Rickie B. Turley

A combined functional and structural genomics approach identified an EST-SSR marker with complete linkage to the Ligon lintless-2 genetic locus in cotton (Gossypium hirsutum L.)

BackgroundCotton fiber length is an important quality attribute to the textile industry and longer fibers can be more efficiently spun into yarns to produce superior fabrics. There is typically a negative correlation between yield and fiber quality traits such as length. An understanding of the regulatory mechanisms controlling fiber length can potentially provide a valuable tool for cotton breeders to improve fiber length while maintaining high yields. 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 a near-isogenic state with a wild-type cotton line, Li2 is a model system with which to study fiber elongation.ResultsTwo near-isogenic lines of Ligon lintless-2 (Li2) cotton, one mutant and one wild-type, were developed through five generations of backcrosses (BC5). An F2 population was developed from a cross between the two Li2 near-isogenic lines and used to develop a linkage map of the Li2 locus on chromosome 18. Five simple sequence repeat (SSR) markers were closely mapped around the Li2 locus region with two of the markers flanking the Li2 locus at 0.87 and 0.52 centimorgan. No apparent differences in fiber initiation and early fiber elongation were observed between the mutant ovules and the wild-type ones. Gene expression profiling using microarrays suggested roles of reactive oxygen species (ROS) homeostasis and cytokinin regulation in the Li2 mutant phenotype. Microarray gene expression data led to successful identification of an EST-SSR marker (NAU3991) that displayed complete linkage to the Li2 locus.ConclusionsIn the field of cotton genomics, we report the first successful conversion of gene expression data into an SSR marker that is associated with a genomic region harboring a gene responsible for a fiber trait. The EST-derived SSR marker NAU3991 displayed complete linkage to the Li2 locus on chromosome 18 and resided in a gene with similarity to a putative plectin-related protein. The complete linkage suggests that this expressed sequence may be the Li2 gene.

Comparative analysis of gene expression between CMS-D8 restored plants and normal non-restoring fertile plants in cotton by differential display

CMS-D8 and its restorer were developed by introducing the cytoplasm and nuclear gene Rf2from the wild diploid Gossypium trilobum (D8) into the cultivated tetraploid Upland cotton (Gossypium hirsutum). No information is available on how the Rf2 gene interacts with CMS-associated genes and how CMS-D8 cytoplasm affects nuclear gene expression. The objective of this study was to identify differentially expressed genes in anther tissues between the non-restoring fertile maintainer ARK8518 (rf2rf2) and its isogenic heterozygous D8 restorer line, ARK8518R (Rf2rf2) with D8 cytoplasm, by mRNA differential display (DD). Out of more than 3,000 DDRT-PCR bands amplified by 31 primer combinations from 12 anchor primers and 8 arbitrary decamer primers, approximately 100 bands were identified as being qualitatively differentially displayed. A total of 38 cDNA fragments including 12 preferentially expressed cDNA bands in anther were isolated, cloned and sequenced. Reverse northern blot analysis showed that only 4 genes, including genes encoding a Cys-3-His zinc finger protein and aminopeptidase, were up-regulated, while 22 genes, including genes for phosphoribosylanthranilate transferase (PAT), starch synthase (SS), 4-coumarate-CoA ligase, electron transporter, calnexin, arginine decarboxylase, and polyubiquitin, were down-regulated in the heterozygous restorer ARK8518R. The down-regulation of SS explains the lack of starch accumulation in sterile rf2 pollen grains in the heterozygous restored plants. The molecular mechanism of CMS and its restoration, specifically the possible roles of SS and PAT genes in relation to restoration of Rf2 to CMS-D8, are discussed. This investigation represents the first account of such an analysis in cotton.

