A genome-wide association study for resistance to Fusarium wilt (Fusarium oxysporum f. sp. vasinfectum) race 4 in diploid cotton (Gossypium arboreum) and resistance transfer to tetraploid Gossypium hirsutum.

Fusarium wilt, caused by the soilborne fungus Fusarium oxysporum f. sp. vasinfectum (FOV), is a devastating disease affecting cotton (Gossypium spp.) worldwide. Understanding the genetic basis of resistance in diploid cotton and successfully transferring the resistance to tetraploid Upland cotton (G. hirsutum) are crucial for developing resistant cotton cultivars. Although numerous studies have been conducted to investigate the genetic basis of Fusarium wilt in tetraploid cotton, little research has been conducted on diploid species. In this study, an association mapping panel consisting of 246 accessions of G. arboreum, was used to identify chromosomal regions for FOV race 4 (FOV4) resistance based on foliar disease severity ratings in four greenhouse tests. Through a genome-wide association study (GWAS) based on 7,009 single nucleotide polymorphic (SNP) markers, 24 FOV4 resistance QTLs, including three major QTLs on chromosomes A04, A06, and A11, were detected. A validation panel consisting of 97 diploid cotton accessions was employed, confirming the presence of several QTLs. Evaluation of an introgressed BC2F7 population derived from G. hirsutum/G. aridum/G. arboreum showed significant differences in disease incidence and mortality rate, as compared to susceptible and resistant controls, suggesting that the resistance in G. arboreum and/or G. aridum was transferred into Upland cotton for the first time. The identification of novel major resistance QTLs, along with the transfer of resistance from the diploid species, expands our understanding of the genomic regions involved in conferring resistance to FOV4 and contributes to the development of resilient Upland cotton cultivars.

Genomic and co-expression network analyses reveal candidate genes for oil accumulation based on an introgression population in Upland cotton (Gossypium hirsutum).

KEY MESSAGE: Integrated QTL mapping and WGCNA condense the potential gene regulatory network involved in oil accumulation. A glycosyl hydrolases gene (GhHSD1) for oil biosynthesis was confirmed in Arabidopsis, which will provide useful knowledge to understand the functional mechanism of oil biosynthesis in cotton. Cotton is an economical source of edible oil for the food industry. The genetic mechanism that regulates oil biosynthesis in cottonseeds is essential for the genetic enhancement of oil content (OC). To explore the functional genomics of OC, this study utilized an interspecific backcross inbred line population to dissect the quantitative trait locus (QTL) interlinked with OC. In total, nine OC QTLs were identified, four of which were novel, and each QTL explained 3.62-34.73% of the phenotypic variation of OC. The comprehensive transcript profiling of developing cottonseeds revealed 3,646 core genes differentially expressed in both inbred parents. Functional enrichment analysis determined 43 genes were annotated with oil biosynthesis processes. Implementation of weighted gene co-expression network analysis showed that 803 differential genes had a significant correlation with the OC phenotype. Further integrated analysis identified seven important genes located in OC QTLs. Of which, the GhHSD1 gene located in stable QTL qOC-Dt3-1 exhibited the highest functional linkages with the other network genes. Phylogenetic analysis showed significant evolutionary differences in the HSD1 sequences between oilseed- and starch- crops. Furthermore, the overexpression of GhHSD1 in Arabidopsis yielded almost 6.78% higher seed oil. This study not only uncovers important genetic loci for oil accumulation in cottonseed, but also provides a set of new candidate genes that potentially influence the oil biosynthesis pathway in cottonseed.

Co-localization and analysis of miR477b with fiber length quantitative trait loci in cotton.

