A deletion/duplication in the Ligon lintless-2 locus induces siRNAs that inhibit cotton fiber cell elongation.

Most cultivated cotton (Gossypium hirsutum L.) varieties have two types of seed fibers: short fuzz fiber strongly adhered to the seed coat, and long lint fiber used in the textile industry. The Ligon lintless-2 (Li2) cotton mutant has a normal vegetative phenotype but produces very short lint fiber on the seeds. The Li2 mutation is controlled by a single dominant gene. We discovered a large structural rearrangement at the end of chromosome D13 in the Li2 mutant based on whole-genome sequencing and genetic mapping of segregating populations. The rearrangement contains a 177-kb deletion and a 221-kb duplication positioned as a tandem inverted repeat. The gene Gh_D13G2437 is located at the junction of the inverted repeat in the duplicated region. During transcription such structure spontaneously forms self-complementary hairpin RNA of Gh_D13G2437 followed by production of small interfering RNA (siRNA). Gh_D13G2437 encodes a Ran-Binding Protein 1 (RanBP1) that preferentially expresses during cotton fiber elongation. The abundance of siRNA produced from Gh_D13G2437 reciprocally corresponds with the abundance of highly homologous (68%-98% amino acid sequence identity) RanBP1 family transcripts during fiber elongation, resulting in a shorter fiber phenotype in the Li2. Overexpression of Gh_D13G2437 in the Li2 mutant recovered the long lint fiber phenotype. Taken together, our findings revealed that siRNA-induced silencing of a family of RanBP1s inhibit elongation of cotton fiber cells in the Li2 mutant.

Mapping-by-sequencing the locus of EMS-induced mutation responsible for tufted-fuzzless seed phenotype in cotton.

Cotton fiber mutants are valuable resources for studying functions of altered genes and their roles in fiber development. The n4t is a recessive tufted-fuzzless seed mutant created through chemical mutagenesis with ethyl methanesulfonate. Genetic analysis indicated that the tufted-fuzzless phenotype is controlled by a single recessive locus. In this study, we developed an F2 population of 602 progeny plants and sequenced the genomes of the parents and two DNA bulks from F2 progenies showing the mutant phenotype. We identified DNA sequence variants between the tufted-fuzzless mutant and wild type by aligning the sequence reads to the reference TM-1 genome and designed subgenome-specific SNP markers. We mapped the n4t locus on chromosome D04 within a genomic interval of about 411 kb. In this region, seven genes showed significant differential expression between the tufted-fuzzless mutant and wild type. Possible candidate genes are discussed in this study. The utilization of the n4t mutant along with other fiber mutants will facilitate our understanding of the molecular mechanisms of cotton fiber cell growth and development.

Elucidation of sequence polymorphism in fuzzless-seed cotton lines.

Most commercially produced cotton cultivars have two types of fibers on the seed coat, short fuzz and long lint. Lint fiber is used in the textile industry, while fuzz is considered an undesirable trait. Both types of fibers are believed to be controlled by the same regulators; however, their mechanisms of actions are still obscure. Cotton fiber mutants provide an excellent system to study the genes that regulate fiber development. Here we described four uncharacterized and three previously reported cotton mutants with fuzzless seed phenotypes. To evaluate whether or not the genes previously associated with fuzzless seed phenotypes have mutations we sequenced whole genomic DNA of seven mutants and wild type varieties. We identified multiple polymorphic changes among the tested genes. Non-synonymous SNPs in the coding region of the MML3-A gene was common in the six mutant lines tested in this study, showing both dominant and recessive fuzzless phenotypes. We have mapped the locus of the causative mutation for one of the uncharacterized fuzzless lines using an F2 population that originated from a cross between the dominant fuzzless mutant and a wild type. Further, we have clarified the current knowledge about the causative n2 mutations by analyzing the sequence data and previously reported mapping data. The key genes and possible mechanisms of fiber differentiation are discussed in this study.