Transcript profiling by microarray and marker analysis of the short cotton (Gossypium hirsutum L.) fiber mutant Ligon lintless-1 (Li1)

BackgroundCotton fiber length is very important to the quality of textiles. Understanding the genetics and physiology of cotton fiber elongation can provide valuable tools to the cotton industry by targeting genes or other molecules responsible for fiber elongation. Ligon Lintless-1 (Li1) is a monogenic mutant in Upland cotton (Gossypium hirsutum) which exhibits an early cessation of fiber elongation resulting in very short fibers (< 6 mm) at maturity. This presents an excellent model system for studying the underlying molecular and cellular processes involved with cotton fiber elongation. Previous reports have characterized Li1 at early cell wall elongation and during later secondary cell wall synthesis, however there has been very limited analysis of the transition period between these developmental time points.ResultsPhysical and morphological measurements of the Li1 mutant fibers were conducted, including measurement of the cellulose content during development. Affymetrix microarrays were used to analyze transcript profiles at the critical developmental time points of 3 days post anthesis (DPA), the late elongation stage of 12 DPA and the early secondary cell wall synthesis stage of 16 DPA. The results indicated severe disruption to key hormonal and other pathways related to fiber development, especially pertaining to the transition stage from elongation to secondary cell wall synthesis. Gene Ontology enrichment analysis identified several key pathways at the transition stage that exhibited altered regulation. Genes involved in ethylene biosynthesis and primary cell wall rearrangement were affected, and a primary cell wall-related cellulose synthase was transcriptionally repressed. Linkage mapping using a population of 2,553 F2 individuals identified SSR markers associated with the Li1 genetic locus on chromosome 22. Linkage mapping in combination with utilizing the diploid G. raimondii genome sequences permitted additional analysis of the region containing the Li1 gene.ConclusionsThe early termination of fiber elongation in the Li1 mutant is likely controlled by an early upstream regulatory factor resulting in the altered regulation of hundreds of downstream genes. Several elongation-related genes that exhibited altered expression profiles in the Li1 mutant were identified. Molecular markers closely associated with the Li1 locus were developed. Results presented here will lay the foundation for further investigation of the genetic and molecular mechanisms of fiber elongation.

Integrated metabolomics and genomics analysis provides new insights into the fiber elongation process in Ligon lintless-2 mutant cotton (Gossypium hirsutum L.)

BackgroundThe length of cotton fiber is an important agronomic trait characteristic that directly affects the quality of yarn and fabric. The cotton (Gossypium hirsutum L.) fiber mutation, Ligon lintless-2, is controlled by a single dominant gene (Li2) and results in extremely shortened lint fibers on mature seeds with no visible pleiotropic effects on vegetative growth and development. The Li2 mutant phenotype provides an ideal model system to study fiber elongation. To understand metabolic processes involved in cotton fiber elongation, changes in metabolites and transcripts in the Li2 mutant fibers were compared to wild-type fibers during development.ResultsPrincipal component analysis of metabolites from GC-MS data separated Li2 mutant fiber samples from WT fiber samples at the WT elongation stage, indicating that the Li2 mutation altered the metabolome of the mutant fibers. The observed alterations in the Li2 metabolome included significant reductions in the levels of detected free sugars, sugar alcohols, sugar acids, and sugar phosphates. Biological processes associated with carbohydrate biosynthesis, cell wall loosening, and cytoskeleton were also down-regulated in Li2 fibers. Gamma-aminobutyric acid, known as a signaling factor in many organisms, was significantly elevated in mutant fibers. Higher accumulation of 2-ketoglutarate, succinate, and malate suggested higher nitrate assimilation in the Li2 line. Transcriptional activation of genes involved in nitrogen compound metabolism along with changes in the levels of nitrogen transport amino acids suggested re-direction of carbon flow into nitrogen metabolism in Li2 mutant fibers.ConclusionsThis report provides the first comprehensive analysis of metabolite and transcript changes in response to the Li2 mutation in elongating fibers. A number of factors associated with cell elongation found in this study will facilitate further research in understanding metabolic processes of cotton fiber elongation.