Cotton is an important cash crop for the textile industry. However, the understanding of natural genetic variation of fiber elongation in relation to miRNA is lacking. A miRNA gene (miR477b) was found to co-localize with a previously mapped fiber length (FL) quantitative trait locus (QTL). The miR477b was differentially expressed during fiber elongation between two backcross inbred lines (BILs) differing in FL and its precursor sequences. Bioinformatics and qRT-PCR analysis were further used to analyse the miRNA genes, which could produce mature miR477b. Cotton plants with virus-induced gene silencing (VIGS) constructs to over-express the allele of miR477b from the BIL with longer fibers had significantly longer fibers as compared with negative control plants, while the VIGS plants with suppressed miRNA expression had significantly shorter fibers. The expression level of the target gene (DELLA) and related genes (RDL1 and EXPA1 for DELLA through HOX3 protein) in the two BILs and/or the VIGS plants were generally congruent, as expected. This report represents one of the first comprehensive studies to integrate QTL linkage mapping and physical mapping of small RNAs with both small and mRNA transcriptome analysis, followed by VIGS, to identify candidate small RNA genes affecting the natural variation of fiber elongation in cotton.

Targeted development of diagnostic SNP markers for resistance to Fusarium wilt race 4 in Upland cotton (Gossypium hirsutum).

Fusarium wilt caused by the soil-borne fungus Fusarium oxysporum f. sp. vasinfectum (FOV) race 4 (FOV4) has become one of the most important emerging diseases in US cotton production. Numerous QTLs have been reported for resistance to FOV; however, no major FOV4-resistance QTL or gene has been identified and used in breeding Upland cotton (Gossypium hirsutum) for FOV4 resistance. In this study, a panel of 223 Chinese Upland cotton accessions was evaluated for FOV4 resistance based on seedling mortality rate (MR) and stem and root vascular discoloration (SVD and RVD). SNP markers were developed based on targeted genome sequencing using AgriPlex Genomics. The chromosome region at 2.130-2.292 Mb on D03 was significantly correlated with both SVD and RVD but not with MR. Based on the two most significant SNP markers, accessions homozygous for AA or TT SNP genotype averaged significantly lower SVD (0.88 vs. 2.54) and RVD (1.46 vs. 3.02) than those homozygous for CC or GG SNP genotype. The results suggested that a gene or genes within the region conferred resistance to vascular discoloration caused by FOV4. The Chinese Upland accessions had 37.22% homozygous AA or TT SNP genotype and 11.66% heterozygous AC or TG SNP genotype, while 32 US elite public breeding lines all had the CC or GG SNP genotype. Among 463 obsolete US Upland accessions, only 0.86% possessed the AA or TT SNP genotype. This study, for the first time, has developed diagnostic SNPs for marker-assisted selection and identified FOV4-resistant Upland germplasms with the SNPs.

Development and validation of allele-specific PCR-based SNP typing in a gene on chromosome D03 conferring resistance to Fusarium wilt race 4 in Upland cotton (Gossypium hirsutum).

Upland cotton (Gossypium hirsutum) is the most important fiber crop for the global textile industry. Fusarium oxysporum f. sp. vasinfectum (FOV) is one of the most destructive soil-borne fungal pathogens in cotton. Among eight pathogenic races and other strains, FOV race 4 (FOV4) is the most virulent race in US cotton production. A single nucleotide polymorphism (SNP) in a glutamate receptor-like gene (GhGLR4.8) on chromosome D03 was previously identified and validated to confer resistance to FOV race 7, and targeted genome sequencing demonstrated that it was also associated with resistance to FOV4. The objective of this study was to develop an easy and convenient PCR-based marker assay. To target the resistance SNP, a forward primer for the SNP with a mismatch in the 3rd position was designed for both the resistance (R) and susceptibility (S) alleles, respectively, with addition of 20-mer T7 promoter primer to the 5' end of the forward primer for the R allele. The two forward primers, in combination with each of five common reverse primers, were targeted to amplify amplicons of 50-260 bp in size with R and S alleles differing in 20 bp. Results showed that each of three common reverse primers in combination with the two forward primers produced polymorphic markers between R and S plants that were consistent with the targeted genome sequencing results. The polymorphism was distinctly resolved using both polyacrylamide and agarose gel electrophoreses. In addition, a sequence comparative analysis between the resistance gene and homologous sequences in sequenced tetraploid and diploid A and D genome species showed that none of the species possessed the resistance gene allele, suggesting its recent origin from a natural point mutation. The allele-specific PCR-based SNP typing method based on a three-primer combination provides a fast and convenient marker-assisted selection method to search and select for FOV4-resistant Upland cotton.

