Identification of fertility restoration candidate genes from a restorer line R186 for Gossypium harknessii cytoplasmic male sterile cotton.

BACKGROUND: The utilization of heterosis based on three-line system is an effective strategy in crop breeding. However, cloning and mechanism elucidation of restorer genes for cytoplasmic male sterility (CMS) in upland cotton have yet been realized. RESULTS: This research is based on CMS line 2074A with the cytoplasm from Gossypium harknessii (D2-2) and restorer line R186. The offspring of 2074A × R186 were used to conduct genetic analysis. The fertility mechanism of 2074A can be speculated to be governed by multiple genes, since neither the single gene model nor the double genes model could be used. The bulked segregant analysis (BSA) for (2074A × R186) F2 determined the genetic interval of restorer genes on a region of 4.30 Mb on chromosome D05 that contains 77 annotated genes. Four genes were identified as candidates for fertility restoration using the RNA-seq data of 2074A, 2074B, and R186. There are a number of large effect variants in the four genes between 2074A and R186 that could cause amino acid changes. Evolutionary analysis and identity analysis revealed that GH_D05G3183, GH_D05G3384, and GH_D05G3490 have high identity with their homologs in D2-2, respectively. Tissue differential expression analysis revealed that the genes GH_D05G3183, GH_D05G3384, and GH_D05G3490 were highly expressed in the buds of the line R186. The predicted results demonstrated that GH_D05G3183, GH_D05G3384 and GH_D05G3490 might interact with GH_A02G1295 to regulate orf610a in mitochondria. CONCLUSION: Our study uncovered candidate genes for fertility restoration in the restorer line R186 and predicted the possible mechanism for restoring the male fertility in 2074A. This research provided valuable insight into the nucleoplasmic interactions.

Plant non-coding RNAs function in pollen development and male sterility.

Male sterility is classified as either cytoplasmic male sterility (CMS) or genic male sterility (GMS). Generally, CMS involves mitochondrial genomes interacting with the nuclear genome, while GMS is caused by nuclear genes alone. Male sterility is regulated by multilevel mechanisms in which non-coding RNAs (ncRNAs), including microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and phased small interfering RNAs (phasiRNAs), which have been proven to be critical elements. The development of high-throughput sequencing technology offers new opportunities to evaluate the genetic mechanism of ncRNAs in plant male sterility. In this review, we summarize the critical ncRNAs that regulate gene expression in ways dependent on or independent of hormones, which involve the differentiation of the stamen primordia, degradation of the tapetum, formation of microspores, and the release of pollen. In addition, the key mechanisms of the miRNA-lncRNA-mRNA interaction networks mediating male sterility in plants are elaborated. We present a different perspective on exploring the ncRNA-mediated regulatory pathways that control CMS in plants and create male-sterile lines through hormones or genome editing. A refined understanding of the ncRNA regulatory mechanisms in plant male sterility for the development of new sterile lines would be conducive to improve hybridization breeding.

Identification of candidate genes involved in salt stress response at germination and seedling stages by QTL mapping in upland cotton.

