Functional Genomics

Upland and Sea Island cotton are two cultivated tetraploid cotton varieties. Upland cotton (Gossypium hirsutum L.) is characterized by its high yield and moderate fiber quality performance, while Sea Island cotton (G. barbadense L.) as its low yield, increased fiber fineness and strength. Chromosome segment introgression lines (CSIL) consist of a battery of near-isogenic lines covering whole genome of crop. Except for one homozygous chromosome segment transferred from donor parent each line, the remaining parts of genome are the same as the recipient parent. It is an ideal material for genome research and especially for QTL mapping. Based on our high dense linkage map constructed between G. hirsutum × G. barbadense, G. barbadense cotton chromosome segment introgression lines in background of genetic standard line of Upland cotton TM-1 were developed with microsatellite assisted backcross. QTLs were mapped by CSIL population. An analysis of variance of the fiber quality traits based on the data from four environments was conducted. A total of 53 additive QTL and 4 epistatic QTL for yield, and 43 additive QTL and 6 epistatic QTLs associated with fiber qualities were detected. Although these QTLs are distributed on all chromosomes, we detected several clustered QTL regions, suggesting the relationship between yield and fiber quality between G. hirsutum and G. barbadense. There were 10 FL QTL, 16 FS QTL and 17 FM QTL based on values from four different environments. The CSILs created and the analyses presented here will enhance the understanding of the genetics of long staple fiber quality traits in G. barbadense and facilitate further molecular breeding of Upland cotton cultivars.

QTL mapping is an important part of marker-assisted selection breeding. There are many studies on the QTL related to cotton fiber yield and quality, but most of them based on a DNA level, which may appear some null QTLs. However, QTL mapping based on transcriptome map at cDNA level can be more reliable. An interspecific transcriptome map of allotetraploid cotton was developed based on an F2 population [Emian22 ×3-79] by amplifying cDNA using EST-SSRs. The map was constructed by using 5DPA developing fiber. This map contained 242 markers which distributed in 32 linkage groups (26 chromosomes). The full length of this map was 1938.72 cM with a mean marker distance of 12.47 cM. Functions of some ESTs have been annotated through searching homologous sequences. Some of the makers related to the differentiation and elongation of cotton fiber, most of them related to basic metabolism. This study proved that constructing transcriptome map by amplifying cDNA using EST-SSRs is a very simple method, and the transcriptome map will be a power tool for mapping eQTLs of fiber quality.

Infertility of anthers under high temperature (HT) has become a critical factor in yield loss in cotton. Previous studies showed altered carbohydrate metabolism or disrupted tapetal cell programmed cell death (PCD) underlie anther sterility. Through large-scale expression profile sequencing we studied the effect of HT on cotton anther development and found that the GhCKI gene, highly homologous with casein kinase I (CKI), was induced by HT in a HT-sensitive cotton line. Our studies showed GhCKI causes glucose (GLU) accumulation in early-stage anthers by inactivating starch synthase via phosphorylation. Subsequent feedback inhibition of glucose assimilation and induction of starch synthesis cause a deficiency in glucose in late-stage anthers. The early accumulation of glucose also promotes excessive amounts of abscisic acid (ABA) in early anthers and alters the balance of reactive oxygen species (ROS) scavenging and accumulation in late anthers, causing delayed PCD in the tapetum leading to anther indehiscence.

Expedited by a decade of fruitful collaboration with several USA institutions, we have developed a well-functioning cotton genomics and bioinformatics research programs in Uzbekistan, a northernmost cotton growing country. The Center of Genomics and Bioinformatics in Tashkent is a well equipped research facility, with a number of well qualified young genomics scientists and molecular breeders. It has already achieved a number of basic and applied research results on cotton germplasm characterization, genetic diversity of cotton gene pools, molecular marker development, genetic mapping of important complex traits, cloning and characterization of several cotton gene families and smallRNA/microRNA pools of various cotton tissues, and transgenomics of de novo characterized cotton genes. Ongoing efforts include an efficient marker-assisted selection program for complex traits such as fiber quality and development of early flowering, early-maturing, highly productive transgenic cotton cultivars with superior fiber quality using RNAi technology and modern transgenomics tools. The successes to date highlight the benefits of these joint efforts and collaborations, and underscore the opportunities for mutually beneficial collaborations and benefit to cotton improvement, production and market share worldwide. Key results on genomics and transgenomics of cotton will be presented.

