Studies On Genetic Diversity Of Gossypium And Interspecific Hybridization Among Four Cultivated Cotton Species

Gossypium species represent a vast resource of genetic diversity for the improvement of cultivated cotton.Gossypium comprises of approximately 45 diploid and five allopolyploid species.The diploid Gossypium species fall into eight groups or genomes,designated A through G,and K.There are four different species having been independently domesticated, including allopolyploids,G.hirsutum L.and G.barbadense L.,and the diploids,G.herbaceum L.and G.arboreum L.and others are 47 wild species.Within these cultivated and wild cotton species,there are many favorable characteristics,which may be available to our development of superior cultivars.For several years allopolyploid Gossypium have been the subject of evolutionary investigations into the genomic mysteries of polyploidy.Despite many intensive study of the tetraploid species of Gossypium,the phylogenetic relationships among these species have remained elusive.Ever since the discovery that tetraploid Gossypium species contain two different genomes,the question,which of the modern species of A-genome and D-genome diploids best serve as models of the progenitor genome donors has stimulated discussion for many years.So research on genetic diversity and genomic relationships among diploid and tetraploid cotton species is necessary for further utilization of extant germplasms and better understanding of tetraploid evolution.In order to better understand the genetic distance and correlation among the Gossypium spp.and to unravel the genetic basis of our own developed quadri-specific hybrid,the present research was undertaken.The brief description of our findings are as follows:1.Genetic diversity of 24 species in Gossypium and its relatives was assessed using Random Amplified Polymorphic DNA(RAPD)and Simple Sequence Repeats(SSRs)for the purpose of relative identification and pedigree classification at DNA level.Respectively 26 and 49 out of 40 and 120 RAPD and SSR primers gave high polymorphism.Jaceard's genetic similarity coefficients were calculated and dendrograms were constructed by the Unweighted Pair Group Method of Arithmetic average(UPGMA)using the NTSYS-pe Version 2.00.The results showed that there was evident genetic diversity among 25 species.Similarity coefficients by RAPD and SSR ranged from 0.69 to 0.96 and from 0.57 to 0.97 respectively.Similarity coefficients comparing results between tetraploid and A,D genome species indicated that G. raimondii might be the closest living relative of the ancestral D-genome donor of tetraploid species.Also the A-genome donor was found to be much similar to the present-day G. herbaceum and G.arboreum,which are phylogenetically equidistant from extant tetraploid cotton species.Moreover,it is evident from the similarity coefficients results that ancient tetraploid cotton spp.were formed by hybridizing and chromosome doubling between them, then different tetraploid cotton species appeared by further geographical and genetic isolation and separating differentiation.The results also showed that SSRs could be an ideal means for the identification of the genetic diversity and relationship of cotton resources at the genomic level.2.In order to further unravel the genomic origin and organization of tetraploid cotton and to provide more direct evidence for the evolution of tetraploid species,we also applied GISH technique using the putative parental A-Genome(G.arboreum or G.herbaceum)and D-Genome(G.raimondii)of tetraploid cotton as a probe or a block respectively,AD-Genome (G.hirsutum and G.barbadense)as a target.The result showed that GISH images gave clear discrimination of two sets of 26 chromosomes and the recombination of two genomes also existed.There were no evident signal differences when using G.herbaceum and G.arboreum as probe,so both of G.arboreum and G.herbaceum are all the A-genome donor.Other D genome species such as G.klotzschianum and G.davidsonii were also used as a probe,only getting less than 26 weak signals than using G.raimondii,so G.raimondii is the sole D-genome donor of tetraploid cotton species.The present work confirms the hybrid origin of tetraploid cotton and supports the hypothesis that the donors of the parental genomes are G. arboreum or G.herbaceum and G.raimondii.GISH was also tested as a superior method for distinguishing between these two putative parental genomes.3.The quadrispecific hybrid F₁(G.arboreum,G.herbaccum,G.hirsutum and G. barbadense),was obtained by crossing the doubled(G.arboreum×G.herbaccum)F₁ with(G. hirsutum×G.barbadense)F₁.The hybrid and its four parents were analysed using SSR markers for its identification and to study the evolutionary mechanisms following hybridization and polyploidization.The results showed that there were 30 pairs of polymorphism primers for total 45 SSR pairs,and 99 polymorphism bands were obtained,with an average number of 3.3 for each SSR locus.The quadrispecific hybrid F₁ had four parents bands in which,20.8% bands come from G.arboreum,22.2%bands from G.herbaccum,15.3%bands from G. hirsutum,and 13.9%from G.barbadense.While the remaining 27.8%bands which did not belong to any of the parent in F₁ hybrid were observed.The results showed that the hybrid possessed the genetic materials of its four parents and recombinations of different parents took place following hybridization and polyploidization.It also revealed that SSR molecular marker was a feasible method to study the genetic relationship and identification in interspecific cotton hybrid.4.The quadri-specific hybrid was high sterile,so it was preserved for 15 years by grafting and self-crossed and back-crossed every year.The characteristic of chromosome behavior,the chromosome configuration during meiosis and the pollen activity were also studied in this research.The results showed that fertile and poor vigor pollens were increasing,but sterile pollens were decreasing following plant preservation,and the fertility of this hybrid was gradually recovered.Some seeds were obtained by backcrossing after 10 years and a few self-crossed seeds were also produced in recent years.The chromosome configuration of this hybrid was 2n=52=4.92â… +14.62â…¡+2.29â…¢+1.56â…£+0.71â…¤+0.19â…¥after 15 years,while 2n=52=8.4â… +8.1â…¡+5.7â…¢+1.9â…£+0.6â…¤15 years ago.It was evident that bivalents increased along with univalents and multivalents decreasing.In conclusion,extending the growth years by grafting was helpful to harmonize the relationship of different chromosome groups from different parents and to balance physiological metabolization,which can lead to the partial fertility recovering of the quadrispecific hybrid.