Oil candidate genes in seeds of cotton (Gossypium hirsutum L.) and functional validation of GhPXN1.

BACKGROUND: Cottonseed oil is a promising edible plant oil with abundant unsaturated fatty acids. However, few studies have been conducted to explore the characteristics of cottonseed oil. The molecular mechanism of cottonseed oil accumulation remains unclear. RESULTS: In the present study, we conducted comparative transcriptome and weighted gene co-expression network (WGCNA) analysis for two G. hirsutum materials with significant difference in cottonseed oil content. Results showed that, between the high oil genotype 6053 (H6053) and the low oil genotype 2052 (L2052), a total of 412, 507, 1,121, 1,953, and 2,019 differentially expressed genes (DEGs) were detected at 10, 15, 20, 25, and 30 DPA, respectively. Remarkably, a large number of the down-regulated DEGs were enriched in the phenylalanine metabolic processes. Investigation into the dynamic changes of expression profiling of genes associated with both phenylalanine metabolism and oil biosynthesis has shed light on a significant competitive relationship in substrate allocation during cottonseed development. Additionally, the WGCNA analysis of all DEGs identified eight distinct modules, one of which includes GhPXN1, a gene closely associated with oil accumulation. Through phylogenetic analysis, we hypothesized that GhPXN1 in G. hirsutum might have been introgressed from G. arboreum. Overexpression of the GhPXN1 gene in tobacco leaf suggested a significant reduction in oil content compared to the empty-vector transformants. Furthermore, ten other crucial oil candidate genes identified in this study were also validated using quantitative real-time PCR (qRT-PCR). CONCLUSIONS: Overall, this study enhances our comprehension of the molecular mechanisms underlying cottonseed oil accumulation.

High-quality genome assembly of Verticillium dahliae VD991 allows for screening and validation of pathogenic genes.

Verticillium dahliae (V. dahliae) is a notorious soil-borne pathogen causing Verticillium wilt in more than 400 dicotyledonous plants, including a wide range of economically important crops, such as cotton, tomato, lettuce, potato, and romaine lettuce, which can result in extensive economic losses. In the last decade, several studies have been conducted on the physiological and molecular mechanisms of plant resistance to V. dahliae. However, the lack of a complete genome sequence with a high-quality assembly and complete genomic annotations for V. dahliae has limited these studies. In this study, we produced a full genomic assembly for V. dahliae VD991 using Nanopore sequencing technology, consisting of 35.77 Mb across eight pseudochromosomes and with a GC content of 53.41%. Analysis of the genome completeness assessment (BUSCO alignment: 98.62%; Illumina reads alignment: 99.17%) indicated that our efforts resulted in a nearly complete and high-quality genomic assembly. We selected 25 species closely related to V. dahliae for evolutionary analysis, confirming the evolutionary relationship between V. dahliae and related species, and the identification of a possible whole genome duplication event in V. dahliae. The interaction between cotton and V. dahliae was investigated by transcriptome sequencing resulting in the identification of many genes and pathways associated with cotton disease resistance and V. dahliae pathogenesis. These results will provide new insights into the pathogenic mechanisms of V. dahliae and contribute to the cultivation of cotton varieties resistant to Verticillium wilt.

Recent progression and future perspectives in cotton genomic breeding.

Upland cotton is an important global cash crop for its long seed fibers and high edible oil and protein content. Progress in cotton genomics promotes the advancement of cotton genetics, evolutionary studies, functional genetics, and breeding, and has ushered cotton research and breeding into a new era. Here, we summarize high-impact genomics studies for cotton from the last 10 years. The diploid Gossypium arboreum and allotetraploid Gossypium hirsutum are the main focus of most genetic and genomic studies. We next review recent progress in cotton molecular biology and genetics, which builds on cotton genome sequencing efforts, population studies, and functional genomics, to provide insights into the mechanisms shaping abiotic and biotic stress tolerance, plant architecture, seed oil content, and fiber development. We also suggest the application of novel technologies and strategies to facilitate genome-based crop breeding. Explosive growth in the amount of novel genomic data, identified genes, gene modules, and pathways is now enabling researchers to utilize multidisciplinary genomics-enabled breeding strategies to cultivate "super cotton", synergistically improving multiple traits. These strategies must rise to meet urgent demands for a sustainable cotton industry.