Salinity is a major abiotic stress at critical stages of seed germination and seedling establishment. Germination rate (GR) and field emergence rate (FER) are the key traits that determine the basic number of plants stand under field conditions. To explore molecular mechanisms in upland cotton under salt stress, a population of 177 recombinant inbred lines, and their parents were evaluated for seed germination traits (GP, germination potential; GR; FW, fresh weight; DW, dry weight; GL, germinal length) and seedling traits (FER; SH, seedling height; NL, number of main stem leaves) in 2016-2018. Based on the linkage map contained 2,859 single nucleotide polymorphism and simple sequence repeat markers, traits under salt stress (E1) and normal conditions (E2), and in the converted relative index (R-value) dataset of 3 years' trials were used to map quantitative trait loci (QTL). A total of 3 QTL and 2 clusters were detected as salt-tolerant QTL. Three QTL (qGR-Chr4-3, qFER-Chr12-3, and qFER-Chr15-1) were detected under salt stress conditions and R-value dataset, which explained variance of phenotype 9.62-13.67%, and 4.2-4.72%, 4.75-8.96%, respectively. Two clusters (Loci-Chr4-2 and Loci-Chr5-4) harboring the QTL for 4 germination traits (GR, FER, GL, and NL) and 6 seedling traits (GR, FER, DW, FW, SH, and NL) were detected related under salt stress. A total of 691 genes were found in the candidate QTL or clusters. Among them, 4 genes (Gh_A04G1106, Gh_A05G3246, Gh_A05G3177, and Gh_A05G3266) showed expression differences between salt-sensitive and -tolerant lines under salt stress conditions, and were assigned as candidate genes in response to salt stress. The consistent salt-tolerance QTL identified in both germination and seedling stages will facilitate novel insights into effective utilization of cotton genetic resources.

Genomic and GWAS analyses demonstrate phylogenomic relationships of Gossypium barbadense in China and selection for fibre length, lint percentage and Fusarium wilt resistance.

Sea Island cotton (Gossypium barbadense) is the source of the world's finest fibre quality cotton, yet relatively little is understood about genetic variations among diverse germplasms, genes underlying important traits and the effects of pedigree selection. Here, we resequenced 336 G. barbadense accessions and identified 16 million SNPs. Phylogenetic and population structure analyses revealed two major gene pools and a third admixed subgroup derived from geographical dissemination and interbreeding. We conducted a genome-wide association study (GWAS) of 15 traits including fibre quality, yield, disease resistance, maturity and plant architecture. The highest number of associated loci was for fibre quality, followed by disease resistance and yield. Using gene expression analyses and VIGS transgenic experiments, we confirmed the roles of five candidate genes regulating four key traits, that is disease resistance, fibre length, fibre strength and lint percentage. Geographical and temporal considerations demonstrated selection for the superior fibre quality (fibre length and fibre strength), and high lint percentage in improving G. barbadense in China. Pedigree selection breeding increased Fusarium wilt disease resistance and separately improved fibre quality and yield. Our work provides a foundation for understanding genomic variation and selective breeding of Sea Island cotton.

Embryogenic Calli Induction and Salt Stress Response Revealed by RNA-Seq in Diploid Wild Species Gossypium sturtianum and Gossypium raimondii.

Wild cotton species can contribute to a valuable gene pool for genetic improvement, such as genes related to salt tolerance. However, reproductive isolation of different species poses an obstacle to produce hybrids through conventional breeding. Protoplast fusion technology for somatic cell hybridization provides an opportunity for genetic manipulation and targeting of agronomic traits. Transcriptome sequencing analysis of callus under salt stress is conducive to study salt tolerance genes. In this study, calli were induced to provide materials for extracting protoplasts and also for screening salt tolerance genes. Calli were successfully induced from leaves of Gossypium sturtianum (C1 genome) and hypocotyls of G. raimondii (D5 genome), and embryogenic calli of G. sturtianum and G. raimondii were induced on a differentiation medium with different concentrations of 2, 4-D, KT, and IBA, respectively. In addition, embryogenic calli were also induced successfully from G. raimondii through suspension cultivation. Transcriptome sequencing analysis was performed on the calli of G. raimondii and G. sturtianum, which were treated with 200 mM NaCl at 0, 6, 12, 24, and 48 h, and a total of 12,524 genes were detected with different expression patterns under salt stress. Functional analysis showed that 3,482 genes, which were differentially expressed in calli of G. raimondii and G. sturtianum, were associated with biological processes of nucleic acid binding, plant hormone (such as ABA) biosynthesis, and signal transduction. We demonstrated that DEGs or TFs which related to ABA metabolism were involved in the response to salt stress, including xanthoxin dehydrogenase genes (ABA2), sucrose non-fermenting 1-related protein kinases (SnRK2), NAM, ATAT1/2, and CUC2 transcription factors (NAC), and WRKY class of zinc-finger proteins (WRKY). This research has successfully induced calli from two diploid cotton species and revealed new genes responding to salt stress in callus tissue, which will lay the foundation for protoplast fusion for further understanding of salt stress responses in cotton.

