A MAGIC population-based genome-wide association study reveals functional association of GhRBB1_A07 gene with superior fiber quality in cotton

Title: Quantile-quantile (Q-Q) Plot of six fiber traits generated from GWAS analysis following mixed linear model (MLM) using GAPIT software. A) Fiber elongation (ELO), B) Micronaire (MIC), C) Short fiber content (SFC), D) Fiber strength (STR), E) Upper half mean fiber length (UHM), and F) Uniformity index (UI). Description of data: Q-Q plots of six fiber traits generated from GWAS analysis following MLM are included in this figure. The X and Y axis have the expected and observed negative logarithm 10 of p value, respectively generated during GWAS analysis. (DOCX 207 kb

Nicotiana sylvestris, a model plant for cell biology: organelle movement and retrotransposon mutagenesis

Nicotiana sylvestris is a diploid tobacco plant that is amenable to laboratory manipulation including facile transformation of nuclear and plastid (chloroplast) genomes. In three separate studies, I used this model organism to observe biological processes with evolutionary and biotechnological implications. The first addresses the mechanisms of horizontal gene transfer by demonstrating cell-to-cell movement of plastids. We grafted Nicotiana sylvestris plants with selectable transgenic plastid genomes to Nicotiana tabacum plants with selectable transgenic nuclear markers. Grafting triggers formation of new cell-tocell contacts, creating an opportunity for organelle movement between the plant cells. I present evidence for cell-to-cell movement of the entire 161-kb plastid genome in these plants, most likely in intact plastids. Acquisition of plastids from neighboring cells provides a mechanism by which cells may be repopulated with functioning organelles. My second objective was to determine whether exceptional pollen transmissionof plastids is accompanied by paternal mitochondria transmission in Nicotiana sylvestris. Plastids and mitochondria in Nicotiana are normally both inherited from the maternal parent. We observed that plastids from the N. sylvestris father were transmitted at a low (~0.002%) frequency via pollen. The plants that inherited paternal plastids did not carry paternal mitochondrial DNA, indicating that leakage of plastids via pollen can produce plant lines with unrelated plastids and mitochondria. My third objective was to observe the behavior of an individual high-copy retrotransposon in N. sylvestris, its native host. Long terminal repeat (LTR) retrotransposons are major components of the nuclear genomes of plants, animals and fungi. The “copy-and-paste” life cycle of retrotransposons accounts for their accumulation in host genomes and permits the assumption that LTRs are identical at the time of insertion. Our objective was to experimentally determine if an introduced synthetic element would interact with native high-copy elements during retrotransposition. I present evidence that S-TNT1 co-packaged with native TNT1 elements to produce hybrid insertions with swapped LTRs and multiple recombinations within the gag-pol gene. We can best explain our observations by dimerization and co-packaging of TNT1 gRNAs in the cytoplasm, followed by template-switching during minus-strand DNA synthesis, which we term the “mixand- paste” pseudodiploid mating system for LTR-retroelements.Ph. D.Includes bibliographical referencesIncludes vitaby Gregory N. Thysse

Phenomics and transcriptomics analyses reveal deposition of suberin and lignin in the short fiber cell walls produced from a wild cotton species and two mutants.

Fiber length is one of the major properties determining the quality and commercial value of cotton. To understand the mechanisms regulating fiber length, genetic variations of cotton species and mutants producing short fibers have been compared with cultivated cottons generating long and normal fibers. However, their phenomic variation other than fiber length has not been well characterized. Therefore, we compared physical and chemical properties of the short fibers with the long fibers. Fiber characteristics were compared in two sets: 1) wild diploid Gossypium raimondii Ulbrich (short fibers) with cultivated diploid G. arboreum L and tetraploid G. hirsutum L. (long fibers); 2) G. hirsutum short fiber mutants, Ligon-lintless 1 (Li1) and 2 (Li2) with their near isogenic line (NIL), DP-5690 (long fibers). Chemical analyses showed that the short fibers commonly consisted of greater non-cellulosic components, including lignin and suberin, than the long fibers. Transcriptomic analyses also identified up-regulation of the genes related to suberin and lignin biosynthesis in the short fibers. Our results may provide insight on how high levels of suberin and lignin in cell walls can affect cotton fiber length. The approaches combining phenomic and transcriptomic analyses of multiple sets of cotton fibers sharing a common phenotype would facilitate identifying genes and common pathways that significantly influence cotton fiber properties

Phenomics and transcriptomics analyses reveal deposition of suberin and lignin in the short fiber cell walls produced from a wild cotton species and two mutants

Fiber length is one of the major properties determining the quality and commercial value of cotton. To understand the mechanisms regulating fiber length, genetic variations of cotton species and mutants producing short fibers have been compared with cultivated cottons generating long and normal fibers. However, their phenomic variation other than fiber length has not been well characterized. Therefore, we compared physical and chemical properties of the short fibers with the long fibers. Fiber characteristics were compared in two sets: 1) wild diploid Gossypium raimondii Ulbrich (short fibers) with cultivated diploid G. arboreum L and tetraploid G. hirsutum L. (long fibers); 2) G. hirsutum short fiber mutants, Ligon-lintless 1 (Li1) and 2 (Li2) with their near isogenic line (NIL), DP-5690 (long fibers). Chemical analyses showed that the short fibers commonly consisted of greater non-cellulosic components, including lignin and suberin, than the long fibers. Transcriptomic analyses also identified up-regulation of the genes related to suberin and lignin biosynthesis in the short fibers. Our results may provide insight on how high levels of suberin and lignin in cell walls can affect cotton fiber length. The approaches combining phenomic and transcriptomic analyses of multiple sets of cotton fibers sharing a common phenotype would facilitate identifying genes and common pathways that significantly influence cotton fiber properties

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

Abstract 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

The P450 gene CYP749A16 is required for tolerance to the sulfonylurea herbicide trifloxysulfuron sodium in cotton (Gossypium hirsutum L.)