Functional divergence of GLP genes between G. barbadense and G. hirsutum in response to Verticillium dahliae infection.

Germin-like proteins (GLPs) play important roles in plant disease resistance but are rarely reported in cotton. We compared the expression of GLPs in Verticillium dahliae inoculate G. hirsutum (susceptible) and G. barbadense (resistant) and enriched 11 differentially expressed GLPs. 2741 GLP proteins identified from 53 species determined that GLP probably originated from algae and could be classified into 7 clades according to phylogenetic analysis, among which Clade I is likely the most ancient. Cotton GLP (two allopolyploids and two diploids) genes within a shared clade were highly conserved. Intriguingly, clade VII genes were mainly located in gene clusters that derived from the expansion of LTR transposons. Clade VII members expressed mainly in root which is the first battle against Verticillium dahlia and could be induced more intensely in G. barbadense than G. hirsutum. The GLP genes are resistant to Verticillium dahliae, which can be further investigated against Verticillium wilt.

Integration of eQTL Analysis and GWAS Highlights Regulation Networks in Cotton under Stress Condition.

The genus Gossypium is one of the most economically important crops in the world. Here, we used RNA-seq to quantify gene expression in a collection of G. arboreum seedlings and performed eGWAS on 28,382 expressed genes. We identified a total of 30,089 eQTLs in 10,485 genes, of which >90% were trans-regulate target genes. Using luciferase assays, we confirmed that different cis-eQTL haplotypes could affect promoter activity. We found ~6600 genes associated with ~1300 eQTL hotspots. Moreover, hotspot 309 regulates the expression of 325 genes with roles in stem length, fresh weight, seed germination rate, and genes related to cell wall biosynthesis and salt stress. Transcriptome-wide association study (TWAS) identified 19 candidate genes associated with the cotton growth and salt stress response. The variation in gene expression across the population played an essential role in population differentiation. Only a small number of the differentially expressed genes between South China, the Yangtze River region, and the Yellow River region sites were located in different chromosomal regions. The eQTLs found across the duplicated gene pairs showed conservative cis- or trans- regulation and that the expression levels of gene pairs were correlated. This study provides new insights into the evolution of gene expression regulation in cotton, and identifies eQTLs in stress-related genes for use in breeding improved cotton varieties.

Genetic and evolution analysis of extrafloral nectary in cotton.

Extrafloral nectaries are a defence trait that plays important roles in plant-animal interactions. Gossypium species are characterized by cellular grooves in leaf midribs that secret large amounts of nectar. Here, with a panel of 215 G. arboreum accessions, we compared extrafloral nectaries to nectariless accessions to identify a region of Chr12 that showed strong differentiation and overlapped with signals from GWAS of nectaries. Fine mapping of an F2 population identified GaNEC1, encoding a PB1 domain-containing protein, as a positive regulator of nectary formation. An InDel, encoding a five amino acid deletion, together with a nonsynonymous substitution, was predicted to cause 3D structural changes in GaNEC1 protein that could confer the nectariless phenotype. mRNA-Seq analysis showed that JA-related genes are up-regulated and cell wall-related genes are down-regulated in the nectary. Silencing of GaNEC1 led to a smaller size of foliar nectary phenotype. Metabolomics analysis identified more than 400 metabolites in nectar, including expected saccharides and amino acids. The identification of GaNEC1 helps establish the network regulating nectary formation and nectar secretion, and has implications for understanding the production of secondary metabolites in nectar. Our results will deepen our understanding of plant-mutualism co-evolution and interactions, and will enable utilization of a plant defence trait in cotton breeding efforts.