Individual Chromosome Cytogenetic Maps And Identification Of The Subgenomes And Oligo Sequences In Cotton Based On FISH

Cotton(Gossypium) is the most important natural fiber crop in the world. Two diploid A, D genome ancestral species were combined to form allotetraploid species through a polyploidization event. As a well-studied polyploidy species, cotton is a model system for studying polyploidization. At present, the cotton genome sequencing work is ongoing in full swing, and partially cotton genome draft sequences have been published. But because of the complexity of cotton genomes, access to high-quality assembly sequence still has a long way to go. Therefore, it is necessary to carry out the relevant basic research work on cotton genome research. Cytogenetic map can integrate genetic loci into physical location of chromosome, which has great potential to guild the assembly of genome sequence. Fluorescence in situ hybridization(FISH) now has been widely used to construct the cytogenetic map. In this study, the cytogenetic maps of homoeologous chromosomes Ah01 and Dh01 of G. hirsutum were constructed by FISH using marker-anchored bacterial artificial chromosomes(BACs). By integration of genetic loci into sequence map, we assessed the quality of the draft genome of G. hirsutum preliminarily. At the same time, we tried the Oligo-FISH in cotton for the first time, which based on the genome sequence data.The main results are as follows:1. Screening of chromosome-specific BAC clones. The chromosome-specific BAC clones of chromosomes Ah01 and Dh01 were screened using SSR markers selected from a whole genome marker map(WGMM). Forty-seven SSR markers were used to screen G. herbaceum var. africenum BAC library and G. barbadense Pima 90-53 BAC library. In total, we got 84 positive BAC clones. Fifteen BAC clones anchored according to 12 SSR markers were mapped on chromosomes Ah01 and(or) Dh01 by FISH. Based on dual-FISH, the order of the two adjacent BACs was determined along the chromosome.2. Construction of the cytogeneric maps of chromosomes Ah01 and Dh01. Relative positons of every BAC clone on chromosome were measured with chromosome analysis software. Then the cytogenetic maps of G. hirsutum chromosomes Ah01 and Dh01 were constructed, including 12 BAC clones respectively.3. Integration and analysis of different maps. After converting the location information of genetic map and sequence maps to relative map positon(RMP), we integrated the two cyotogenetic maps with the genetic map and genome sequence maps respectively.(1) The integration of cytogenetic and genetic maps showed that the orders of most SSR markers tested are colinear with corresponding BAC FISH signals. One exception was found, that is, the genetic distance between markers NAU3433 and BNL2921 is 11.2% of total genetic distance of chromosomes Ah01 and Dh01 in the genetic map, but the physical distances between these two markers are 47% and 59.4% of total length of the chromosomes Ah01 and Dh01 respectively.(2) By integration of cyotogenetic maps with sequence maps of two chromosomes, we inferred the locations of two scaffolds, that is, scaffold183_A01 should be mapped to 3.4%~9.6%(90268610~96488204 bp) of chromosome Ah01, and scaffold3710_D01 should be mapped to 6145600~9387374 bp of chromosome Dh01.(3) The concordant orders and RMP were revealed by comparison sequence map with the physical map based on FISH.(4) During the process of sequence assembly, some homologous sequences were removed as repeats, and only partial sequences information with homology were kept on one of the two homoeologous chromosomes. Only 4 of 11 mutual SSRs on the two cyotogenetic maps of Ah01 and Dh01 chromosomes were mapped on the two corresponding sequences maps, the other 7 were mapped on one of the two homoeologous chromosomes respectively.(5) The coverage of the two chromosome sequence maps was estimated based on the locations of BAC clones 378J07 and 400L15, which were mapped on 8.0% and 96% of the cytogenetic map, the sequence locations of corresponding SSR markers were 3.4% and 97.8% from the two distal ends.4. Perfection of the upland cotton pachytene chromosome preparation technique. We confirmed the main cytological characteristics of pollen mother cells(PMC) in the pachytene phase to ensure the efficient selection of materials; The appropriate time of digestion with higher-concentration mixed enzyme solution(4% cellulose+1% pectolyase+1% helicase, 3 h) was applied; After crushing in 60% acetic acid, baking and acid soluting(50 ℃, 1?2 min) are necessary for ideal pachyteen chromosome preparation.5. Bioinformatic analysis of a repeats-enched BAC clone. During the screening of the 1th chromosome-specific BACs from the BAC library of G. herbaceum var. africanum, a genome-specific BAC clone 57I23 was obtained. Given its specificity, we sequenced the BAC clone and analysized its composition. One hundred and twenty nine repetitive elements, which account for more than 62% of the assembled BAC sequence, were identified from the BAC sequences. A type of Gypsy-48_GR-I-like LTR-RT was the key element in the BAC 57I23, which also can be used to partly explain the size gap between A and D genome. By comparing FISH with BLASTN results, we infered the assembly of the identified repetitive sequences in AD1-NBI draft genome has better matchup on their chromosome belonging.6. According to the publicated cotton genome sequence, we designed the oligonucleotides(oligos)library, obtained the oligo probe based on emulsion PCR, T7 in vitro transcription, and reverse transcription. FISH verification was performed using corresponding cotton genome DNA as target. Cotton Oligo-FISH technology system was set up for the first time, which provided a new approach for further dissection of cotton genome.