Genome-Wide Analysis of DA1-Like Genes in Gossypium and Functional Characterization of GhDA1-1A Controlling Seed Size.

Cotton (Gossypium spp.) is an economically important crop grown for natural fiber and seed oil production. DA1 is a ubiquitin receptor that determines final seed and organ size by restricting the period of cell proliferation. In the present study, we identified 7 DA1-like genes each in cultivated tetraploid (AADD) G. hirsutum and G. barbadense, and 4 and 3 DA1-like genes in their ancestral diploid G. arboreum (A2A2) and G. raimondii (D5D5), respectively. The 7 GhDA1 genes were confirmed to be distributed on four At and three Dt subgenome chromosomes in G. hirsutum. GhDA1-1A showed a high sequence similarity to AtDA1 in Arabidopsis, and they possessed the same functional domains, suggesting conserved functions. The overexpression of GhDA1-1A R301K in Arabidopsis significantly increased seed size and seed weight, indicating that GhDA1-1A is a promising target for cotton improvement. This study provides information on the molecular evolutionary properties of DA1-like genes in cotton, which will be useful for the genetic improvement of cotton.

Analysis of the MIR160 gene family and the role of MIR160a_A05 in regulating fiber length in cotton.

MAIN CONCLUSION: The MIR160 family in Gossypium hirsutum and G. barbadense was characterized, and miR160a_A05 was found to increase cotton-fiber length by downregulating its target gene (ARF17) and several GH3 genes. Cotton fiber is the most important raw material for the textile industry. MicroRNAs are involved in regulating cotton-fiber development, but a role in fiber elongation has not been demonstrated. In this study, miR160a was found to be differentially expressed in elongating fibers between two interspecific (between Gossypium hirsutum and G. barbadense) backcross inbred lines (BILs) with different fiber lengths. The gene MIR160 colocalized with a previously mapped fiber-length quantitative trait locus. Its target gene ARF17 was differentially expressed between the two BILs during fiber elongation, but in the inverse fashion. Bioinformatics was used to analyze the MIR160 family in both G. hirsutum and G. barbadense. Moreover, qRT-PCR analysis identified MIR160a as the functional MIR160 gene encoding the miR160a precursor during fiber elongation. Using virus-induced gene silencing and overexpression, overexpressed MIR160a_A05 resulted in significantly longer fibers compared with wild type, whereas suppression of miR160 resulted in significantly shorter fibers. Expression levels of the target gene auxin-response factor 17 (ARF17) and related genes GH3 in the two BILs and/or the virus-infected plants demonstrated similar changes in response to modulation of miR160a level. Finally, overexpression or suppression of miR160 increased or decreased, respectively, the cellular level of indole-3-acetic acid, which is involved in fiber elongation. These results describe a specific regulatory mechanism for fiber elongation in cotton that can be utilized for future crop improvement.

A Comparative Genome-Wide Analysis of the R2R3-MYB Gene Family Among Four Gossypium Species and Their Sequence Variation and Association With Fiber Quality Traits in an Interspecific G. hirsutum × G. barbadense Population.

Cotton (Gossypium spp.) is the most important natural fiber crop in the world. The R2R3-MYB gene family is a large gene family involved in many plant functions including cotton fiber development. Although previous studies have reported its phylogenetic relationships, gene structures, and expression patterns in tetraploid G. hirsutum and diploid G. raimondii, little is known about the sequence variation of the members between G. hirsutum and G. barbadense and their involvement in the natural quantitative variation in fiber quality and yield. In this study, a comprehensive genome-wide comparative analysis was performed among the four Gossypium species using whole genome sequences, i.e., tetraploid G. hirsutum (AD1) and G. barbadense (AD2) as well as their likely ancestral diploid extants G. raimondii (D5) and G. arboreum (A2), leading to the identification of 406, 393, 216, and 213 R2R3-MYB genes, respectively. To elucidate whether the R2R3-MYB genes are genetically associated with fiber quality traits, 86 R2R3-MYB genes were co-localized with quantitative trait loci (QTL) hotspots for fiber quality and yield, including 42 genes localized within the fiber length QTL hotspots, in interspecific G. hirsutum × G. barbadense populations. There were 20 interspecific nonsynonymous single-nucleotide polymorphism (SNP) sites between the two tetraploid cultivated species, of which 16 developed from 11 R2R3-MYB genes were significantly correlated with fiber quality and yield in a backcross inbred population (BIL) of G. hirsutum × G. barbadense in at least one of the four field tests. Taken together, these results indicate that the sequence variation in these 11 R2R3-MYB genes is associated with the natural variation (i.e., QTL) in fiber quality and yield. Moreover, the functional SNPs of five R2R3-MYB allele pairs from the AD1 and AD2 genomes were significantly correlated with the gene expression related to fiber quality in fiber development. The results will be useful in further elucidating the role of the R2R3-MYB genes during fiber development.