QTL controlling fiber quality traits under salt stress in upland cotton (Gossypium hirsutum L.).

KEY MESSAGE: QTL for fiber quality traits under salt stress discerned candidate genes controlling fatty acid metabolism. Salinity stress seriously affects plant growth and limits agricultural productivity of crop plants. To dissect the genetic basis of response to salinity stress, a recombinant inbred line population was developed to compare fiber quality in upland cotton (Gossypium hirsutum L.) under salt stress and normal conditions. Based on three datasets of (1) salt stress, (2) normal growth, and (3) the difference value between salt stress and normal conditions, 51, 70, and 53 QTL were mapped, respectively. Three QTL for fiber length (FL) (qFL-Chr1-1, qFL-Chr5-5, and qFL-Chr24-4) were detected under both salt and normal conditions and explained 4.26%, 9.38%, and 3.87% of average phenotypic variation, respectively. Seven genes within intervals of two stable QTL (qFL-Chr1-1 and qFL-Chr5-5) were highly expressed in lines with extreme long fiber. A total of 35 QTL clusters comprised of 107 QTL were located on 18 chromosomes and exhibited pleiotropic effects. Thereinto, two clusters were responsible for improving five fiber quality traits, and 6 influenced FL and fiber strength (FS). The QTL with positive effect for fiber length exhibited active effects on fatty acid synthesis and elongation, but the ones with negative effect played passive roles on fatty acid degradation under salt stress.

Mitochondrial proteomic analysis reveals that proteins relate to oxidoreductase activity play a central role in pollen fertility in cotton.

Cotton (Gossypium hirsutum L.) is an important economic crop. Cytoplasm male sterility (CMS) has been used to develop hybrid system and to produce hybrid seeds in cotton, but the molecular mechanism of CMS remains unclear. Mitochondria are semi-autonomous organelles, which play an important role in the reproduction of flowering plants. Male sterility has been proved associated with mitochondrial dysfunction in plants. In present study, a new strategy of proteomic sequencing data-independent acquisition (DIA) was used to analysis protein abundance across CMS lines 2074A (cytoplasm of Gossypium harknessii, D2-2) and 2074S (cytoplasm of G. hirsutum, AD1), and their maintainer 2074B. Comparing with transcriptome results showed that there is little consistence between proteome and transcriptome. A total of 2095 protein species were identified in three materials, and 186 and 161 differentially proteins were detected in the comparisons of 2074A vs 2074B, and 2074S vs 2074B, respectively. Among them, 49 and 50 proteins were specific existed in anther, and mainly participated in oxidoreductase activity, carbohydrate metabolism, fatty acid metabolism, cell aging, wax or cutin deposition and signal transduction. Gh_A07G0770 and Gh_D05G1908 were specific up-regulated in sterility lines, and the other genes Gh_D08G1196, Gh_D12G1971, Gh_A11G1250, Gh_D08G0388 were down-regulated, which presented similar expression tendency verified by qRT-PCR, transcriptome and proteome results. These six genes related to lipid synthesis, response to oxidative stress and cell aging, suggested them being involved in CMS occurrence. Using virus-induced gene silencing (VIGS) system, sterility obtained demonstrated the silencing Gh_A11G1250 in maintainer 2074B led to partial anthers abortion. Gh_A11G1250 encoded a mitochondrial localization of peroxisomal-like protein, participated in response to reactive oxygen species (ROS). Twenty-two proteins interacting with Gh_A11G1250 mainly related to chlorophyll biosynthetic process, photoperiodism and flowering, which showed different expression pattern between the male sterile line 2074A and maintainer 2074B. This novel research based on mitochondrial proteomics comparison confirmed that DAPs related to oxidative stress are critical to pollen abortion. BIOLOGICAL SIGNIFICANCE: Cytoplasm male sterility (CMS) system is utilized widely for hybrid production in cotton. However, the genetic and molecular mechanisms of CMS still need to be further elucidated. Up till now, fewer comprehensive comparisons of the mitochondrial proteomes from cotton CMS line and maintainer line have been reported. In this study, we performed a novel comparison of mitochondrial protein profiles in two CMS lines and their common maintainer line. Based on our results, we found a potential protein related to oxidative stress led to the anthers abortion. These results accumulate data to interpret the molecular mechanisms of CMS in cotton.

