Germplasm and Genetic Stocks

Drought in conjunction with high temperature is an important environmental constraint to cotton production. Development of cotton varieties with increased fitness to adverse environmental conditions has been proposed as economical and durable strategy for getting reliable yields. Molecular marker technology holds great promise for crop improvement exercise particularly in cotton where repeated cycles of directional selection for high yield and earliness might have caused loss of genetic diversity. In the present study, 11 SSR primers were used to estimate genetic divergence for water stress tolerance among 22 cotton genotypes. These genotypes were previously selected on the basis of drought induced morpho-physiological responses. Two SSR primers (JESPR-247 and JESPR-291) out of 11 were able to discriminate cotton varieties thus revealing informative primers to be 18%. Total detected loci were 33 with an average of 3 loci per primer. The number of amplified fragments detected at each locus ranged from one (JESPR-284) to six (JESSPR-302). Allelic diversity found in the experimental material was low i.e. 0.1. In this study, genetic similarity coefficients ranged from 0.87 to 1.00 where 17 varieties possessed similarity coefficient 1.00, thus showing that major portion of the genome was similar due to related genetic background among genotypes. The cluster analysis arranged 21 genotypes in two main groups whilst CIM-109 remained ungrouped being divergent from remaining varieties. Clustering of 17 genotypes in one major cluster confirmed similarity coefficients to be 1.00. Overall,low genetic diversity was found in 22 cotton genotypes. It had been suggested to apply more polymorphic SSR markers to explore the workable genetic variation among the screened cotton genotypes.

The anatomical structure of integuments of seed of wild and cultivated representatives of cotton species G.herbaceum L., G.arboreum L., G.hirsutum L. and G.barbadense L. it has been studied. This is study have purpose determine a traits hardness for spermoderm, which depend on early. The hardness for spermoderm depends on common thickness of spermoderm and cause of dimensions cell external epidermis and height cell palisade. Also the hardness depends on correlation between mechanical layer and parenchyma layer, degree pigmentation of integuments and hardness of cell wall. The thickness of spermoderm in mature age it is not always index of earliness, but more depend on ripe and duration increase cells of integuments.

Salinity is one of the major environmental factors that limit the worldwide productivity and distribution of crops. In plants one of the main mechanisms to overcome unfavorable environmental factors, such as water deficit and high salinity, is the accumulation of organic compounds of low molecular weight known as compatible solutes. Which biosynthetic pathways one of them is glycinebetaine have been characterized in higher plants and in microorganisms. In Uzbekistan salinity is one of the major problem for plant breeders. Taking the above into consideration, development of salt tolerant crops in Uzbekistan is of great importance and development of local commercial salt tolerant crop varieties prioritized. Moreover, improved tolerance of glycinebetaine-biosynthetic transgenic plants (cotton, maize and wheat) have been observed only to water stress. There are no reports about improved tolerance of GB transgenic plants (mentioned above) to salinity. Since a number of salt tolerance genes have been characterized in some plant species. We designed degenerative primers for plant salt tolerance genes (SOS1, SOS 3 and HKT 1) to identify and characterize their homologs in genomes of cotton species. Moreover, genetic construct of modified codA gene from A.globiformis is under development. This construct will be used for development of local salt tolerant crop varieties.

Despite intensive enough research of species of genus Gossypim L. still there are disputable questions of their systematization and phylogeny, a number of representatives are insufficiently known and especially Australian cotton group (subgenus Sturtia Tod.). Methods of obtainment of different fructifying hybrids on the basis of their genetic interrelations and degree of phylogenetic relationship are not developed. Investigations showed that for the first time it was cleared out the phylogenetic relationship between Australian (C genome) cotton species and degree of genetic relationship with the American (D genome) cotton by using the methods of comparative morphology, hybridizations and cytogenetics. It was made ware accurate systematically condition of some representatives of species G.sturtianum, confirmed independence of species G.sturtianum var. nandewarense selected to the species rank as G.nandewarense. On the ground of distinctions it was offered to bring out species G.bickii into separate subsection. In the result of studying of inherit of morphological traits of some intragenomic cotton hybrids F2 revealed direct correlational dependence of traits as color nectary of leaf blades with the polytocous. Worked out the schemes of phylogenetic relationship of the Australian cotton species with the representatives of the American cotton species. Worked out schemes make easy selection of initial material and provide effectiveness for obtaining new hybrids forms with setted traits. Obtained hybrids forms with the useful economic traits vitally full filled cotton genepool and also it is valuable initial material for further investigations of genus Gossypium L species in the field of systematics genetics, cytology and practical breeding.