A Rapid and Reliable Method for Evaluating Cotton Resistance to Fusarium Wilt Race 4 Based on Taproot Rot at the Seed Germination Stage.

Fusarium oxysporum f. sp. vasinfectum race 4 (FOV4) is a soilborne fungal pathogen threatening U.S. cotton production. The objective of this study was to develop a reliable and efficient method to evaluate cotton for FOV4 resistance based on taproot rot during seed germination through five growth chamber tests and two greenhouse tests. Seeds from eight cotton cultivars (Set 1) were germinated in a paper towel for 6 days, and taproots were inoculated with a FOV4 conidial suspension using three inoculation methods (i.e., taproot dipping, taproot wounding, and paper towel drenching), in addition to seed soaking before germination. The taproot rot-based disease incidence (DI) and disease severity rating (DSR), seed germination percentage (SGP), and plant fresh weight (PFW) were measured 7 days after inoculation. Taproot dipping was the most efficient and reliable evaluation method. The SGP and PFW were not significantly correlated with the DI and DSR, making them inappropriate to use in resistance evaluation. Pima DP 359 RF and PHY 881 RF were the most resistant with the lowest root rot. The taproot dipping method was repeated in another test and confirmed in two tests using another set of eight cultivars (Set 2). The taproot rot-based DSR at germination was significantly correlated with the DSR at the seedling stage in the greenhouse in both sets and with previous results in seedling mortality in the greenhouse and field in Set 2. The results suggest that the response to FOV4 infections at the seed germination stage is overall congruent with that at the seedling stage.

Mapping of dynamic QTLs for resistance to Fusarium wilt (Fusarium oxysporum f. sp. vasinfectum) race 4 in a backcross inbred line population of Upland cotton.

KEY MESSAGE: A backcross inbred line population of cotton was evaluated for Fusarium wilt race 4 resistance at different days after inoculation (DAI). Both constitutively expressed and developmentally regulated QTLs were detected. The soil-borne fungus Fusarium oxysporum f. sp. vasinfectum (FOV) race 4 (FOV4) causes Fusarium wilt including seedling mortality in cotton. A backcross inbred line (BIL) population of 181 lines, derived from a bi-parental cross of moderately resistant non-recurrent Hai 7124 (Gossypium barbadense) and recurrent parent CCRI 36 (G. hirsutum), was evaluated under temperature-controlled conditions for FOV4 resistance with artificial inoculations. Based on three replicated tests evaluated at 7, 14, 21, and 28 days after inoculation (DAI), only 2-5 BILs showed lower disease severity ratings (DSR) than the parents while 22-50 BILs were more susceptible, indicating transgressive segregation toward susceptibility. Although DSR were overall congruent between DAI, there were many BILs displaying different responses to FOV4 across DAI. Genetic mapping using 7709 SNP markers identified 42 unique QTLs for four evaluation parameters- disease incidence (DI), DSR, mortality rate (MR), and area under disease progress curve (AUDPC), including 26 for two or more parameters. All five QTLs for AUDPC were co-localized with QTLs for DI, DSR, and/or MR at one or two DAI, indicating the unnecessary use of AUDPC in QTL mapping for FOV4 resistance. Those common QTLs explained the significant positive associations between parameters observed. Ten common QTLs with negative or positive additive effects were detected between DAI. DAI-specific and consistent QTLs were detected between DAI in cotton for the first time, suggesting the existence of both constitutively expressed and developmentally regulated QTLs for FOV4 resistance and the importance of evaluating genetic populations for FOV4 resistance at different growth stages.

Expressed genes and their new alleles identification during fibre elongation reveal the genetic factors underlying improvements of fibre length in cotton.

