Germplasm and Genetic Stocks

The cotton germplasm maintained at TNAU were evaluated for drought tolerance in two different pot experiments using physio-morphological and biochemical traits. The selected cotton entries were further evaluated under field conditions and six germplasm lines were selected from these studies that have shown contrasting drought tolerance and superior fiber quality traits for mapping population development. Genetic linkage maps were constructed using different mapping population types such as F2, BC1 and RILs derived from Gossypium hirsutum cv. KC3 (drought tolerant) and G. barbadense cv. Suvin (drought susceptible). QTL analysis identified several QTLs including two major QTLs on chromosome 19 and 25 for fiber quality traits under drought stress and such QTLs were also reported across the world using different types of mapping populations and genetic backgrounds. Consistent major QTLs linked to fiber quality traits under water limited environments are being fine mapped with the new set of markers. Further, the mapping populations were evaluated under rainfed and controlled irrigation at different cotton growing regions and seasons to validate the QTLs and confirm the potential of these QTLs in MAS. To this end, hybrids are being developed to produce backcross progenies through MAS. Simultaneously, an attempt was also made to understand molecular response of cotton to water stress through gene expression studies. Negative regulations of gene expression by endogenous, non-coding microRNAs (miRNAs) have been shown to play important role in abiotic stress responses in plants. Though miRNAs with their targets have been widely studied in cotton, limited knowledge is available on the miRNA population of cotton in response to water stress. Differentially expressed miRNAs and their targets were identified in response to drought under natural field conditions through high-throughput small RNA sequencing by comparing leaf samples of KC3 and Suvin. Comparison of miRNAs expressed under water stressed conditions between KC3 and Suvin, have resulted 7494 miRNA reads with at least two folds of differential expression and interestingly, almost all of the miRNAs identified in this study were up regulated at least by two folds. Our results reveal the presence of numerous conserved and unidentified, novel miRNAs in cotton in response to drought under the field conditions and first time we have demonstrated that there are huge numbers of water stress related miRNAs in cotton that sequentially and complexly involved in gene regulation that ultimately lead to drought resistance in cotton. Further, this is the first report on experimentally identifying and validating miRNAs in G. barbadense. Characterization of these miRNAs might have enormous potential in elucidating the molecular mechanism and more importantly genetic improvement of drought tolerance in cotton.

Due to global warming, temperature of the world is increasing day by day and it appeared as one of the major abiotic stress in the world specifically for agricultural production. According to estimates about 65-70 % yield losses have been observed in cotton due to heat stress in Pakistan. Heat also reduces the expression of Cry gene in Bt cottons and heat stress also cause square and flower shedding as well as pollen sterility in cotton. An experiment was conducted in the fields of University of Agriculture, Faisalabad to screen eighty cotton accessions for heat tolerance. To calculate RCI%, two samples of leaf disc were collected from each genotype grown in cotton field; one from one side of the mid rib of the leaf and second from the other side of the mid rib of the same leaf. One sample was used as control at room temperature in distilled water filled (10cc) falcon tubes while the second sample was also kept in similar falcon tubes at 50oC in water bath. Then both the samples were kept in incubator for 24 hours at 22oC then EC1 and EC2 were measured for each sample separately, before and after autoclave treatment. By using EC1 and EC2 values, RCI% of eighty genotypes were calculated. The genotypes VH-142 and VH-259 were found to be heat tolerant with RCI% values of 48 and 54.9 respectively while VH-282 and DNH-40 genotypes were found to be heat susceptible with RCI% values of 93.2 and 94.5 respectively in these studies. The identified heat tolerant in present investigation can be used for the development of heat tolerant and high yielding cotton genotypes.