A binary plasmid vector harboring Bacillus thuringiensis Cry 9C gene was introduced into an elite cotton cultivar Simian-3 by Agrobacterium tumefaciens -mediated transformation for control of Spodoptera litura. Integration and expression of the Cry 9C genes in three transgenic lines were confirmed by polymerase chain reaction (PCR) and reverse transcription-polymerase chain reaction (RT-PCR). Among these transgenic lines, line 16 (L-16) with normal phenotypes and higher expression level were selected by ELISA and subjected to insect bioassay. The ELISA data revealed that the expression level of Cry 9C in the T1 population ( L-16) ranged from 29-45 μg/g fresh leaf. The insect bioassay showed that transgenic S3-35S::Cry9C cotton plants exhibited moderate toxicity to Heliothis armigera but strong toxicity to Spodoptera litura compared to the transgenic plants with Cry 1Ac gene. We pyramided Cry 9C gene with Cry 2A or Cry 1Ac by crossing two parents plants with single Bt gene each. The Cry 9C protein expression in the F1 progeny has a similar level as the parent plants. The higher heritability of Bt gene between parents and progeny made it possible to pyramid different Bt genes by sexual crossing. Progeny from Cry 9C ×Cry 2A and Cry 9C ×Cry 1Ac exhibited higher resistance to Spodoptera litura, which is the most destructive insect to the Cry1Ac Bt cotton in most cotton cultivation region of China. These transgenic plants may be used for field test of resistance management strategies involving gene pyramiding and serve as novel insect-resistant resources in cotton breeding.

Pakistan is an agricultural country and cotton is one of the major crop playing a pivotal role in boosting the national economy. Both biotic and abiotic stresses affect cotton crop which results in severe yield losses. Cotton leaf curl virus and heat stress are the major threats which limit cotton production in Pakistan. To tackle both these threats a breeding programme was initiated to develop cotton varieties which can perform well even in the prevalence of both these calamities. A special programme was designed to select parental lines which can tolerate cotton leaf curl virus and high temperature as well. During the selection procedure intensive care was taken regarding fiber quality of these parental lines. For the development of virus tolerant cultivars hot spot areas were chosen for the evaluation of promising material. For heat stress tolerance boll retention percentage and CMT% was considered as selection criteria. The genotype FH-142 performed very well throughout Pakistan both in terms of heat stress and virus tolerance and already approved for general cultivation in Punjab covering about 25% of the cultivated area in Punjab. Other strains like FH-Lalazar and FH-312 is under the process of approval. It is expected that in future more improved germplasm will be available which will enhance the cotton productivity in Pakistan.

Assessing genetic diversity of Gossypium hirsutum L. cultivars used in breeding contributed to studying genetic relationships among diverse cultivars, and detecting SSR markers associated with agronomic traits contributed to molecular assisted selection in breeding, both of which can improve the efficiency of traditional breeding. In this study, 172 upland cotton cultivars used in recent years of breeding practice were genotyped using 74 microsatellite markers. Genetic cluster and population structure were estimated using NTSYSpc-2.20 and Structure2.3.4 software, respectively. Association analysis between SSR markers and 3 agronomic traits were performed using TASSEL 2.1（general linear model, GLM）program. The results showed that 74 SSR markers revealed 246 alleles belonging to 148 loci, ranging from 2 to 7 with the average of 3.32 alleles per SSR marker. The polymorphic information content（PIC）for per marker is 0.0281—0.3733,with the average of 0.2730. The genetic similarity ranged from 0.2816—1,with the mean of 0.5369. The cluster analysis showed that all the accessions could be divided into twelve subgroups, which was in agreement with pedigree but not with origin. No significant correlation between geographic distance and genetic distance was observed. Low levels of genetic diversity for upland cotton in China were observed, with an average genetic similarity of 0.5369. However, some materials from America and Xinjiang region of China showed abundant genetic variation, which implying the necessarity of strengthening the exchange of gemplasms between different regions and expanding the genetic basis of upland cotton of China by introducing new resources from foreign countries. Structure analysis divided the whole cotton panel into three subpopulation. Association analysis detected 30 SSR loci associated with boll weight, lint, and Verticilium wilt resistance, respectively. These results have provided valuable information for the association mapping of important agronomic traits, as well as for the breeding and exploit of new cotton germplasms. Key words: upland cotton; associate analysis; SSR; genetic diversity; population structure