Genome-wide association study of the oil content in upland cotton (Gossypium hirsutum L.) and identification of GhPRXR1, a candidate gene for a stable QTLqOC-Dt5-1.

Cottonseed oil is one of the most important renewable resources for edible oil and biodiesel. To detect QTLs associated with cottonseed oil content (OC) and identify candidate genes that regulate oil biosynthesis, a panel of upland cotton germplasm lines was selected among those previously used to perform GWASs in China. In the present study, 13 QTLs associated with 53 common SNPs on 13 chromosomes were identified in multiple environments based on 15,369 polymorphic SNPs using the Cotton63 KSNP array. Of these, the OC QTL qOC-Dt5-1 delineated by nine SNPs occurred in a confidence interval of 4 SSRs with previously reported OC QTLs. A combined transcriptome and qRT-PCR analysis revealed that a peroxidase gene (GhPRXR1) was predominantly expressed during the middle-late stage (20-35 days post anthesis) of ovule development. The overexpression of GhPRXR1 in yeast significantly increased the OC by 20.01-37.25 %. Suppression of GhPRXR1 gene expression in the virus-induced gene-silenced cotton reduced the OC by 18.11%. Our results contribute to identifying more OC QTLs and verifying a candidate gene that influences cottonseed oil biosynthesis.

QTL analysis and candidate gene identification for plant height in cotton based on an interspecific backcross inbred line population of Gossypium hirsutum × Gossypium barbadense.

KEY MESSAGE: We constructed the first high-quality and high-density genetic linkage map for an interspecific BIL population in cotton by specific-locus amplified fragment sequencing for QTL mapping. A novel gene GhPIN3 for plant height was identified in cotton. Ideal plant height (PH) is important for improving lint yield and mechanized harvesting in cotton. Most published genetic studies on cotton have focused on fibre yield and quality traits rather than PH. To facilitate the understanding of the genetic basis in PH, an interspecific backcross inbred line (BIL) population of 250 lines derived from upland cotton (Gossypium hirsutum L.) CRI36 and Egyptian cotton (G. barbadense L.) Hai7124 was used to construct a high-density genetic linkage map for quantitative trait locus (QTL) mapping. The high-density genetic map harboured 7,709 genotyping-by-sequencing (GBS)-based single nucleotide polymorphism (SNP) markers that covered 3,433.24 cM with a mean marker interval of 0.67 cM. In total, ten PH QTLs were identified and each explained 4.27-14.92% of the phenotypic variation, four of which were stable as they were mapped in at least two tests or based on best linear unbiased prediction in seven field tests. Based on functional annotation of orthologues in Arabidopsis and transcriptome data for the genes within the stable QTL regions, GhPIN3 encoding for the hormone auxin efflux carrier protein was identified as a candidate gene located in the stable QTL qPH-Dt1-1 region. A qRT-PCR analysis showed that the expression level of GhPIN3 in apical tissues was significantly higher in four short-statured cotton genotypes than that in four tall-statured cotton genotypes. Virus-induced gene silencing cotton has significantly increased PH when the expression of the GhPIN3 gene was suppressed.

A genome-wide analysis of the phospholipid: diacylglycerol acyltransferase gene family in Gossypium.