The fiber is considered the basic product of cotton plant is inherited polygenecally along with other quantitative traits. The fiber output is related with following traits, as: fiber index, fiber length, fiber output, number of seeds per boll and 1000 seeds weight. Inheritance of the trait of fiber output at the interspecific hybrids F1 received at crossing of intraspecific varieties of the species G.arboreum L. (ssp. obtusifolium, ssp. obtusifolium var. indicum, ssp. perenne (Blanco) Mauer, ssp. nanking (Meyen) (with a brown and white fiber)) and Australian (G.australe F.Muell., G.nelsonii Fryx.) cotton species has been studied. It is necessary to notice that the intraspecific varieties of the species G.arboreum L. and Australian wild species on a fiber output strongly differ from each other. Average value of a fiber output basically make up 28,4-29,9%, except for subspecies ssp. obtusifolium at which the fiber output make up 20,5%. The Australian wild cotton species are characterized by low indicators of a fiber output. Average value of studied trait at them make up 6,0-6,5%. The analysis of results of a fiber output at the interspecific hybrids F1 has shown that the studied trait is inherited with a bias towards the best parent and average value of a fiber output has made up 11,4-25,2%. At hybrid combination F1 G.australe х G.arboreum ssp. obtusifolium the fiber output has made up 20,6%. The high amplitude of variability of a trait within 14,3-28,6% was noted. The domination coefficient was equal hp = 1,0 where dominant inheritance of the trait was observed. It is necessary to notice that the reciprocal combination of the given hybrid (F1 G.arboreum ssp. obtusifolium х G.australe) have appeared sterile, for definition of the reasons of sterility demands additional cytoem-bryological research. At the reciprocal hybrids F1 G.arboreum ssp. obtusifolium var. indicum х G.australe and G.australe х G.arboreum ssp. obtusifolium var. indicum the fiber output has made up 24,6-25,2%, the amplitude of variability was equal 16,7-31,3% and 20,0-28,6% accordingly. Intermediate inheritance of the trait (hp = 0,6 and hp = 0,5 accordingly) was observed. At the interspecific hybrids F1 received at crossing of the intraspecific varieties of the species G.arboreum L. with species G.nelsonii the intermediate inheritance of the trait with a bias towards the best parent was revealed basically. Thus, as a result of studying of the inheritance a fiber output at the interspecific hybrids F1 received at the crossing of the intraspecific varieties of an old-world cotton species G.arboreum L. and wild-growing Australian species, dominant and intermediate inheritance of a trait of «fiber output» was established. It is necessary to note that, the received interspecific hybrid combinations will enrich the cotton genepool and can be used in the further genetic and breeding research as the donors combining useful traits and properties of the Indochinese and Australian species.

Quantitative trait loci (QTL) mapping was the foundation of marker-assisted selection (MAS) for lint yield and fiber quality in cotton breeding. Boll-related traits have impact on yield and fiber quality. Here, detection of QTLs related to boll traits and fiber quality traits were was performed using intraspecific F2 and F2:3 populations of AQ × 08-10604 (Gossypium hirsutum L). Totally, 91 QTLs were mapped for 17 traits related to boll and fiber quality traits in F2 and F2:3 populations, 39 QTLs were significant. Six pairs of common QTLs were found, two pairs for boll coat weight (BCW) were found in the same or neighbored position on Chr. A10 and D1. Two pairs of QTLs for boll length (BL) were found in the neighbored position on Chr. A10 and A13 with a higher percentage of PV. Two pairs of QTLs for boll diameter (BD) were found in the neighbored position on Chr. A10 and D1. It can be speculated that BCW, BL and BD are three of the traits with a higher level of stability. Five QTL clusters for the same traits or different traits were also identified in Chr. A10 (2), A13, D1, D5. Total of 64 epistatic QTLs were detected, playing important rules in genetic variation for boll-related traits. Correlation analysis showed that significant positive correlation existed between seed yield and boll number per plant and between boll weight and BCW, BL, and BD. The results presented here will enhance the understanding of the genetic basis of boll-related traits and enable further MAS in upland cotton.

The extra long staple cotton (G. barbadense) is characterized by its excellent fiber qualities and low yield, while Upland cotton (G. hirsutum) is characterized by its high yield and moderate fiber quality performance. In this study, we developed G. hirsutum chromosome segment substitution lines (CSSL) cotton in G. barbadense cv. Xinhai 25 by repeatedly backcrossing and selfing. Difference between these lines and their recipient parent is very small at genomic level. It is an ideal material for genome research especially for cotton yield QTL mapping. We investigated yield traits of these lines in 2013 in Aksu, Xinjiang Uygur Autonomous Region, and used molecular marker-assisted selection (MAS) to screen out several G. hirsutum chromosome segment substitution lines which have significantly better yield traits than the recipient parent in boll weight (BW) or lint percentage (LP). Two lines with boll weight (BW) and lint percentage (LP) higher than their recipient parent Xinhai #25 by 17.9% and 10.8%, respectively, and as the same as the extra long staple fiber qualities as Xinhai #25 were developed. How genomic selection to increase G. barbadense yield by introgressed G. hirsutum segments is discussed.