The diploid Asiatic cotton is a fiber-producing species that shows a reduced level of genome complexity in comparison to the allotetraploid cotton. The fuzzless mutant is of great applicable value for identification and characterization of the genes related to fiber development in cotton. The filter arrays of cDNAs were performed to identify the differentiation expressed genes between the wild-type DPL971 and its fuzz mutant line DPL972 in Gossypium arboreumL. Some genes related to the initiation and development of the cotton fiber were isolated, and part of them have been verified by RT-PCR , QRT-PCR, immunolocalization , Southern blotting and Northern blotting techniques, And the induction of cotton ovule culture fibre branching by translate the fiber trichome genes were carried out in this study. The main results as following: 1. Some genes related to fiber initiation and development in Gossypium arboreum were identified, and seven of them, KAK(DR461366), MYB5(ES812048), TTG1(ES811600), MYB23(DR453866), CSLD3(DR459646), RHD2(DR461821), ZWI(ES791383), were highly homologous to the Arabodopsis proteins which played important roles in the initiation of the trichomes development in Arabidopsis. The 5 genes of MYB23,MYB5, TTG1, CSLD3, and RHD2had significantly different expressions in +3DPA ovules between the mutant and its wild line. 2. Two adenylyl cyclase-associated protein (CAP) homologs from wild-type and its mutant (DPL972) isolated. The Cap genes contain nine introns and an open reading frame (ORF) encoding a 471 amino acid protein. The expression of GaCAP reached the highest level in both wild-type and mutant fibers during 1 to 4 d postanthesis (DPA), then gradually declined in the mutant after 4 DPA. A polar substitution was observed at a conserved position between wild-type and mutant. This suggested that GaCAP may play a functional role during early stages of fiber development. 3. The homologs GaMYB23(a homolog of GL1), GaDEL65(a homolog of GL3),GaTTG1, GaCPC and GaTRY were identified in Gossypium arboreum. In situ analysis showed that GaMYB23, GaGL2, GaDEL65, and GaTRY were predominantly expressed in fuzz fiber, but GaTRY proteins were primarily found in undeveloped epidermal cells. G. arboretum fuzzless mutant with consistently high level GaMYB23 transcript has lost the detectable GaMYB23 promoter of GaGL2 complex, corresponding to sharply reduced transcription of GaGL2. 4. The nuclear DNA content was constant in fuzz, whereas a limited and reversible change occurred in lint after initiation. Gossypeum arboreum BRANCHLESS TRICHOMES(GaBLT) was not transcribed in fibres. The homologue of STICHEL(STI), which is essential for trichome branching, was a pseudogene in Gossypium. Targeted expression of GaBLT, Arabidopsis STI, and the cytokinesis-repressing GaSIAMESE in G. hirsutum fibre cells cultured in vitro resulted in branching. The highly elongated single-celled cotton fiber was similar to the Arabidopsis trichome. Some key Arabidopsis trichome branching genes in Gossypium arboreum were identificated and characterized here. These findings not only lay a base for the initiation of the fiber in diploid cotton, but also deepened our understanding of the molecular basis of cotton fiber development.