BACKGROUND: Cotton (Gossypium spp.) is the most important natural fiber crop worldwide, and cottonseed oil is its most important byproduct. Phospholipid: diacylglycerol acyltransferase (PDAT) is important in TAG biosynthesis, as it catalyzes the transfer of a fatty acyl moiety from the sn-2 position of a phospholipid to the sn-3 position of sn-1, 2-diacylglyerol to form triacylglycerol (TAG) and a lysophospholipid. However, little is known about the genes encoding PDATs involved in cottonseed oil biosynthesis. RESULTS: A comprehensive genome-wide analysis of G. hirsutum, G. barbadense, G. arboreum, and G. raimondii herein identified 12, 11, 6 and 6 PDATs, respectively. These genes were divided into 3 subfamilies, and a PDAT-like subfamily was identified in comparison with dicotyledonous Arabidopsis. All GhPDATs contained one or two LCAT domains at the C-terminus, while most GhPDATs contained a preserved single transmembrane region at the N-terminus. A chromosomal distribution analysis showed that the 12 GhPDAT genes in G. hirsutum were distributed in 10 chromosomes. However, none of the GhPDATs was co-localized with quantitative trait loci (QTL) for cottonseed oil content, suggesting that their sequence variations are not genetically associated with the natural variation in cottonseed oil content. Most GhPDATs were expressed during the cottonseed oil accumulation stage. Ectopic expression of GhPDAT1d increased Arabidopsis seed oil content. CONCLUSIONS: Our comprehensive genome-wide analysis of the cotton PDAT gene family provides a foundation for further studies into the use of PDAT genes to increase cottonseed oil content through biotechnology.

Genetic variation of dynamic fiber elongation and developmental quantitative trait locus mapping of fiber length in upland cotton (Gossypium hirsutum L.).

BACKGROUND: In upland cotton (Gossypium hirsutum L.), genotypes with the same mature fiber length (FL) might possess different genes and exhibit differential expression of genes related to fiber elongation at different fiber developmental stages. However, there is a lack of information on the genetic variation influencing fiber length and its quantitative trait loci (QTLs) during the fiber elongation stage. In this study, a subset of upland cotton accessions was selected based on a previous GWAS conducted in China and grown in multiple environments to determine the dynamic fiber length at 10, 15, 20, and 25 days post-anthesis (DPA) and maturity. The germplasm lines were genotyped with the Cotton 63 K Illumina single-nucleotide polymorphism (SNP) array for GWAS. RESULTS: A total of 25, 38, 57, 89 and 88 SNPs showed significant correlations with fiber length at 10, 15, 20 and 25 DPA and maturity, respectively. In addition, 60 more promising SNPs were detected in at least two tests and two FL developmental time points, and 20 SNPs were located within the confidence intervals of QTLs identified in previous studies. The fastest fiber-length growth rates were obtained at 10 to 15 DPA in 69 upland cotton lines and at 15 to 20 DPA in 14 upland cotton accessions, and 10 SNPs showed significant correlations with the fiber-length growth rate. A combined transcriptome and qRT-PCR analysis revealed that two genes (D10G1008 and D13G2037) showed differential expression between two long-fiber genotypes and two short-fiber genotypes. CONCLUSIONS: This study provides important new insights into the genetic basis of the time-dependent fiber-length trait and reveals candidate SNPs and genes for improving fiber length in upland cotton.

Overexpression of the Wheat (Triticum aestivum L.) TaPEPKR2 Gene Enhances Heat and Dehydration Tolerance in Both Wheat and Arabidopsis.

Wheat (Triticum aestivum L.) yield and quality are adversely affected by heat, drought, or the combination of these two stresses in many regions of the world. A phosphoenolpyruvate carboxylase kinase-related kinase gene, TaPEPKR2, was identified from our previous heat stress-responsive transcriptome analysis of heat susceptible and tolerant wheat cultivars. Based on the wheat cultivar Chinese Spring genome sequence, TaPEPKR2 was mapped to chromosome 5B. Expression analysis revealed that TaPEPKR2 was induced by heat and polyethylene glycol treatment. To analyze the function of TaPEPKR2 in wheat, we transformed it into the wheat cultivar Liaochun10, and observed that the transgenic lines exhibited enhanced heat and dehydration stress tolerance. To examine whether TaPEPKR2 exhibits the same function in dicotyledonous plants, we transformed it into Arabidopsis, and found that its overexpression functionally enhanced tolerance to heat and dehydration stresses. Our results imply that TaPEPKR2 plays an important role in both heat and dehydration stress tolerance, and could be utilized as a candidate gene in transgenic breeding.