The comparison of four mitochondrial genomes reveals cytoplasmic male sterility candidate genes in cotton.

BACKGROUND: The mitochondrial genomes of higher plants vary remarkably in size, structure and sequence content, as demonstrated by the accumulation and activity of repetitive DNA sequences. Incompatibility between mitochondrial genome and nuclear genome leads to non-functional male reproductive organs and results in cytoplasmic male sterility (CMS). CMS has been used to produce F1 hybrid seeds in a variety of plant species. RESULTS: Here we compared the mitochondrial genomes (mitogenomes) of Gossypium hirsutum sterile male lines CMS-2074A and CMS-2074S, as well as their restorer and maintainer lines. First, we noticed the mitogenome organization and sequences were conserved in these lines. Second, we discovered the mitogenomes of 2074A and 2074S underwent large-scale substitutions and rearrangements. Actually, there were five and six unique chimeric open reading frames (ORFs) in 2074A and 2074S, respectively, which were derived from the recombination between unique repetitive sequences and nearby functional genes. Third, we found out four chimeric ORFs that were differentially transcribed in sterile line (2074A) and fertile-restored line. CONCLUSIONS: These four novel and recombinant ORFs are potential candidates that confer CMS character in 2074A. In addition, our observations suggest that CMS in cotton is associated with the accelerated rates of rearrangement, and that novel expression products are derived from recombinant ORFs.

Exploration of miRNAs and target genes of cytoplasmic male sterility line in cotton during flower bud development.

Cytoplasmic male sterility (CMS) lines provide crucial material to harness heterosis for crop plants, which serves as an important strategy for hybrid seed production. However, the molecular mechanism remains obscure. Although microRNAs (miRNAs) play important roles in vegetative growth and reproductive growth, there are few reports on miRNAs regulating the development of male sterility in Upland cotton. In present study, 12 small RNA libraries were constructed and sequenced for two development stages of flower buds from a CMS line and its maintainer line. Based on the results, 256 novel miRNAs were allocated to 141 new miRNA families, and 77 known miRNAs belonging to 54 conserved miRNA families were identified as well. Comparative analysis revealed that 61 novel and 10 conserved miRNAs were differentially expressed. Further transcriptome analysis identified 232 target genes for these miRNAs, which participated in cellular developmental process, cell death, pollen germination, and sexual reproduction. In addition, expression patterns of typical miRNA and the negatively regulated target genes, such as PPR, ARF, AP2, and AFB, were verified by qRT-PCR in cotton flower buds. These targets were previously reported to be related to reproduction development and male sterility, suggesting that miRNAs might act as regulators of CMS occurrence. Some miRNAs displayed specific expression profiles in special developmental stages of CMS line and its fertile hybrid (F1). Present study offers new information on miRNAs and their related target genes in exploiting CMS mechanism, and revealing the miRNA regulatory networks in Upland cotton.

Rapid evolutionary divergence of diploid and allotetraploid Gossypium mitochondrial genomes.

