The Construction Of Genetic Map And Molecular Cytogenetic Researches In Allotetraploid Cotton

The construction of genetic map based on double haploid (DH) population is in favor of thecontinuous construction of map and get the integrated map. More detailed and correctedresults about common genotype can be achieved because it can be easily self-pollinated toproduce large numbers of progeny which facilitates the communication performed bydifferent researchers. Pervious researches have constructed a genetic map for allotetraploidcotton based on a DH population which derived from the crossing of G. hirsutum L.'TM-1' and G. barbadense L. 'Hai7124'. It was developed by means of vSg, virescentlymarked semigamy line in Sea Island cotton which was characterized by a cytologicalmechanism for developing haploids. But this population is too small that only contains 58individual plants, and the genetic map has lots of gaps and needs to fill by new markers.According to this, we developed a new DH population that contains 73 plants based on thesame strategy, and then a new genetic map was constructed. This map comprised 444simple-sequence repeats (SSR) markers mapped to 40 linkage groups (LGs) with anaverage distance of 7.35 cM between markers, covering 3262.9 cM. Twenty-nine linkagegroups were assigned to all 19 identified chromosomes (chromosomes 1-6, 9-12, 14-18,20, 22, 23, 25 and 26) ,10 to A or D subgenomes (LGA01, LGA02, LGA03, LGD02,LGD03 and LGD08) , and 1 was unassigned (LGU01).To compare mapping efficiency between different populations, and facilitate thecomparative genome research, a genetic map constructed based on a backcross populationwhich developed from the same cross (BC) of G. hirsutum L. 'TM-1' and G. barbadense L.'Hai7124', was selected to compared with pervious DH population map. The results shownthat fairly good collinearity of marker order was observed along most of the chromosomesor linkage groups. Significant differences in recombination between maps were observed atthe chromosomal and genomic level and possible reasons were discussed. These resultsindicate that the BC₁ population of 140 individuals has higher capacity in estimating maximum recombination fraction than relatively smaller DH population. Map comparisonand combined data provided an essential basis for further mapping of interested genes andQTLs and for studies of compare mapping between defferent Gossypium species.Significant progress has been made in the construction of genetic maps in the tetraploidcotton Gossypium hirsutum, in that several genetic maps including several thousands ofmarkers have been constructed. However, six linkage groups have still not been assigned tospecific chromosomes, which is a hindrance for integrated genetic map construction. In thepresent research, specific bacterial artificial chromosome (BAC) clones for these six LGswere identified by screening the BAC library using linkage group-specific SSR markers.Based on a meiotic fluorescence in situ hybridization (FISH) methods, we assigned the sixLGs to the corresponding chromosomes. During this study, a set of relative techniquesabout it was developed and most of which were first published.First, a high throughput approach for BAC-DNA isolation was developed to permitPCR-based screening of a cotton BAC for SSR genetic markers. We optimized bacterialgrowth in 96 deep-well plates and the procedure of BAC-DNA isolation. Due to utilizing aprogrammable robotic liquid handing system to process liquid pipetting, 960 BACminipreps can be completed in 8 h. A two-dimensional BAC screening strategy andmultiplexed amplification were also used to increase the efficiency of SSR markersscreening. The approach is high throughput, accurate and no sample cross-contamination.Second, protocols of BAC-FISH in cotton mitotic and meiotic cells were introduced inthis paper. The clear signals were detected in most metaphase cells derived from differentsliders, and it indicated that the results were correct and repeatable performing by thisprotocol.Finally, linkage group-specific BACs constructed in G. hirsutum acc. TM-1 for these sixLGs (A01, A02, A03, D02, D03 and D08) were identified by screening the BAC libraryusing linkage group-specific SSR markers. These BACs were hybridized to tentranslocation heterozygotes of G. hirsutumL, as BAC-FISH probes, which allowed us toassign these six LGs A01, A02, A03, D02, D03, and D08 to chromosomes 13, 8, 11, 21, 24,and 19, respectively. Therefore, the 13 homeologous chromosome pairs have beenestablished, and we have proposed a new chromosome nomenclature for allotetraploidcotton. The A subgenome chromosomes were assigned as A1 through A13, the same as theformer assignment, but the D subgenome chromosomes were reassigned as D1 throughD13 to comply with the order of the A homeology. Chromosome identification is the base of karyotype analysis, and also critical for futureadvances in cytogenetics. However, because of the large number and small size of thechromosomes, as well as the absence of suitable cytogenetic markers like bands, routineand unambiguous identification of individual chromosomes based on their morphology isalmost impossible in cotton. BAC-FISH has shown the powerful characteristic for plantchromosome identification, especially for those species with large number of smallchromosomes. Based on previous research, one set of tetraploid cotton Gossypium hirsutum26 chromosome-specific BAC clones were developed using the chromosome-specific SSRmarkers, which selected from a cotton genetic map, to screen another BAC libraryconstructed by a restorer line 0-613-2R of a cytoplasmic male sterility. The FISH signalsused as cytogentic markers enabled identification of all 26 cotton mitotic metaphasechromosomes. It also the first one complete set of chromosome-specific BACs forpolyploid species in plant. In the mean time, BAC-FISH provided a cyto-genomic approachto correlate molecular maps to cytological maps or chromosomes. By evaluating therelative positions of FISH markers through comparisons of physical positions of markers inthe chromosomes and linkage maps, we found that the FISH markers tend toward locatingnear the ends of chromosomes, although their linkage markers were defined interiorposition in respective linkage group. It may indicate that the recombination active regionsof cotton chromosomes are primarily in the distal segment. FISHing of BAC clones isolatedby markers from the ends of linkage groups has been used to evaluate grossly the degreewhether a linkage map provides good coverage. Here, two BACs 47N15 and 75F07selected with end markers from linkage A6 were physically located the end of thechromosome. It indicates that the physical coverage of the chromosome is nearly touchingthe ends for linkage group A6.Sequence-specific primers Y2232 were designed from an EST which was relative withcotton heterosis, and a BAC clone, 36D03, was identified by screening library. FISHanalysis showed that BAC 36D03 is located on long arm of chromosome A7 consistentwith that of genetic mapping. Because the genome clones are usually more than 100kb,they are much easier to be detected than using small single-copy sequence directly in FISH,and present a powerful tool and new method for target sequence physical location.Differential DNA synteny existed in homeologous chromosomes in allotetraploid cottonwere detected by BAC-FISH. Results shown that the two alleles produced by one SSRprimer were most likely derived from the homeologous chromosomes. Another finding shown that these does exist large homologous segment between homeologous chromosomepairs even though standing long period evolution. All these provided the new evidences forthe homeologous relationship in allotetraploid cotton and provide new chance for theresearches of cotton polyploid and evolution.