Interspecific breeding in cotton takes advantage of genetic recombination among desirable genes from different parental lines. However, the expression new alleles (ENAs) from crossovers within genic regions and their significance in fibre length (FL) improvement are currently not understood. Here, we generated resequencing genomes of 191 interspecific backcross inbred lines derived from CRI36 (Gossypium hirsutum) × Hai7124 (Gossypium barbadense) and 277 dynamic fibre transcriptomes to identify the ENAs and extremely expressed genes (eGenes) potentially influencing FL, and uncovered the dynamic regulatory network of fibre elongation. Of 35 420 eGenes in developing fibres, 10 366 ENAs were identified and preferentially distributed in chromosomes subtelomeric regions. In total, 1056-1255 ENAs showed transgressive expression in fibres at 5-15 dpa (days post-anthesis) of some BILs, 520 of which were located in FL-quantitative trait locus (QTLs) and GhFLA9 (recombination allele) was identified with a larger effect for FL than GhFLA9 of CRI36 allele. Using ENAs as a type of markers, we identified three novel FL-QTLs. Additionally, 456 extremely eGenes were identified that were preferentially distributed in recombination hotspots. Importantly, 34 of them were significantly associated with FL. Gene expression quantitative trait locus analysis identified 1286, 1089 and 1059 eGenes that were colocalized with the FL trait at 5, 10 and 15 dpa, respectively. Finally, we verified the Ghir_D10G011050 gene linked to fibre elongation by the CRISPR-cas9 system. This study provides the first glimpse into the occurrence, distribution and expression of the developing fibres genes (especially ENAs) in an introgression population, and their possible biological significance in FL.

A GWAS identified a major QTL for resistance to Fusarium wilt (Fusarium oxysporum f. sp. vasinfectum) race 4 in a MAGIC population of Upland cotton and a meta-analysis of QTLs for Fusarium wilt resistance.

KEY MESSAGE: A major QTL conferring resistance to Fusarium wilt race 4 in a narrow region of chromosome D02 was identified in a MAGIC population of 550 RILs of Upland cotton. Numerous studies have been conducted to investigate the genetic basis of Fusarium wilt (FW, caused by Fusarium oxysporum f. sp. vasinfectum, FOV) resistance using bi-parental and association mapping populations in cotton. In this study, a multi-parent advanced generation inter-cross (MAGIC) population of 550 recombinant inbred lines (RILs), together with their 11 Upland cotton (Gossypium hirsutum) parents, was used to identify QTLs for FOV race 4 (FOV4) resistance. Among the parents, Acala Ultima, M-240 RNR, and Stoneville 474 were the most resistant, while Deltapine Acala 90, Coker 315, and Stoneville 825 were the most susceptible. Twenty-two MAGIC lines were consistently resistant to FOV4. Through a genome-wide association study (GWAS) based on 473,516 polymorphic SNPs, a major FOV4 resistance QTL within a narrow region on chromosomes D02 was detected, allowing identification of 14 candidate genes. Additionally, a meta-analysis of 133 published FW resistance QTLs showed a D subgenome and individual chromosome bias and no correlation between homeologous chromosome pairs. This study represents the first GWAS study using a largest genetic population and the most comprehensive meta-analysis for FW resistance in cotton. The results illustrated that 550 lines were not enough for high resolution mapping to pinpoint a candidate gene, and experimental errors in phenotyping cotton for FW resistance further compromised the accuracy and precision in QTL localization and identification of candidate genes. This study identified FOV4-resistant parents and MAGIC lines, and the first major QTL for FOV4 resistance in Upland cotton, providing useful information for developing FOV4-resistant cultivars and further genomic studies towards identification of causal genes for FOV4 resistance in cotton.

Transcriptome analysis and identification of genes associated with oil accumulation in upland cotton.

Cotton is not only the most important fiber crop but also the fifth most important oilseed crop in the world because of its oil-rich seeds as a byproduct of fiber production. By comparative transcriptome analysis between two germplasms with diverse oil accumulation, we reveal pieces of the gene expression network involved in the process of oil synthesis in cottonseeds. Approximately, 197.16 Gb of raw data from 30 RNA sequencing samples with 3 biological replicates were generated. Comparison of the high-oil and low-oil transcriptomes enabled the identification of 7682 differentially expressed genes (DEGs). Based on gene expression profiles relevant to triacylglycerol (TAG) biosynthesis, we proposed that the Kennedy pathway (diacylglycerol acyltransferase-catalyzed diacylglycerol to TAG) is the main pathway for oil production, rather than the phospholipid diacylglycerol acyltransferase-mediated pathway. Using weighted gene co-expression network analysis, 5312 DEGs were obtained and classified into 14 co-expression modules, including the MEblack module containing 10 genes involved in lipid metabolism. Among the DEGs in the MEblack module, GhCYSD1 was identified as a potential key player in oil biosynthesis. The overexpression of GhCYSD1 in yeast resulted in increased oil content and altered fatty acid composition. This study may not only shed more light on the underlying molecular mechanism of oil accumulation in cottonseed oil, but also provide a set of new gene for potential enhancement of oil content in cottonseeds.