IDENTIFICATION OF A DELTA -TIP CDNA CLONE AND DETERMINATION OF RELATED A AND D GENOME SUBFAMILIES IN GOSSYPIUM SPECIES

Tonoplast intrinsic proteins (TIPs) have been implicated in the process of cell elongation, such as occurs in the developing cotton fiber. We have isolated a cDNA clone (997 bp in length) from a cotton (Gossypium hirsutum L.) library which putatively encodes a protein of 248 residues (Mr 25079) with 85% identity to Arabidopsis δ-TIP. The derived amino acid sequence included two conserved sequences associated with major intrinsic proteins (SGxHxNPA at residues 78 to 85, NPA residues at 197 to 199) and a cysteine residue at 116 which is reported to bind mercury in Arabidopsis δ-TIP. The polymerase chain reaction was used to generate partial genomic clones of the cotton δ-TIP. In comparison to other genomic TIP sequences, the number (two) and position of the introns were conserved in cotton. Comparing the TIP sequences from cotton revealed two subfamilies, which were consistently distinguished by a Tsp45I restriction site polymorphism. This polymorphism was used to demonstrate that TIP subfamilies were specific to either the A or D genomes of Gossypium. When δ-TIP DNA fragments were amplified from cDNA of fiber 14 days after anthesis, the A and D were found, indicating the presence of δ-TIP transcripts in these elongating cells.

Next generation genetic mapping of the Ligon-lintless-2 ( Li 2 ) locus in upland cotton ( Gossypium hirsutum L.)

Key messageMapping-by-sequencing and novel subgenome-specific SNP markers were used to fine map the Ligon-lintless 2 (Li2) short-fiber gene in tetraploid cotton. These methodologies will accelerate gene identification in polyploid species.AbstractNext generation sequencing offers new ways to identify the genetic mechanisms that underlie mutant phenotypes. The release of a reference diploid Gossypium raimondii (D5) genome and bioinformatics tools to sort tetraploid reads into subgenomes has brought cotton genetic mapping into the genomics era. We used multiple high-throughput sequencing approaches to identify the relevant region of reference sequence and identify single nucleotide polymorphisms (SNPs) near the short-fiber mutant Ligon-lintless 2 (Li2) gene locus. First, we performed RNAseq on 8-day post-anthesis (DPA) fiber cells from the Li2 mutant and its wild type near isogenic line (NIL) Gossypium hirsutum cv. DP5690. We aligned sequence reads to the D5 genome, sorted the reads into A and D subgenomes with PolyCat and called SNPs with InterSNP. We then identified SNPs that would result in non-synonymous substitutions to amino acid sequences of annotated genes. This step allowed us to identify a 1-Mb region with 24 non-synonymous SNPs, representing the introgressed region that differentiates Li2 from its NIL. Next, we sequenced total DNA from pools of F2 plants, using a super bulked segregant analysis sequencing (sBSAseq) approach. The sBSAseq predicted 82 non-synonymous SNPs among 3,494 SNPs in a 3-Mb region that includes the region identified by RNAseq. We designed subgenome-specific SNP markers and tested them in an F2 population of 1,733 individuals to construct a genetic map. Our resulting genetic interval contains only one gene, an aquaporin, which is highly expressed in wild-type fibers and is significantly under-expressed in elongating Li2 fiber cells.

The inheritance model for the fiberless trait in upland cotton (Gossypium hirsutum L.) line SL1-7-1: variation on a theme

Segregating populations were developed to evaluate the inheritance of the fiberless seed phenotype of upland cotton (Gossypium hirsutum L.) line SL1-7-1. We report the inheritance of fuzzy, fuzzless and fiberless seed from crosses of SL1-7-1 with wildtype DP5690, Mexican fuzzless seed UA 3-3 (accession 143), Ballard fuzzless seed (accession 243), and MD17. Results from the F1, F2 and F2:3 progeny derived from the SL1-7-1 X DP5690 indicated that the expression of the fiberless phenotype fit a three loci model with one locus being the dominant fuzzless seed allele N1. The other two loci were tested to verify whether they were allelic to either recessive fuzzless seed alleles n2 or n3. Using the segregation ratios of the F2 progeny derived from the 143 X SL1-7-1 cross and F2-derived F3 families from SL1-7-1 X DP5690 with fuzzy seed (lacked N1), it is proposed that SL1-7-1 lacks the recessive n2 allele, but contains the n3 allele in the genotype of SL1-7-1. The third locus was previously not characterized and has been designated as fl1 (fiberless), therefore, the genotype for the fiberless phenotype of SL1-7-1 is N1N1fl1fl1n3n3. Fiberless lines MD17 X SL1-7-1 were crossed to verify similarities in genotypes between line and the genotype model predictability. Various combinations of the homozygous and heterozygous expression of N1, n2, n3 and fl1 allele produced plants with lower lint percentages.