Degree of a research level of evolution and genetic interrelation of cotton species and varieties has great value for the further use of the big genetic diversity at development of the elite cultivars possessing useful agricultural traits caused variety of germplasm. For development of early and highly productive, resistant to different diseases and pests of cultivars, it is necessary to study an initial breeding material and it a taxonomic accessory. Information on systematization, phylogeny of the intraspecific representatives of the species data are rather limited and isolated, have chaotic character. Therefore, questions of studying of systematization, phylogenetic interrelations and genetic potential of an intraspecific diversity of species G.barbadense L. and G.darwinii Watt. till now remain opened and actual. The ruderal, cultivated-tropical and subtropical varieties and forms of the species G.barbadense L., and also species G.darwinii Watt. conserved in the genepool of the Institute of Genetics and Plant Experimental Biology of the Uzbek Academy of Sciences were objects of the research. The identification of phylogenetic relationship congeniality of intraspecific varieties of G.barbadense L. and interspecific congeniality with the species G.darwinii Watt. and determination of possibilities to use them in breeding was purpose given investigations. The interspecific and intraspecific hybridization, comparative morphology, genetic and static analysis methods was used during this research. Application of research work using complex methods, resulted in obtaining, for the first time, new data on phylogenetic congeniality, morphological features and nature of inheritance of biological and economy-valuable traits of intraspecific varieties of the species G.barbadense L. and G.darwinii Watt. and their intraspecific and interspecific hybrids F1, F2, F1B1. New hybrid forms-donors of valuable economic traits were obtained. A new phylogenetic scheme was developed and a systematic position of intraspecific varieties of Peruvian cotton species was specified. It was proposed for the first time to introduce ruderal form of spp. ruderale f. pisco to the composition of wild forms, on the basis of morphological diversities (by branching type, sizes of blooms and bolls, weight of 1000 seeds and seed cotton, length of vegetation period, photoperiodic reaction), crossing ability and nature of inheritance of morphological features in F1, F2, F1B1. It is necessary to notice that the intraspecific hybrids will enrich cotton germplasm collection and might be used as donors of valuable biological and agronomic traits in cotton breeding programs. Therefore, the new scheme of phylogenetic propinquity of the intraspecific varieties of Peruvian species will facilitate the selection of starting material and ensure efficiency of nurture of new hybrid forms for genetic and selection researches to produce competitive fine-stapled cotton in the world market.

The importance of an initial material for breeding consists available a biological variety of forms of plants and in their genetic identification on a complex of valuable traits. In this connection studying and an all-round estimation of germplasm on morphological and biological and economic-valuable traits and properties defined its topicality. At studying of morphological variability of cotton had been made attempt to find out adaptive value of some traits, and also to compare a trait of wild and cultural species that should be used at finding-out of their relations. Subjects of research were the intraspecific diversity of species G.herbaceum L. and G.arboreum L. Subject of the research was intraspecific and interspecific phylogenetic relations of an intraspecific diversity of the species G.herbaceum L. and G.arboreum L. Purpose of given investigation was to specify the systematic position and identify phylogenetic relationship congeniality and the genetic potential of the wild, ruderal, tropical and subtropical varieties and forms of polymorphic species of G.herbaceum L. and G.arboreum L. of genus Gossypium for their utilization in practical breeding. Interspecific and intraspecific hybridization, comparative morphology, genetic and statistical methods was used in this research. Application of the research worked using integrated methods, resulted in obtaining, for the first time, new data on phylogenetic relationship congeniality, the systematic position of intraspecific genetic varieties and the forms of polymorphic species G.herbaceum L. and G.arboreum L. was specified. For the reasons of geographical distribution, the results of an intraspecific and interspesific hybridization, and the distinct morphological and biological traits, it is proposed to elevate the taxonomic rank of f. harga (G.herbaceum ssp. pseudoarboreum) and f. sangiuneum (G.arboreum ssp. neglectum) to subspecies. The nature of inheritance and the degree of correlative interconnection of the studied morphological and biological, and were revealed economically valuable traits of the F1 and F2 plants of intra- and interspecific hybrids. The chart of phylogenetic relationship was developed congeniality of intra- and interspecific varieties of G.herbaceum L. and G.arboreum L. It is necessary to notice that the intra- and interspecific transgressive hybrid forms will be used in breeding as donors of valuable agronomic traits. Received forms substantial replenish and enrich of cotton gene pool with new germplasm. Therefore, the new chart of phylogenetic relationship congeniality of the intraspecific and interspecific varieties of G.herbaceum L. and G.arboreum L. facilitates selection of source material and ensure the efficiency for development of new hybrid forms for genetic and breeding researches to produce commercially competitive cotton varieties in the world market.