Analysis of the MIR396 gene family and the role of MIR396b in regulating fiber length in cotton.

Cotton fiber is one of the most important natural raw materials in the world textile industry. Improving fiber yield and quality has always been the main goal. MicroRNAs, as typical small noncoding RNAs, could affect fiber length during different stages of fiber development. Based on differentially expressed microRNA in the two interspecific backcross inbred lines (BILs) with a significant difference in fiber length, we identified the miR396 gene family in the two tetraploid cotton genomes and found MIR396b_D13 as the functional precursor to produce mature miR396 during the fiber elongation stage. Among 46 target genes regulated by miR396b, the GROWTH-REGULATING FACTOR 5 gene (GRF5, Gh_A10G0492) had a differential expression level in the two BILs during fiber elongation stage. The expression patterns indicated that the miR396b-GRF5 regulatory module has a critical role in fiber development. Furthermore, virus-induced gene silencing (VIGS) of miR396b significantly produced longer fiber than the wild type, and the expression level of GRF5 showed the reverse trends of the miR396b expression level. The analysis of co-expression network for the GRF5 gene suggested that a cytochrome P450 gene functions as an allene oxide synthase (Gh_D06G0089, AOS), which plays a critical role in jasmonate biosynthetic pathway. In conclusion, our results revealed that the miR396b-GRF5 module has a critical role in fiber development. These findings provide a molecular foundation for fiber quality improvement in the future.

Comparative Analysis of Infection Process in Pima Cotton Differing in Resistance to Fusarium Wilt Caused by Fusarium oxysporum f. sp. vasinfectum Race 4.

Fusarium oxysporum f. sp. vasinfectum race 4 (FOV4) causes an early season cotton disease including seedling deaths. This study compared two Pima cottons (Gossypium barbadense) in the infection process of FOV4 using a confocal and a scanning electron microscope. Seedlings were grown in a hydroponic system and inoculated with a virulent local FOV4 isolate. As compared with the susceptible Pima S-7, the resistant Pima PHY 841 RF had significantly fewer conidia attached and germinated on the root surface. FOV4 penetration into the root epidermis of PHY 841 RF was delayed until 24 h postinoculation (hpi) as compared with 8 hpi in Pima S-7. In Pima S-7, hyphae progressed to the xylem through the cortex between 5 and 7 days postinoculation. However, hyphae grew much slower in the cortex with no apparent hyphae observed in the xylem of PHY 841 RF. At plant maturity, no FOV4 was detected through fungal isolation and PCR in the stem of PHY 841 RF and its resistance donor parents PHY 800 and Pima S-6, as compared with Pima S-7 and DP 744 with positive results. The results demonstrate that PHY 841 RF is resistant to FOV4, due to delayed infection, reduced fungal growth and reproduction, and prevention of the fungus from invading the xylem.

GWAS reveals consistent QTL for drought and salt tolerance in a MAGIC population of 550 lines derived from intermating of 11 Upland cotton (Gossypium hirsutum) parents.

