH system was formed based on the DNA fiber-FISH which was successfully combined with the mitosis metaphase chromosome FISH and pachytene chromosome FISH.The technique is so simple,rapid,low cost and easy-to-use that it is bound to provide powerful technique for cotton genome sequencing.6.Researching on application of cotton FISH systems.There are two-pair large signals on the mitosis metaphase chromosomes and pachytene chromosomes of G.arboretum Shixiya 1 hybridized with 45S rDNA probe.The signals look like noncontinuous beads on the DNA fiber,which consist of multi-copy and arrange tandemly.Fiber-FISH results with 45S rDNA probe showed that an average signal length is about 3～11μm in many tandem copy sequence(measure number,n=8),so we estimated the size of each copy to be approximately 11～30kb in cotton genome.There are dual signals on each end of the mitosis metaphase chromosomes and pachytene chromosomes hybridized with telomere DNA probe.The signals also look like non-continuous beads on the DNA fiber,the length is about 1～9μm,so we estimated the size is about 4～27kb.The signals almost covered the whole mitosis metaphase chromosomes and pachytene chromosomes of G.arboretum Shixiya 1 hybridized with gDNA probe,the euchromatin zone and heterochromatin zone were identified clearly on pachytene chromosomes,and the signals also look like non-continuous beads.Two BAC clones 150-D-24 and 182-F-9 in DNA BAC library of Pima90 were selected as probe to hybridize with mitosis metaphase chromosomes,pachytene chromosomes and DNA fiber of G.raimondii.The results indicate that the distance between the two signals of BAC clone is 0.21μm on mitosis metaphase chromosomes,3.8μm on pachytene chromosomes.The signals on DNA fibers are also non-continuous-like beads.The length of 150-D-24 is about 32.7μm and that of 182-F-9 is about 37.9μm. The distance of two BAC clone is 128.4μm.According to a stretching degree of approximately 3.27 kb/μm, the size of 150-D-24 is about 114kb,the size of 182-F-9 is about 107kb,and the distance between 150-D-24 and 182-F-9 is about 419.8kb.Due to accumulation and fracture of DNA fiber,the actual size may be larger.