Effects of fuzzless cottonseed phenotype on cottonseed nutrient composition in near isogenic cotton (Gossypium hirsutum L.) mutant lines under well-watered and water stress conditions.

There is no information available on the effect of fuzzless seed trait on cottonseed nutrient composition (minerals, N, S, protein, and oil) under drought stress. The objective of this research was to investigate the effect of the fuzzless seed trait on cottonseed nutrients using five sets of near-isogenic lines (NILs). Each set consists of two lines that share the same genetic background, but differ in seed fuzziness (fuzzy, F; fuzzless, N). The near isogenic lines will enable us to compare the effect of the trait without confounding the genotypic background effects. We hypothesized that since the fuzzless trait involved in fiber initiation development, and was reported to be involved in biochemical, molecular, and genetic processes, this trait may also alter cottonseed nutrient composition. Results showed that NIL sets accumulated different levels of minerals in seeds and leaves, and the fuzzless trait (N) in most of the lines altered seed and leaf mineral accumulations when compared with fuzzy lines (F) or the control line. For example, K, P, Mg, Cu, and Na concentrations in seeds were higher in MD N and STV N than in their equivalent MD F and STV F lines. Leaf concentrations of Ca, K, Mg, S, B, Cu, and Fe in MD N lines were higher than MD F line. Lower levels of nutrients in seeds and leaves were observed under water stress conditions, especially Ca, Mg, N, and B in seeds.Generally and with few exceptions, seed protein was higher in fuzzy lines than in fuzzless lines; however, seed oil was higher in fuzzless lines than in fuzzy lines. Our research demonstrated that fuzzless trait altered the composition and level of nutrients in seed and leaves in well watered and water stressed plants. Differences in protein and oil between fuzzy and fuzzless seeds may indicate alteration in nitrogen and carbon fixation and metabolism. The differential accumulation of seed nutrients in this germplasm could be used by cotton breeders to select for higher cottonseed quality.

Cotton benzoquinone reductase: Up-regulation during early fiber development and heterologous expression and characterization in Pichia pastoris

Benzoquinone reductase (BR; EC 1.6.5.7) is an enzyme which catalyzes the bivalent redox reactions of quinones without the production of free radical intermediates. Using 2D-PAGE comparisons, two proteins were found to be up-regulated in wild-type cotton ovules during the fiber initiation stage but not in the fiberless line SL 1-7-1. These proteins were excised from the gel, partially sequenced and identified to be BR isoforms. PCR was used to amplify both full length coding regions of 609bp and once cloned, the restriction enzyme HindIII was used to distinguish the clones encoding the BR1 (one site) and BR2 (two sites) isoforms. Both deduced protein sequences had 203 residues which differed at 14 residues. The molecular mass and pIs were similar between the measured protein (2D-PAGE) and the theoretical protein (deduced). Heterologous proteins BR1 and BR2 were produced for further study by ligating the BR1 and BR2 clones in frame into the alpha-factor secretion sequence in pPICZalphaA vector and expressed with Pichia pastoris. Both BR1 and BR2 were approximately 26.5kDa and did enzymatically reduce 2,6-dimethoxybenzoquinone similar to the fungal BR.

Ontogeny of Cotton Seeds: Gametogenesis, Embryogenesis, Germination, and Seedling Growth

Production of a viable seed is of paramount importance to the survival of a plant species. Many plants, including cotton, amass large reserves of storage protein and oil in their seeds (oilseeds) to use during dormancy, germination, and seedling growth. These reserves are mobilized after imbibition when environmental conditions are favorable for growth. Due to their rich supply of protein and oil, e.g., approximately 80% of the dry weight of cottonseed kernels consists of protein and oil reserves (Doman et al., 1982), oilseeds have also been used as a human food source. The nutritional value of each oilseed variety or species differs; therefore, minor changes in either protein or oil composition could result in major nutritional improvements and increased economic value of the crop. Improving seed quality is not restricted to increasing its nutritional value; it can also be accomplished by increasing seed vigor, viability, cold tolerance, or resistance to disease. A better understanding of the cellular processes involved in the ontogeny of cottonseeds will lead to new approaches in seed improvement. Discussion of developmental events from gametogenesis to seedling growth will be presented and will focus on important processes involved in producing a viable cottonseed and plant.