Genome-Scale Analysis of the WRI-Like Family in Gossypium and Functional Characterization of GhWRI1a Controlling Triacylglycerol Content.

Cotton (Gossypium spp.) is the most important natural fiber crop and the source of cottonseed oil, a basic by-product after ginning. AtWRI1 and its orthologs in several other crop species have been previously used to increase triacylglycerols in seeds and vegetative tissues. In the present study, we identified 22, 17, 9, and 11 WRI-like genes in G. hirsutum, G. barbadense, G. arboreum, and G. raimondii, respectively. This gene family was divided into four subgroups, and a more WRI2-like subfamily was identified compared with dicotyledonous Arabidopsis. An analysis of chromosomal distributions revealed that the 22 GhWRI genes were distributed on eight At and eight Dt subgenome chromosomes. Moreover, GhWRI1a was highly expressed in ovules 20-35 days after anthesis and was selected for further functional analysis. Ectopic expression of GhWRI1a rescued the seed phenotype of a wri1-7 mutant and increased the oil content of Arabidopsis seeds. Our comprehensive genome-wide analysis of the cotton WRI-like gene family lays a solid foundation for further studies.

Unconventional splicing of wheat TabZIP60 confers heat tolerance in transgenic Arabidopsis.

Conditions that disrupt protein folding, such as heat stress, can overwhelm the capacity of cells to fold proteins, thus causing endoplasmic reticulum (ER) stress. In Arabidopsis thaliana and other plants, inositol-requiring enzyme-1 mediated unconventional splicing of bZIP60 plays a crucial role in the heat and ER stress responses. However, little is known about this pathway in wheat (Triticum aestivum), especially its importance in heat tolerance. Here, we found that heat stress induced upregulation and unconventional splicing of TabZIP60 occurred in wheat seedlings. Constitutive expression of the spliced form of TabZIP60 (TabZIP60s) enhanced heat tolerance in Arabidopsis, but overexpression of the unspliced form (TabZIP60u) did not. RNA-sequencing analysis revealed ER stress related genes involved in heat responses in TabZIP60s-overexpression transgenic Arabidopsis. Chromatin immunoprecipitation-qPCR showed that TabZIP60s directly binds to 17 target genes including AtbZIP60. Also, the 26S proteasome pathway post-translationally regulates TabZIP60s levels during heat stress responses. Our findings suggest that unconventional splicing of TabZIP60 could contribute to heat tolerance in transgenic plants by modulating the expression of ER stress-related genes.

A genome-wide analysis of the small auxin-up RNA (SAUR) gene family in cotton.

BACKGROUND: Small auxin-up RNA (SAUR) gene family is the largest family of early auxin response genes in higher plants, which have been implicated in the regulation of multiple biological processes. However, no comprehensive analysis of SAUR genes has been reported in cotton (Gossypium spp.). RESULTS: In the study, we identified 145, 97, 214, and 176 SAUR homologous genes in the sequenced genomes of G. raimondii, G. arboreum, G. hirsutum, and G. barbadense, respectively. A phylogenetic analysis revealed that the SAUR genes can be classified into 10 groups. A further analysis of chromosomal locations and gene duplications showed that tandem duplication and segmental duplication events contributed to the expansion of the SAUR gene family in cotton. An exon-intron organization and motif analysis revealed the conservation of SAUR-specific domains, and the auxin responsive elements existed in most of the upstream sequences. The expression levels of 16 GhSAUR genes in response to an exogenous application of IAA were determined by a quantitative RT-PCR analysis. The genome-wide RNA-seq data and qRT-PCR analysis of selected SAUR genes in developing fibers revealed their differential expressions. The physical mapping showed that 20 SAUR genes were co-localized with fiber length quantitative trait locus (QTL) hotspots. Single nucleotide polymorphisms (SNPs) were detected for 12 of these 20 genes between G. hirsutum and G. barbadense, but no SNPs were identified between two backcross inbred lines with differing fiber lengths derived from a cross between the two cultivated tetraploids. CONCLUSIONS: This study provides an important piece of genomic information for the SAUR genes in cotton and lays a solid foundation for elucidating the functions of SAUR genes in auxin signaling pathways to regulate cotton growth.

Ectopic expression of TaOEP16-2-5B, a wheat plastid outer envelope protein gene, enhances heat and drought stress tolerance in transgenic Arabidopsis plants.