Fibre is an important raw material of cotton for fibre industries. To meet ever changing fibre norms of textile industry, understanding the fibre genomics and the biological function of the genes involved in fibre development is essential. We studied differentially expressed genes between fibreless mutant and normal diploid cottons at 0 and 10 days after pollination through transcriptome studies. Functional annotation of these differentially expressed genes was carried out by comparison with tair data base version 10 of A. thaliana.

In West Texas, the annual cotton crop rarely produces lint fibers that meet the cultivar’s full genetic potential. Late season cool temperature during boll and fiber maturation greatly slows the deposition of cellulose in the secondary cell wall. This produces immature fibers that greatly affect the agronomic performance of cotton by lowering yield and quality. The negative impact of low micronaire values is lower prices and a reduced demand for West Texas cotton on the world market. We have developed transgenic cotton plants that increase expression of the alternative oxidase enzyme (AOX) which has been shown to increase cellular temperatures in some thermogenic plants. Our hypothesis is that by elevating AOX production in cotton, specifically in the boll, it will mitigate adverse effect of cool temperature on lint quality measures. In theory, AOX over-expression would serve to create an optimum and stable temperature environment for fiber development under adverse temperatures. To test this hypothesis, a gene efficacy experiment was conducted on two AOX lines (66-6 and 94-20), their non-transgenic “null” lines (66-2, and 94-3), and Coker 312-17. Agrobacterium mediated transformation was used to create transgenic Coker 312-17 lines to constitutively express the tobacco Aox1 gene. All five lines were evaluated in field trials at the Texas Tech Research Farm in Lubbock, Texas during the 2010, 2011, and 2012 seasons. A time-course assessment of fiber development was conducted each season by tagging flowers daily then harvesting open boll according to the week it flowered. Lint samples that represented each week were independently analyzed using HVI and AFIS testing. A statistical analysis of each trait, using a mixed model in SAS, is being used to assess gene efficacy of AOX.

Studies have revealed that most, if not all, of traits, especially those of agronomic importance and quantitative inheritance, are the consequence of interactions among a number of genes. Therefore, it is crucial to understanding the molecular basis of the traits and developing molecular toolkits enabling effective manipulation of the traits in enhanced breeding to isolate the genes controlling the traits and to determine how they work together to make the traits. To rapidly clone the genes and QTLs controlling traits or biological processes of importance in different species, we have developed a high-throughput gene and QTL cloning and studying system based on natural or artificial genetic variation of the target traits using maize. Using the system, thousands of genes controlling several agronomical traits such as those controlling yield and quality traits can be cloned within a few years by a scientist. Its gene and QTL cloning efficiency is approximately thousand-fold higher than the currently used gene and QTL cloning methods such as map-based cloning, T-DNA or transposon insertional mutagenesis, chemically-induced mutagenesis and RNA interference. We have been working toward cloning of all major genes controlling cotton fiber yield and quality traits using a new RIL population developed from a cross between G. hirsutum (94L-25) and G. barbadense (NMSI 1331) with the high-throughput gene and QTL cloning system since 2009. We have phenotyped the genetic variation of six major fiber yield and quality traits, including lint percentage, lint yield, fiber length, fiver strength, fiber uniformity and micronaire from 2009 – 2011 and collected necessary tissues from the field trials. We are currently analyzing the tissues using modern molecular technologies. The project is expected to be completed within this year, which will allow isolation of thousands of genes controlling the fiber yield and quality traits. The fiber genes will then be used to study the molecular basis of the fiber traits and to design toolkits desirable for enhanced cotton fiber breeding.