An EMS-induced mutation in a tetratricopeptide repeat-like superfamily protein gene (Ghir_A12G008870) on chromosome A12 is responsible for the liy short fiber phenotype in cotton.

KEY MESSAGE: The EMS-induced threonine/isoleucine substitution in a tetratricopeptide repeat-like superfamily protein encoded by gene Ghir_A12G008870 is responsible for the Ligon-lintless-y (liy) short fiber phenotype in cotton. A short fiber mutant Ligon-lintless-y was created through treating the seeds of the cotton line MD15 with ethyl methanesulfonate. Genetic analysis indicated that the short fiber phenotype is controlled by a single recessive locus designated liy. From F2 populations derived from crosses between the mutant and its wild type (WT), we selected 132 short fiber progeny (liy/liy) and made two DNA bulks. We sequenced these DNA bulks along with the two parents of the population. The liy locus was located on chromosome A12. Using multiple F2 populations and F3 progeny plants, we mapped the liy locus within a genomic region of 1.18 Mb. In this region, there is only one gene, i.e., Ghir_A12G008870 encoding a tetratricopeptide repeat-like superfamily protein that has a non-synonymous mutation between the liy mutant and its WT. Analysis of a SNP marker representing this gene in the F2 and F3 progeny plants demonstrated its complete linkage with the liy short fiber phenotype. We further analyzed this SNP marker in a panel of 384 cotton varieties. The mutant allele is absent in all varieties analyzed. RNAseq and RT-qPCR analysis of the gene Ghir_A12G008870 during fiber development showed a significant expression difference between the liy mutant and its WT in developing fiber cells beginning at 12 days post-anthesis. Virus-induced gene silencing of the gene Ghir_A12G008870 significantly reduced the fiber length of the WT cotton line MD15. Taken together, our results suggest that the gene Ghir_A12G008870 is involved in the cotton fiber cell elongation process and is a promising candidate gene responsible for the liy short fiber phenotype.

Genetic and transcriptomic dissection of the fiber length trait from a cotton (Gossypium hirsutum L.) MAGIC population.

BACKGROUND: Improving cotton fiber length without reducing yield is one of the major goals of cotton breeding. However, genetic improvement of cotton fiber length by breeding has been a challenge due to the narrow genetic diversity of modern cotton cultivars and negative correlations between fiber quality and yield traits. A multi-parent advanced generation inter-cross (MAGIC) population developed through random mating provides an excellent genetic resource that allows quantitative trait loci (QTL) and causal genes to be identified. RESULTS: An Upland cotton MAGIC population, consisting of 550 recombinant inbred lines (RILs) derived from eleven different cultivars, was used to identify fiber length QTLs and potential genes that contribute to longer fibers. A genome wide association study (GWAS) identified a cluster of single nucleotide polymorphisms (SNPs) on chromosome (Chr.) D11 that is significantly associated with fiber length. Further evaluation of the Chr. D11 genomic region among lines of the MAGIC population detected that 90% of RILs have a D11 haplotype similar to the reference TM-1 genome (D11-ref), whereas 10% of RILs inherited an alternative haplotype from one of the parents (D11-alt). The average length of fibers of D11-alt RILs was significantly shorter compared to D11-ref RILs, suggesting that alleles in the D11-alt haplotype contributed to the inferior fiber quality. RNAseq analysis of the longest and shortest fiber length RILs from D11-ref and D11-alt populations identified 949 significantly differentially expressed genes (DEGs). Gene set enrichment analysis revealed that different functional categories of genes were over-represented during fiber elongation between the four selected RILs. We found 12 genes possessing non-synonymous SNPs (nsSNPs) significantly associated with the fiber length, and three that were highly significant and were clustered at D11:24-Mb, including D11G1928, D11G1929 and D11G1931. CONCLUSION: The results of this study provide insights into molecular aspects of genetic variation in fiber length and suggests candidate genes for genetic manipulation for cotton improvement.