Abiotic stresses, such as heat and drought, are major environmental factors restricting crop productivity and quality worldwide. A plastid outer envelope protein gene, TaOEP16-2, was identified from our previous transcriptome analysis [1,2]. In this study, the isolation and functional characterization of the TaOEP16-2 gene was reported. Three homoeologous sequences of TaOEP16-2 were isolated from hexaploid wheat, which were localized on the chromosomes 5A, 5B and 5D, respectively. These three homoeologues exhibited different expression patterns under heat stress conditions, TaOEP16-2-5B was the dominant one, and TaOEP16-2-5B was selected for further analysis. Compared with wild type (WT) plants, transgenic Arabidopsis plants overexpressing the TaOEP16-2-5B gene exhibited enhanced tolerance to heat stress, which was supported by improved survival rate, strengthened cell membrane stability and increased sucrose content. It was also found that TaOEP16-2 was induced by drought stress and involved in drought stress tolerance. TaOEP16-2-5B has the same function in ABA-controlled seed germination as AtOEP16-2. Our results suggest that TaOEP16-2-5B plays an important role in heat and drought stress tolerance, and could be utilized in transgenic breeding of wheat and other crop plants.

Overexpression of wheat ferritin gene TaFER-5B enhances tolerance to heat stress and other abiotic stresses associated with the ROS scavenging.

BACKGROUND: The yield of wheat (Triticum aestivum L.), an important crop, is adversely affected by heat stress in many regions of the world. However, the molecular mechanisms underlying thermotolerance are largely unknown. RESULTS: A novel ferritin gene, TaFER, was identified from our previous heat stress-responsive transcriptome analysis of a heat-tolerant wheat cultivar (TAM107). TaFER was mapped to chromosome 5B and named TaFER-5B. Expression pattern analysis revealed that TaFER-5B was induced by heat, polyethylene glycol (PEG), H2O2 and Fe-ethylenediaminedi(o-hydroxyphenylacetic) acid (Fe-EDDHA). To confirm the function of TaFER-5B in wheat, TaFER-5B was transformed into the wheat cultivar Jimai5265 (JM5265), and the transgenic plants exhibited enhanced thermotolerance. To examine whether the function of ferritin from mono- and dico-species is conserved, TaFER-5B was transformed into Arabidopsis, and overexpression of TaFER-5B functionally complemented the heat stress-sensitive phenotype of a ferritin-lacking mutant of Arabidopsis. Moreover, TaFER-5B is essential for protecting cells against heat stress associated with protecting cells against ROS. In addition, TaFER-5B overexpression also enhanced drought, oxidative and excess iron stress tolerance associated with the ROS scavenging. Finally, TaFER-5B transgenic Arabidopsis and wheat plants exhibited improved leaf iron content. CONCLUSIONS: Our results suggest that TaFER-5B plays an important role in enhancing tolerance to heat stress and other abiotic stresses associated with the ROS scavenging.

A wheat lipid transfer protein 3 could enhance the basal thermotolerance and oxidative stress resistance of Arabidopsis.

Wheat (Triticum aestivum L.) is one of the major grain crops, and heat stress adversely affects wheat production in many regions of the world. Previously, we found a heat-responsive gene named Lipid Transfer Protein 3 (TaLTP3) in wheat. TaLTP3 was deduced to be regulated by cold, ABA, MeJA, Auxin and oxidative stress according to cis-acting motifs in its promoter sequences. In this study, we show that TaLTP3 is responsive to prolonged water deficit, salt or ABA treatment in wheat seedlings. Also, TaLTP3 accumulation was observed after the plant suffered from heat stress both at the seedling and the grain-filling stages. TaLTP3 protein was localized in the cell membrane and cytoplasm of tobacco epidermal cells. Overexpression of TaLTP3 in yeast imparted tolerance to heat stress compared to cells expressing the vector alone. Most importantly, transgenic Arabidopsis plants engineered to overexpress TaLTP3 showed higher thermotolerance than control plants at the seedling stage. Further investigation indicated that transgenic lines decreased H2O2 accumulation and membrane injury under heat stress. Taken together, our results demonstrate that TaLTP3 confers heat stress tolerance possibly through reactive oxygen species (ROS) scavenging.