Cotton fiber is the most prevalent natural raw material used in the textile industry. Cotton seed fibers are highly elongated single-celled trichomes that differentiate from the outer epidermis (protoderm) of the ovule. The fiber cells elongate for about 27-39 days past anthesis (DPA) and the secondary cell wall is formed from 17 to 53 DPA depending of the cotton species, cultivar and environment. The length of fiber is ranging from 25 mm to 40 mm. The environment determines whether the fiber properties reach the genetic potential of the cotton cultivar. Good quality of cotton consists of long, fine and strong fiber. The length of the fiber is one of the most important characteristics, which determines the quality of the resulting yarn. One of the major limitations in genetic improvement of fiber is the lack of information at the molecular level about genes controlling fiber development. Competition with synthetic fibers has forced cotton industry to invest heavily in research to develop higher-quality fibers; however one of the major limitations in genetic improvement of fiber is the lack of information at the molecular level about genes controlling fiber development. Elucidating the cellular and molecular basis of fiber elongation beside its importance in basic cell biology could also identify potential targets for genetic manipulation of fiber length. Genetic mutants are useful tools for studying gene function. The cotton (Gossypium hirsutum L.) fiber mutation Ligon lintless-2 is controlled by a single dominant gene (Li2) that results in significantly shorter fibers than a wild-type. In this study we identified transcript and metabolic responses associated with fiber elongation using Li2 near isogenic lines. Significant changes in the relative abundance of multiple identified metabolites were observed between near isogenic lines which are the result of genetic reprogramming of primary metabolism in response to Li2 mutation.

Cotton fibers are unicellular seed trichomes and consist of almost pure cellulose. During the transition from elongation to secondary cell wall (SCW) biosynthesis stage in developing cotton fibers, cellulose biosynthesis is significantly increased. Cellulose synthase catalytic subunits (CesAs) play an important role for cellulose biosynthesis. Although the first two cellulose synthase catalytic subunits (CesAs) were isolated from developing cotton fibers, it is not clear how many CesAs are involved in SCW biosynthesis in fibers, or how these genes are regulated during cell wall biogenesis. Based on diploid Arabidopsis genome data that a trio of CesAs is required for SCW biosynthesis, a minimum of three pairs of homeologous CesA genes are expected to be expressed during SCW biosynthesis in allotetraploid G. hirsutum. To date, two pairs of homeologous SCW CesA genes (GhCesA1-Dt, GhCesA4 or GhCesA1-At, GhCesA2-Dt, and GhCesA2-At) have been characterized from G. hirsutum fibers. We have characterized a pair of homeologous GhCesA2 genes and analyzed promoter activity of GhCesA4. Temporal and spatial expressions, transcriptional regulation and potential alternative splice of SCW GhCesAs will be presented. Kim et al. (2012) Cloning and characterization of homeologous cellulose synthase catalytic subunit 2 genes from allotetraploid cotton (Gossypium hirsutum L.). Gene, 494: 181-189. Kim et al. (2011) Functional analysis of Gossypium hirsutum cellulose synthase catalytic subunit 4 promoter in transgenic Arabidopsis and cotton tissues. Plant Science, 180: 323-332.

Previously we have screened the Gossypium hirsutum L. USDA germplasm collection for accessions that were more tolerant of heat stress using a chlorophyll fluorescence assay. Seven accessions were identified that appeared to be significantly more thermotolerant than the DeltaPine 90 (DP90) variety used as the thermosensitive control. Of these accessions, five accessions TX337, TX1427, TX2287, TX2288, and TX2454 have been used to produce segregating F2 populations. Leaves from greenhouse grown plants of these five accessions along with DP90 and a second commercial variety SureGrow 747 (SG747) were used for preparation of small RNA libraries either from control (0 hr heat stress), 4 hr heat stress, or 24 hr heat stress at 42 C (growth chamber). RNA-seq was conducted on these small RNA libraries derived from total RNA from these samples (note that the 4 and 24 hr HS samples were combined for library sequencing generating 14 leaf libraries) using the ABI Solid platform with single-end sequencing, and the resulting sequence libraries were submitted to a small RNA library pipeline (based on MIREAP) that we have developed. Normalized abundance of each small RNA found in each library was determined. The CLC Genomics Workbench platform was utilized to statistically determine the heat stress up- and down-regulated miRNAs in each of the 7 genotypes. The subset of miRNAs that are differentially expressed differently in control versus 1 or more accessions were selected for further analysis. Where possible these were mapped to D-genome chromosome scaffolds and A-genome larger contigs. By then assembling transcriptome libraries for each genotype to the defined miRNA precursor sequences a set of SNPs has been defined which we will subsequently utilize to follow these miRNA precursor sequences in segregating F2 populations. Heat stress tolerance of individual plants the F2 populations will be correlated with the SNPs to validate a role for specific miRNA genes in heat stress tolerance and hopefully generate a set of useful markers that can be utilized in subsequent breeding work.