Molecular analysis of genetic diversity and population structure of 288 Gossypium barbadense L. worldwide cotton varieties from Uzbek cotton germplasm collection was done by using 750 SSR primer pairs. Among the utilized SSRs 108 (14.4%) were informative and produced 302 polymorphic loci. PIC values of SSR markers varied from 0.02 to 0.65 with an average of 0.27 (SD=0.17) and could discriminate between all Pima cotton varieties. The minimum number of loci was 2 and maximum – 5 with an average 2, 8 loci per marker. Presence of minor alleles (MAF) was detected in 21% (41) of loci. Average heterozygosity (H) of SSRs was H=0,31 (SD=0,2) with maximum and minimum value of Н=0,02 and Н=0,72 respectively. Phylogenetic tree were generated by UPGMA and revealed genetic distances (GD) among 288 Pima cotton varieties which varied from GD=0.01 to GD=0.65 with an average of GD=0.2. The lowest genetic similarity (0,76±0,13) was observed in African cotton varieties whereas the highest genetic similarity observed in Uzbek and US varieties (0,81±0,11 and 0,81±0,10 respectively). Analysis of population structure identified two distinct subpopulations according to Q-matrix generated by STRUCTURE software. A highest genetic diversity (0,35) was identified inside the first subpopulation, consisting of 16 Pima varieties. In the second subpopulation, presented by 272 Pima varieties, genetic diversity was 0,15. Genetic variation in the first and the second subpopulation was 32,8% and 23,6% respectively. The Fst value from AMOVA between two subpopulations was 0,58 (р≤0,0001). Our further studies aimed to investigate genome wide linkage disequilibrium and association mapping of cotton fiber traits among germplasm collection of Pima cotton varieties.

A BC1 population containing 115 individuals from a cross between Gossypium hirsutum cv. CCRI8 and G. barbadense cv. Pima 90-53 was established and 519 SSR markers, two conserved intron scanning primers (CISP) and transcript-derived fragments (TDF) amplified from 156 Apo I/Taq I selective primer combinations were used to construct a genetic linkage map. The map consisted of 579 markers distributed on 56 linkage groups. Accounting for 83.4% of the cotton genome, it covered 4,168.72 cM, with an average distance of 7.19 cM between markers. Based on this newly constructed map of tetraploid cotton, we performed quantitative trait loci (QTL) mapping of fiber quality traits from the BC1 and its derived BC1F2 lines. A total of 46 fiber quality QTL were detected on 17 chromosomes, explaining 7.72% to 23.73% of the phenotypic variation. The Pima 90-53 offered 15 QTL alleles with positive additive effects and four with negative additive effects for fiber quality traits. Of these, the qFS9-1 and qFS9-2 explaining 15.71% and 14.42% of the phenotypic variation were detected in the common interval from NAU1092a to NAU6101b in BC1 population and its derived BC1F2 lines, grown at Baoding suburb in 2007 and Xinji suburb in 2008. In order to better utilize fiber strength of Pima 90-53, the stable QTL of fiber strength was validated by multi-populations, multi-generations, multi-sites and multi-years. Three populations (five generations) were employed to validate QTL for fiber strength in cotton grown at 4 years representing six different environments. The first one comprising 131 BC1F2 population developed by backcrossing with CCRI 8 and two its derived family lines was grown at Baoding suburb in 2010, Qingxian county and Guangzong county in 2011, Qingxian county and Nangong county in 2012. The second and third, including 176 BC1 population and 350 F2 population derived from a cross G. hirsutum cv. Han 208 and Pima 90-53 were grown at Sanya suburb in 2013. Fortunately, this QTL was further dissected into two QTL at this region in BC1F2 population. The qFS9-3 and qFS9-4 explaining 16.27% and 17.85% of the phenotypic variation were detected in the common interval from NAU 2395 to NAU1092a in BC1F2 and its derived BC1F2:3 lines at two environments, including Baoding suburb in 2010 and Qingxian county in 2011, respectively. Also, The qFS9-5 and qFS9-6 explaining 12.13% and 14.75% of the phenotypic variation were detected in the common range from NAU 2395 to NAU1092 in BC1 and F2 population at Sanya in 2013. All QTL showed positive additive effects except the qFS9-5. So, this is a very stable and common QTL with the same interval markers, and we proposed qFSA9 as the name for these QTL. A CI of qFSA9 was 1.37-5.92 cM with two SSR markers (NAU2395 and NAU1092) and the largest LOD value was 5.00 for this QTL in BC1 grown at Baoding suburb in 2007. The SSR markers linked to the QTL can be utilized to marker-assisted selection of fiber strength.