Abstract Background Weed management is critical to global crop production and is complicated by rapidly evolving herbicide resistance in weeds. New sources of herbicide resistance are needed for crop plants so that applied herbicides can be rotated or combined to thwart the evolution of resistant weeds. The diverse family of cytochrome P450 proteins has been suggested to be a source of detoxifying herbicide metabolism in both weed and crop plants, and greater understanding of these genes will offer avenues for crop improvement and novel weed management practices. Results Here, we report the identification of CYP749A16 (Gh_D10G1401) which is responsible for the natural tolerance exhibited by most cotton, Gossypium hirsutum L., cultivars to the herbicide trifloxysulfuron sodium (TFS, CGA 362622, commercial formulation Envoke). A 1-bp frameshift insertion in the third exon of CYP749A16 results in the loss of tolerance to TFS. The DNA marker designed from this insertion perfectly co-segregated with the phenotype in 2145 F2 progeny of a cross between the sensitive cultivar Paymaster HS26 and tolerant cultivar Stoneville 474, and in 550 recombinant inbred lines of a multi-parent advanced generation inter-cross population. Marker analysis of 382 additional cotton cultivars identified twelve cultivars containing the 1-bp frameshift insertion. The marker genotypes matched perfectly with phenotypes in 188 plants from the selected twelve cultivars. Virus-induced gene silencing of CYP749A16 generated sensitivity in the tolerant cotton cultivar Stoneville 474. Conclusions CYP749A16 located on chromosome D10 is required for TFS herbicide tolerance in cotton. This finding should add to the repertoire of tools available to farmers and breeders for the advancement of agricultural productivity

Multi-omics analysis of pigmentation related to proanthocyanidin biosynthesis in brown cotton (Gossypium hirsutum L.)

Naturally-colored brown cotton (NBC) fiber is an environmentally friendly raw source of fiber for textile applications. The fiber of some NBC cultivars exhibits flame-retardant properties, which can be used in textiles that require flame resistance. Proanthocyanidins or their derivatives are responsible for the brown pigment in NBC; however, how flame retardancy is related to pigmentation in NBC is poorly understood. To gain insight into brown pigment biosynthesis, we conducted comparative transcripts and metabolites profiling analysis of developing cotton fibers between the brown (MC-BL) and white (MC-WL) cotton near-isogenic lines (NILs), genetically different only in the Lc1 locus. In this study, mass spectrometry was used to detect metabolites in BL and WL developing fibers at 8, 12, 16, 20, 24, 36, and 40 days post anthesis (DPA) and mature fibers. Transcripts analysis was performed at two critical fiber developmental points, 8 DPA (fiber elongation) and 20 DPA (secondary cell wall deposition). We found 5836 (ESI MS positive mode) and 4541 (ESI MS negative mode) metabolites significantly different accumulated between BL and WL. Among them, 142 were known non-redundant metabolites, including organic acids, amino acids, and derivatives of the phenylpropanoid pathway. Transcript analysis determined 1691 (8 DPA) and 5073 (20 DPA) differentially expressed genes (DEGs) between BL and WL, with the majority of DEGs down-regulated at 20 DPA. Organic acids of the citric acid cycle were induced, while most of the detected amino acids were reduced in the MC-BL line. Both cis- and trans-stereoisomers of flavan-3-ols were detected in developing MC-WL and MC-BL fibers; however, the gallocatechin and catechin accumulated multiple times higher. Gas chromatography-mass spectrometry (GC-MS) analysis of fatty acids determined that palmitic acid long-chain alcohols were the main constituents of waxes of mature fibers. Energy-dispersive X-ray spectrometry (EDS) analysis of mature fibers revealed that potassium accumulated three times greater in MC-BL than in MC-WL mature fibers. This study provides novel insights into the biosynthesis of pigments and its association with flame retardancy in NBC fibers

The Immature Fiber Mutant Phenotype of Cotton (Gossypium hirsutum) Is Linked to a 22-bp Frame-Shift Deletion in a Mitochondria Targeted Pentatricopeptide Repeat Gene

Cotton seed trichomes are the most important source of natural fibers globally. The major fiber thickness properties influence the price of the raw material, and the quality of the finished product. The recessive immature fiber (im) gene reduces the degree of fiber cell wall thickening by a process that was previously shown to involve mitochondrial function in allotetraploid Gossypium hirsutum. Here, we present the fine genetic mapping of the im locus, gene expression analysis of annotated proteins near the locus, and association analysis of the linked markers. Mapping-by-sequencing identified a 22-bp deletion in a pentatricopeptide repeat (PPR) gene that is completely linked to the immature fiber phenotype in 2837 F2 plants, and is absent from all 163 cultivated varieties tested, although other closely linked marker polymorphisms are prevalent in the diversity panel. This frame-shift mutation results in a transcript with two long open reading frames: one containing the N-terminal transit peptide that targets mitochondria, the other containing only the RNA-binding PPR domains, suggesting that a functional PPR protein cannot be targeted to mitochondria in the im mutant. Taken together, these results suggest that PPR gene Gh_A03G0489 is involved in the cotton fiber wall thickening process, and is a promising candidate gene at the im locus. Our findings expand our understanding of the molecular mechanisms that modulate cotton fiber fineness and maturity, and may facilitate the development of cotton varieties with superior fiber attributes