The Genomic Constitution And Evolution Revealed By Fluresence In Situ Hybridization In Gossypium

The cotton is the most important fiber economic crop in the world.Gossypium contains 4 cultivated species and 46 wild species which fall into nine different cytotypes designated A, B,C,D,E,F,G;K and AD.Although such abundant germplasm resouses offer the number of practicable material for genetics studing,it makes the research work inconvenient contrarily which about proving interspecific relationship and evolutional rule of the Gossypium.Otherwise it is difficult to research the cotton in cytogenetics because of the large number and small size of the cotton chromosomes,as well as the absence of suitable cytogenetic markers,identification of individual chromosomes based on their morphology is almost impossible.Fluorescence in situ hybridization(FISH) got the foregoing problem to be readily sovled,furthmore it turn into an important facility what with possessing many trait such as fast-speed detecting,high-sensitivity,wide-applicable and so on in molecular cytogenetics. This paper mainly presented the genomic constitution of cultivated Gossypium arboreum, interspecific relationship between the allotetraploid Gossypium hirsutum accession and the wild diploid specis through the FISH derivative technology:BAC-FISH;genomic in situ hybridization(GISH) and Multi-color GISH(McGISH).The research finding is provided with scientific significance toward clarifying the genomic constitution,evolution and heredity improvement in cotton.The main results were as follows:1.The G arboreum chromosome karyotype by BAC-FISH method:Based on previous research,twenty bacterial artificial chromosome(BAC) clones that could produce bright signals and no or very low fluorescence in situ hybridization(FISH) background were identified from G arboreum cv.JLZM,and G.hirsutum acc.TM-1 and 0-613-2R. Combining with 45S and 5S rDNA,a 22-probe cocktail that could identify all 26 G arboreum chromosomes simultaneously was developed.According to their homology with tetraploid cotton,the G.arboreum chromosomes were designated as A1-A13,and a standard karyotype analysis of G arboreum was presented.The current descending order of the metaphase lengths is A5(including the rDNA section),A7(including the rDNA section), A3,A4,A6,A1,A12,A2,A10,A8,A13,A11,and A9.These results demonstrated an application for multiple BAC-FISH in cotton cytogenetic studies and a technique to overcome the problem of simultaneous chromosome recognition.2.Establishment of the system for the Multi-color GISH technique:Increased stringency condition was employed to establish the GISH technique for cotton by optimizing ratios of block DNA to probe,the formation concentration in wash solution, wash temperature and duration in wash solution,after DNA hybridization using mitotic metaphase chromosome of somatic cell of upland cotton standard line TM-1(G.hirsutum L.) as target,genomic DNA form TM-1 and G.sturticmum Willis originated from Australia as bocking DNA and labbled probe,respectively.The results showed that the proper stringency conditions were the combination of four factors,including the ratios of blocking DNA to labbled probe being 100:1,60%of formamide wash solution,43℃of wash temperature and duration of 13 min for wash,ender which there is just no probe signal on the target chromosome.Under the proper stringency conditions,GISH technique established was tested to discern chromosome of G.sturtianum(C₁ genome) from intergenomic triploid F₁(2n=3X=A_tD_tC₁=39) between upland cotton and G.sturtianum. The results indicated that every donor chromosome of the two species showed different color and were readily distinguishes.So it is proposed that the GISH technique establishes was feasible for chromosome identification in intergenomic cotton hybrid.Bases on the above results,the Multi-color GISH was developed using two specis DNA from G.stuitianum and G herbaceum L.var.africanum(A₁ genome) as labeled probes simultaneously and DNA from G.davidsonii(D_3-d genome) as blocking DNA,to simultaneouely discriminate three genome of above intergenomic hybrid.The results also indicated that the three genome,A_t,D_t and C₁,each set of chromosome were vividly recognized in different color repetitively.The power of the Multi-color GISH has been proven in analysis hexaploid hybrid(kindly provided by CSIOR of Australia) and each of three geomes,A_t,D_t and G₂,of chromosomes display one color.It is believed that the powerful Multi-color GISH technique established in the research could be applied extensively in analysis component in polyploidy and precisely identification of alien chromosome in the recipient progenies,which will greatly assist to transfer much more economically important traits from wild species into upland cotto in the future.3.Studying on the evolution of the cotton species by GISH:To research the diploid progenitor of D subgenome of the G hirsutum,using mitotic metaphase chromosome of upland cotton standard line TM-1(G hirsutum L.)as target,11 diploid wild cotton species as probes,they are respectively G.davidsonii,G raimondii,G thurberi,G gossypiodies,G armourianum,G.trilobum,G laxum,G harknessii,G schwendimanii,G lobatum and G.klotzschianum.The GISH results showed that G trilobum had the stongest hybrid signal and the largest coverage with chromosomes.It indicated G trilobum was the highest homology with the D subgenome of the G hirsutum and provided new evidence in the cotton origin,evolution research.Moreover,G anomalum(B genome),G sturtianum(C genome),G somalense(E genome),G longicalyx(F genome) and G australe(G genome) were used as probes to study the genetic relationship between the diploid wild species and cultivated tetraploid G.hirsutum.The conculsion is B,C,E,F and G genome had a nearly relationship with A subgenome of the G hirsutum than its D subgenome.Meanwhile there presented the strong signals from G.anomalum,G sturtianum,G somalense,G longicalyx but G australe generated the weak.We also found it may be some different from the others that the repeated sequence G.sturtianum including as it's the unique probe appearing the red signal on the D subgenome of the G.hirsutum.