BACKGROUND: Cotton (Gossypium spp.) is commonly grouped into eight diploid genomic groups and an allotetraploid genomic group, AD. The mitochondrial genomes supply new information to understand both the evolution process and the mechanism of cytoplasmic male sterility. Based on previously released mitochondrial genomes of G. hirsutum (AD1), G. barbadense (AD2), G. raimondii (D5) and G. arboreum (A2), together with data of six other mitochondrial genomes, to elucidate the evolution and diversity of mitochondrial genomes within Gossypium. RESULTS: Six Gossypium mitochondrial genomes, including three diploid species from D and three allotetraploid species from AD genome groups (G. thurberi D1, G. davidsonii D3-d and G. trilobum D8; G. tomentosum AD3, G. mustelinum AD4 and G. darwinii AD5), were assembled as the single circular molecules of lengths about 644 kb in diploid species and 677 kb in allotetraploid species, respectively. The genomic structures of mitochondrial in D group species were identical but differed from the mitogenome of G. arboreum (A2), as well as from the mitogenomes of five species of the AD group. There mainly existed four or six large repeats in the mitogenomes of the A + AD or D group species, respectively. These variations in repeat sequences caused the major inversions and translocations within the mitochondrial genome. The mitochondrial genome complexity in Gossypium presented eight unique segments in D group species, three specific fragments in A + AD group species and a large segment (more than 11 kb) in diploid species. These insertions or deletions were most probably generated from crossovers between repetitive or homologous regions. Unlike the highly variable genome structure, evolutionary distance of mitochondrial genes was 1/6th the frequency of that in chloroplast genes of Gossypium. RNA editing events were conserved in cotton mitochondrial genes. We confirmed two near full length of the integration of the mitochondrial genome into chromosome 1 of G. raimondii and chromosome A03 of G. hirsutum, respectively, with insertion time less than 1.03 MYA. CONCLUSION: Ten Gossypium mitochondrial sequences highlight the insights to the evolution of cotton mitogenomes.

Molecular evolution of the plastid genome during diversification of the cotton genus.

Cotton (Gossypium spp.) is commonly grouped into eight diploid genomic groups, designated A-G and K, and one tetraploid genomic group, namely AD. To gain insight into the phylogeny of Gossypium and molecular evolution of the chloroplast genome duringdiversification, chloroplast genomes (cpDNA) from 6 D-genome and 2 G-genome species of Gossypium (G. armourianum D2-1, G. harknessii D2-2, G. davidsonii D3-d, G. klotzschianum D3-k, G. aridum D4, G. trilobum D8, and G. australe G2, G. nelsonii G3) were newly reported here. In combination with the 26 previously released cpDNA sequences, we performed comparative phylogenetic analyses of 34 Gossypium chloroplast genomes that collectively represent most of the diversity in the genus. Gossypium chloroplasts span a small range in size that is mostly attributable to indels that occur in the large single copy (LSC) region of the genome. Phylogenetic analysis using a concatenation of all genes provides robust support for six major Gossypium clades, largely supporting earlier inferences but also revealing new information on intrageneric relationships. Using Theobroma cacao as an outgroup, diversification of the genus was dated, yielding results that are in accord with previous estimates of divergence times, but also offering new perspectives on the basal, early radiation of all major clades within the genus as well as gaps in the record indicative of extinctions. Like most higher-plant chloroplast genomes, all cotton species exhibit a conserved quadripartite structure, i.e., two large inverted repeats (IR) containing most of the ribosomal RNA genes, and two unique regions, LSC (large single sequence) and SSC (small single sequence). Within Gossypium, the IR-single copy region junctions are both variable and homoplasious among species. Two genes, accD and psaJ, exhibited greater rates of synonymous and non-synonymous substitutions than did other genes. Most genes exhibited Ka/Ks ratios suggestive of neutral evolution, with 8 exceptions distributed among one to several species. This research provides an overview of the molecular evolution of a single, large non-recombining molecular during the diversification of this important genus.