SNP markers representing whole Gossypium hirsutum genome were developed in Taqman MGB format and validated their functionality using diverse germplasm lines including commercial inbred lines. Template sequences of total number of 96 Taqman SNPs were acquired from EST sequencing conducted in Monsanto Company. Based on in silico comparative mapping approach using public D genome sequence, these 96 template sequences were expected to be distributed across 26 chromosomes in both A and D genomes. Polymorphism information content value of these 96 Taqman SNPs ranged between 0.310 and 0.375. Genetic diversity among germplasm lines was revealed based on SNP allele calls and marker-based genetic diversity was also compared to phenotype based approach. The result showed that SNP marker-based genetic diversity was informative and can assist with variety identification purposes such as enforcement of intellectual property rights or determination of genetic conformity resolution of essentially derived variety (EDV) or in the examination of Distinctness. Monsanto Company is releasing these 96 SNP markers to CottonGEN (http://www.cottongen.org/) for cotton genomics community to use them as a genetic diversity assay tool.

Drought is the major limiting factor for cotton production in the US. Climate change will likely make cotton production even more difficult. Making cotton resistant to drought in order to maintain sustainable fiber production in arid and semiarid areas is extremely important for Southwestern USA. Among the efforts to achieve such a goal, up regulating cytokinin production by ectopically expressing the IPT gene to delay the drought induced senescence appears to be the most promising. Despite consistent data showing improved tolerance to drought by doing so, we noticed that the timing of the water deficit stress might be very important to this trait. We analyzed IPT-transgenic cotton lines in greenhouse for two rounds to check the impact of drought stress timing on their final yields in comparing to wild-type plants and segregated non-transgenic lines. Our data shows that IPT-transgenic plants out-performed non-transgenic controls dramatically only when water deficit stress took place earlier in their growth period. If the water-deficit stress was applied 55 days after germination, no significant differences were observed in the final yields between IPT-transgenic plants and non-transgenic controls. These results indicate that the IPT gene driven by the SARK promoter is more effective during early stages of cotton growth and development in regarding increasing final yield after drought treatment.

Cotton is one of the most important crops for its natural textile fibers in the world. However, it often suffered from drought stress during its growth and development, resulting in a drastic reduction in cotton productivity. Therefore, study on molecular mechanism of cotton drought-tolerance is very important for increasing cotton production. To investigate molecular mechanism of cotton drought-resistance, we employed RNA-Seq technology to identify differentially expressed genes in the leaves of two different cultivars (drought-resistant cultivar J-13 and drought-sensitive cultivar Lu-6) of cotton. The results indicated that there are about 13.38% to 18.75% of all the unigenes differentially expressed in drought-resistant sample and drought-sensitive control, and the number of differentially expressed genes was increased along with prolonged drought treatment. DEG (differentially expression gene) analysis showed that the normal biophysical profiles of cotton (cultivar J-13) were affected by drought stress, and some cellular metabolic processes (including photosynthesis) were inhibited in cotton under drought conditions. Furthermore, the experimental data revealed that there were significant differences in expression levels of the genes related to abscisic acid signaling, ethylene signaling and jasmonic acid signaling pathways between drought-resistant cultivar J-13 and drought-sensitive cultivar Lu-6, implying that these signaling pathways may participate in cotton response and tolerance to drought stress.