Cotton is grown in arid and semi-arid regions where abiotic stresses such as drought and salt are prevalent. There is a lack of studies that simultaneously address the genetic and genomic basis of tolerance to drought and salt stress. In this study, a multi-parent advanced generation inter-cross (MAGIC) population of 550 recombinant inbred lines (RILs) together with their 11 Upland cotton parents with a total of 473,516 polymorphic SNP markers was used to identify quantitative trait loci (QTL) for drought tolerance (DT) and salt tolerance (ST) at the seedling stage based on two replicated greenhouse tests. Transgressive segregation occurred in the MAGIC-RILs, indicating that tolerant and sensitive alleles recombined for tolerance to the abiotic stress during the intermating process for the population development. A total of 20 QTL were detected for DT including 13 and 7 QTL based on plant height (PH) and dry shoot weight (DSW), respectively; and 23 QTL were detected for ST including 12 and 11 QTL for PH and DSW, respectively. There were several chromosomes with QTL clusters for abiotic stress tolerance including four QTL on chromosome A13 and three QTL on A01 for DT, and four QTL on D08 and three QTL on A11 for ST. Nine QTL (21% of the 43 QTL) detected were in common between DT and ST, indicating a common genetic basis for DT and ST. The narrow chromosomal regions for most of the QTL detected in this study allowed identification of 53 candidate genes associated with responses to salt and drought stress and abiotic stimulus. The QTL identified for both DT and ST have significantly augmented the repertoire of QTL for abiotic stress tolerance that can be used for marker-assisted selection to develop cultivars with resilience to drought and/or salt and further genomic studies towards the identification of drought and salt tolerance genes in cotton.

Evaluation and genome-wide association study of resistance to bacterial blight race 18 in U.S. Upland cotton germplasm.

Bacterial blight (BB), caused by Xanthomonas citri pv. malvacearum (Xcm), is a destructive disease to cotton production in many countries. In the U.S., Xcm race 18 is the most virulent and widespread race and can cause serious yield losses. Planting BB-resistant cotton cultivars is the most effective method of controlling this disease. In this study, 335 U.S. Upland cotton accessions were evaluated for resistance to race 18 using artificial inoculations by scratching cotyledons on an individual plant basis in a greenhouse. The analysis of variance detected significant genotypic variation in disease incidence, and 50 accessions were resistant including 38 lines with no symptoms on either cotyledons or true leaves. Many of the resistant lines were developed in the MAR (multi-adversity resistance) breeding program at Texas A&M University, whereas others were developed before race 18 was first reported in the U.S. in 1973, suggesting a broad base of resistance to race 18. A genome-wide association study (GWAS) based on 26,301 single nucleotide polymorphic (SNP) markers detected 11 quantitative trait loci (QTL) anchored by 79 SNPs, including three QTL on each of the three chromosomes A01, A05 and D02, and one QTL on each of D08 and D10. This study has identified a set of obsolete Upland germplasm with resistance to race 18 and specific chromosomal regions delineated by SNPs for resistance. The results will assist in breeding cotton for BB resistance and facilitate further genomic studies in fine mapping resistance genes to enhance the understanding of the genetic basis of BB resistance in cotton.

The cellulose synthase (CesA) gene family in four Gossypium species: phylogenetics, sequence variation and gene expression in relation to fiber quality in Upland cotton.

Cellulose synthases (CesAs) are multi-subunit enzymes found on the plasma membrane of plant cells and play a pivotal role in cellulose production. The cotton fiber is mainly composed of cellulose, and the genetic relationships between CesA genes and cotton fiber yield and quality are not fully understood. Through a phylogenetic analysis, the CesA gene family in diploid Gossypium arboreum and Gossypium raimondii, as well as tetraploid Gossypium hirsutum ('TM-1') and Gossypium barbadense ('Hai-7124' and '3-79'), was divided into 6 groups and 15 sub-groups, with each group containing two to five homologous genes. Most CesA genes in the four species are highly collinear. Among the five cotton genomes, 440 and 1929 single nucleotide polymorphisms (SNPs) in the CesA gene family were identified in exons and introns, respectively, including 174 SNPs resulting in amino acid changes. In total, 484 homeologous SNPs between the A and D genomes were identified in diploids, while 142 SNPs were detected between the two tetraploids, with 32 and 82 SNPs existing within G. hirsutum and G. barbadense, respectively. Additionally, 74 quantitative trait loci near 18 GhCesA genes were associated with fiber quality. One to four GhCesA genes were differentially expressed (DE) in ovules at 0 and 3 days post anthesis (DPA) between two backcross inbred lines having different fiber lengths, but no DE genes were identified between these lines in developing fibers at 10 DPA. Twenty-seven SNPs in above DE CesA genes were detected among seven cotton lines, including one SNP in Ghi_A08G03061 that was detected in four G. hirsutum genotypes. This study provides the first comprehensive characterization of the cotton CesA gene family, which may play important roles in determining cotton fiber quality.