A Gly65Val substitution in an actin, GhACT_LI1, disrupts cell polarity and F-actin organization resulting in dwarf, lintless cotton plants.

Actin polymerizes to form part of the cytoskeleton and organize polar growth in all eukaryotic cells. Species with numerous actin genes are especially useful for the dissection of actin molecular function due to redundancy and neofunctionalization. Here, we investigated the role of a cotton (Gossypium hirsutum) actin gene in the organization of actin filaments in lobed cotyledon pavement cells and the highly elongated single-celled trichomes that comprise cotton lint fibers. Using mapping-by-sequencing, virus-induced gene silencing, and molecular modeling, we identified the causative mutation of the dominant dwarf Ligon lintless Li1 short fiber mutant as a single Gly65Val amino acid substitution in a polymerization domain of an actin gene, GhACT_LI1 (Gh_D04G0865). We observed altered cell morphology and disrupted organization of F-actin in Li1 plant cells by confocal microscopy. Mutant leaf cells lacked interdigitation of lobes and F-actin did not uniformly decorate the nuclear envelope. While wild-type lint fiber trichome cells contained long longitudinal actin cables, the short Li1 fiber cells accumulated disoriented transverse cables. The polymerization-defective Gly65Val allele in Li1 plants likely disrupts processive elongation of F-actin, resulting in a disorganized cytoskeleton and reduced cell polarity, which likely accounts for the dominant gene action and diverse pleiotropic effects associated with the Li1 mutation. Lastly, we propose a model to account for these effects, and underscore the roles of actin organization in determining plant cell polarity, shape and plant growth.

Genome-wide analysis of gene expression of EMS-induced short fiber mutant Ligon lintless-y (liy) in cotton (Gossypium hirsutum L.).

In this work we describe a chemically-induced short fiber mutant cotton line, Ligon-lintless-y (liy), which is controlled by a single recessive locus and affects multiple traits, including height of the plant, and length and maturity of fiber. An RNAseq analysis was used to evaluate global transcriptional changes during cotton fiber development at 3, 8 and 16days post anthesis. We found that 613, 2629 and 3397 genes were significantly down-regulated, while 2700, 477 and 3260 were significantly up-regulated in liy at 3, 8 and 16 DPA. Gene set enrichment analysis revealed that many metabolic pathways, including carbohydrate, cell wall, hormone metabolism and transport were substantially altered in liy developing fibers. We discuss perturbed expression of genes involved in signal transduction and biosynthesis of phytohormones, such as auxin, abscisic acid, gibberellin and ethylene. The results of this study provide new insights into transcriptional regulation of cotton fiber development.

Small RNA sequencing and degradome analysis of developing fibers of short fiber mutants Ligon-lintles-1 (Li 1 ) and -2 (Li 2 ) revealed a role for miRNAs and their targets in cotton fiber elongation.

BACKGROUND: The length of cotton fiber is an important agronomic trait that directly affects the quality of yarn and fabric. Understanding the molecular basis of fiber elongation would provide a means for improvement of fiber length. Ligon-lintless-1 (Li 1 ) and -2 (Li 2 ) are monogenic and dominant mutations that result in an extreme reduction in the length of lint fiber on mature seeds. In a near-isogenic state with wild type cotton these two short fiber mutants provide an effective model system to study the mechanisms of fiber elongation. Plant miRNAs regulate many aspects of growth and development. However, the mechanism underlying the miRNA-mediated regulation of fiber development is largely unknown. RESULTS: Small RNA libraries constructed from developing fiber cells of the short fiber mutants Li 1 and Li 2 and their near-isogenic wild type lines were sequenced. We identified 24 conservative and 147 novel miRNA families with targets that were detected through degradome sequencing. The distribution of the target genes into functional categories revealed the largest set of genes were transcription factors. Expression profiles of 20 miRNAs were examined across a fiber developmental time course in wild type and short fiber mutations. We conducted correlation analysis between miRNA transcript abundance and the length of fiber for 11 diverse Upland cotton lines. The expression patterns of 4 miRNAs revealed significant negative correlation with fiber lengths of 11 cotton lines. CONCLUSIONS: Our results suggested that the mutations have changed the regulation of miRNAs expression during fiber development. Further investigations of differentially expressed miRNAs in the Li 1 and Li 2 mutants will contribute to better understanding of the regulatory mechanisms of cotton fiber development. Four miRNAs negatively correlated with fiber length are good candidates for further investigations of miRNA regulation of important genotype dependent fiber traits. Thus, our results will contribute to further studies on the role of miRNAs in cotton fiber development and will provide a tool for fiber improvement through molecular breeding.