Detection and Characterization of Fusarium Wilt (Fusarium oxysporum f. sp. vasinfectum) Race 4 Causing Fusarium Wilt of Cotton Seedlings in New Mexico.

Fusarium wilt (FW), caused by Fusarium oxysporum f. sp. vasinfectum (Atk.) W.C. Snyder & H.N. Hans (FOV), is one of the most destructive diseases of cotton (Gossypium spp.) worldwide. FOV race 4 (FOV4) is a highly virulent nominal race of this pathogen and a significant threat to cotton production in the western and southwestern USA and, potentially, the entire Cotton Belt. A field survey to identify FOV4 was performed in three southern counties of New Mexico in 619 cotton fields from 2018 to 2020. From 132 samples of cotton plants that exhibited wilt symptoms, Fusarium spp. were the most frequently isolated group of fungal species, with an isolation frequency of 57.4%. Eighty-four Fusarium spp. isolates were subsequently characterized by a DNA sequence analysis of three genes, EF-1α, PHO, and BT, encoding for translation elongation factor, phosphate permease, and ß-tubulin, respectively. Forty-two isolates from 10 cotton fields were identified as FOV4 and confirmed with a positive 500-bp fragment diagnostic for FOV4. Twenty-six (62%) of the 42 FOV4 isolates were T type and the remainder (38%) were null type with and without a Tfo1 insertion in PHO, respectively. Each FOV4-infested field contained the same FOV4 genotype. Ten representative FOV4 isolates (one each from the 10 FOV4-infested fields) were evaluated for their pathogenicity on resistant Pima PHY 841 RF and susceptible Upland PHY 725 RF at 7, 14, 21, and 28 days after inoculation under temperature-controlled conditions at 21 to 22°C. Based on the disease severity rating, mortality rate, and area under the disease progress curve value, all 10 isolates were pathogenic to both cotton cultivars and differed in virulence; four isolates of the T genotype as a whole were more virulent than the six isolates of the N genotype. PHY 841 RF had significantly higher levels of resistance than PHY 725 RF to all FOV4 isolates. The results provide the first comprehensive account of the occurrence, distribution, and virulence of FOV4 in cotton production in New Mexico and will be useful for developing an effective strategy to manage FW in the state of New Mexico and the entire western and southwestern Cotton Belt.

A genome-wide analysis of pentatricopeptide repeat (PPR) protein-encoding genes in four Gossypium species with an emphasis on their expression in floral buds, ovules, and fibers in upland cotton.

Cotton is the most important natural fiber used in textiles. Breeding for "three-lines", i.e., cytoplasmic male sterility (CMS)-based sterile (A), maintainer (B), and restorer (R) line, is a promising approach to harness hybrid vigor in cotton. Pentatricopeptide repeat (PPR) protein-encoding genes play an important role in plant growth and development including restoration of CMS plants to male fertility. However, PPRs, especially those contributing to CMS and fiber development, remain largely unknown in cotton. In this study, a genome-wide identification and characterization of PPR gene family in four Gossypium species with genome sequences (G. arboreum, G. raimondii, G. hirsutum, and G. barbadense) were performed, and expressed PPR genes in developing floral buds, ovules, and fibers were compared to identify possible PPRs related to CMS restoration and fiber development. A total of 539, 558, 1032, and 1055 PPRs were predicted in the above four species, respectively, which were further mapped to chromosomes for a synteny analysis. Through an RNA-seq analysis, 86% (882) PPRs were expressed in flowering buds of upland cotton (G. hirsutum); however, only 11 and 6 were differentially expressed (DE) between restorer R and its near-isogenic (NI) B and between R and its NI A line, respectively. Another RNA-seq analysis identified the expression of only 54% (556) PPRs in 0 and 3 day(s) post-anthesis (DPA) ovules and 24% (247) PPRs in 10 DPA fibers; however, only 59, 6, and 27 PPRs were DE in 0 and 3 DPA ovules, and 10 DPA fibers between two backcross inbred lines (BILs) with differing fiber length, respectively. Only 2 PPRs were DE between Xuzhou 142 and its fiberless and fuzzless mutant. Quantitative RT-PCR analysis confirmed the validity of the RNA-seq results for the gene expression pattern. Therefore, only a very small number of PPRs may be associated with fertility restoration of CMS and genetic differences in fiber initiation and elongation. These results lay a foundation for understanding the roles of PPR genes in cotton, and will be useful in the prioritization of candidate PPR gene functional validation for cotton CMS restoration and fiber development.