Methoprene and temperature effects on caste differentiation and protein composition in the Formosan Subterranean termite, Coptotermes formosanus.

The utilization of multiple castes is a shared feature of social insects. In termites, multiple extrinsic factors have been shown to impact caste differentiation; for example, increased temperature has been shown to increase soldier production. Also, application of exogenous methoprene has also been demonstrated to increase soldier production. The objective of this investigation was to examine and correlate the effects of temperature variation and methoprene treatments on termite caste differentiation, and identify the resulting changes in protein levels. Our results indicate that worker-to-soldier differentiation is modulated by temperature, where a greater number of soldiers developed at a higher rate at higher temperatures compared to lower temperatures. We analyzed total protein by sodium dodecyl sulfate Polyacrylamide gel electrophoresis and N-terminal sequencing and found several changes. Specifically, four proteins affected by temperature change were identified: Hexamerin-1, Hexamerin-2, Endo-beta 1,4 glucanase, and myosin. These proteins were further examined for their response to temperature, assay length (time), and exposure to the juvenile hormone analog methoprene. Hexamerin-1 protein showed a temperature-and assay length-dependent effect, while Hexamerin-2, Endo-beta 1, 4 glucanase, and myosin protein levels were all affected by temperature, assay length, and exposure to methoprene. Our analysis allows the correlation of temperature, assay length, and presence of methoprene with specific changes in protein levels that occur during caste differentiation. These results can be directly applied to better understand the complex developmental factors that control termite differentiation and guide the use of juvenile hormone analogs to maximize efficiency of termite eradication in the field.

Role of xyloglucan in cotton (Gossypium hirsutum L.) fiber elongation of the short fiber mutant Ligon lintless-2 (Li2).

Xyloglucan is a matrix polysaccharide found in the cell walls of all land plants. In growing cells, xyloglucan is thought to connect cellulose microfibrils and regulate their separation during wall extension. Ligon lintless-2 (Li2) is a monogenic dominant cotton fiber mutation that causes extreme reduction in lint fiber length with no pleiotropic effects on vegetative growth. Li2 represents an excellent model system to study fiber elongation. To understand the role of xyloglucan in cotton fiber elongation we used the short fiber mutant Li2 and its near isogenic wild type for analysis of xyloglucan content and expression of xyloglucan-related genes in developing fibers. Accumulation of xyloglucan was significantly higher in Li2 developing fibers than in wild type. Genes encoding enzymes for nine family members of xyloglucan biosynthesis were identified in the draft Gossypium hirsutum genome. RNAseq analysis revealed that most differentially expressed xyloglucan-related genes were down-regulated in Li2 fiber cells. RT-qPCR analysis revealed that the peak of expression for the majority of xyloglucan-related genes in wild type developing fibers was 5-16days post anthesis (DPA) compared to 1-3 DPA in Li2 fibers. Thus, our results suggest that early activation of xyloglucan-related genes and down regulation of xyloglucan degradation genes during the elongation phase lead to elevated accumulation of xyloglucan that restricts elongation of fiber cells in Li2.

Cross-reaction between Formosan termite (Coptotermes formosanus) proteins and cockroach allergens.

Cockroach allergens can lead to serious allergy and asthma symptoms. Termites are evolutionarily related to cockroaches, cohabitate in human dwellings, and represent an increasing pest problem in the United States. The Formosan subterranean termite (Coptotermes formosanus) is one of the most common species in the southern United States. Several assays were used to determine if C. formosanus termite proteins cross-react with cockroach allergens. Expressed sequence tag and genomic sequencing results were searched for homology to cockroach allergens using BLAST 2.2.21 software. Whole termite extracts were analyzed by mass-spectrometry, immunoassay with IgG and scFv antibodies to cockroach allergens, and human IgE from serum samples of cockroach allergic patients. Expressed sequence tag and genomic sequencing results indicate greater than 60% similarity between predicted termite proteins and German and American cockroach allergens, including Bla g 2/Per a 2, Bla g 3/Per a 3, Bla g 5, Bla g 6/Per a 6, Bla g 7/Per a 7, Bla g 8, Per a 9, and Per a 10. Peptides from whole termite extract were matched to those of the tropomyosin (Bla g 7), arginine kinase (Per a 9), and myosin (Bla g 8) cockroach allergens by mass-spectrometry. Immunoblot and ELISA testing revealed cross-reaction between several proteins with IgG and IgE antibodies to cockroach allergens. Several termite proteins, including the hemocyanin and tropomyosin orthologs of Blag 3 and Bla g 7, were shown to crossreact with cockroach allergens. This work presents support for the hypothesis that termite proteins may act as allergens and the findings could be applied to future allergen characterization, epitope analysis, and clinical studies.

Myosin Gene Expression and Protein Abundance in Different Castes of the Formosan Subterranean Termite (Coptotermes formosanus).

The Formosan subterranean termite (Coptotermes formosanus) is an important worldwide pest, each year causing millions of dollars in structural damage and control costs. Termite colonies are composed of several phenotypically distinct castes. Termites utilize these multiple castes to efficiently perform unique roles within the colony. During the molting/caste differentiation process, multiple genes are believed to be involved in the massive reorganization of the body plan. The objective of this research was to analyze the muscle gene, myosin, to further understand the role it plays in C. formosanus development. We find that comparing worker vs. solider caste myosin gene expression is up-regulated in the soldier and a myosin antibody-reactive protein suggests changes in splicing. Comparison of body regions of mature soldier and worker castes indicates a greater level of myosin transcript in the heads. The differential expression of this important muscle-related gene is anticipated considering the large amount of body plan reorganization and muscle found in the soldier caste. These results have a direct impact on our understanding of the downstream genes in the caste differentiation process and may lead to new targets for termite control.

Diet-mediated inter-colonial aggression in the formosan subterranean termite Coptotermes formosanus.

In most social insects, intercolonial and interspecific aggression are expressions of territoriality. In termites, cuticular hydrocarbons (CHCs) have been extensively studied for their role in nestmate recognition and aggressive discrimination of nonnest-mates. More recently, molecular genetic techniques have made it possible to determine relatedness between colonies and to investigate the influence of genetics on aggression. In the Formosan subterranean termite, Coptotermes formosanus, however, the role of CHCs and genetic relatedness in inter-colony aggression has been ambiguous, suggesting the involvement of additional factors in nest-mate recognition. In this study we assess the range of aggression in this termite species and characterize the influence of genetic relatedness, CHC profiles and diet on aggression levels. We collected four colonies of C. formosanus, feeding either on bald cypress or birch, from three locations in Louisiana. Inter-colony aggression ranged from low to high. Differences in CHC profiles, as well as genetic distances between colonies determined by using microsatellite DNA markers, showed no significant correlation with aggression. However, termite diet (host tree) played a significant role in determining the level of aggression. Thus, two distantly related colonies, each feeding on different diets, showed high aggression that significantly diminished if they were fed on the same wood in the laboratory (spruce). Using headspace solid phase microextraction, we found three compounds from workers fed on birch that were absent in workers fed on spruce. Such diet-derived chemicals may be involved in the complex determination of nest-mate recognition in C. formosanus.