A genome-wide association study uncovers consistent quantitative trait loci for resistance to Verticillium wilt and Fusarium wilt race 4 in the US Upland cotton.

KEY MESSAGE: A high-resolution GWAS detected consistent QTL for resistance to Verticillium wilt and Fusarium wilt race 4 in 376 U.S. Upland cotton accessions based on six independent replicated greenhouse tests. Verticillium wilt (VW, caused by Verticillium dahliae Kleb.) and Fusarium wilt (FOV, caused by Fusarium oxysporum f.sp. vasinfectum Atk. Sny & Hans) are the most important soil-borne fungal diseases in cotton. To augment and refine resistance quantitative trait loci (QTL), we conducted a genome-wide association study (GWAS) using high-density genotyping with the CottonSNP63K array. Resistance of 376 US Upland cotton accessions to a defoliating VW and virulent FOV4 was evaluated in four and two independent replicated greenhouse tests, respectively. A total of 15 and 13 QTL for VW and FOV4 resistances were anchored by 30 (on five chromosomes) and 56 (on six chromosomes) significant single nucleotide polymorphic (SNPs) markers, respectively. QTL on c8, c10, c16, and c21 were consistent in two or more tests for VW resistance, while two QTL on c8 and c14 were consistent for FOV4 resistance in two tests. Two QTL clusters on c16 and c19 were observed for both VW and FOV4 resistance, suggesting that these genomic regions may harbor genes in response to both diseases. Using BLAST search against the sequenced TM-1 genome, 30 and 35 candidate genes were identified on four QTL for VW resistance and on three QTL for FOV4 resistance, respectively. These genomic regions were rich in NBS-LRR genes presented in clusters. The results create opportunities for further studies to determine the correlations of field resistance with these QTL, molecular examinations of VW and FOV4 resistances, marker-assisted selection (MAS) and eventual cloning of QTL for disease resistance in cotton.

Examining two sets of introgression lines across multiple environments reveals background-independent and stably expressed quantitative trait loci of fiber quality in cotton.

KEY MESSAGE: Background-independent (BI) and stably expressed (SE) quantitative trait loci (QTLs) were identified using two sets of introgression lines across multiple environments. Genetic background more greatly affected fiber quality traits than environmental factors. Sixty-one SE-QTLs, including two BI-QTLs, were novel and 48 SE-QTLs, including seven BI-QTLs, were previously reported. Cotton fiber quality traits are controlled by QTLs and are susceptible to environmental influence. Fiber quality improvement is an essential goal in cotton breeding but is hindered by limited knowledge of the genetic basis of fiber quality traits. In this study, two sets of introgression lines of Gossypium hirsutum × G. barbadense were used to dissect the QTL stability of three fiber quality traits (fiber length, strength and micronaire) across environments using 551 simple sequence repeat markers selected from our high-density genetic map. A total of 76 and 120 QTLs were detected in the CCRI36 and CCRI45 backgrounds, respectively. Nine BI-QTLs were found, and 78 (41.71%) of the detected QTLs were reported previously. Thirty-nine and 79 QTLs were SE-QTLs in at least two environments in the CCRI36 and CCRI45 backgrounds, respectively. Forty-eight SE-QTLs, including seven BI-QTLs, were confirmed in previous reports, and 61 SE-QTLs, including two BI-QTLs, were considered novel. These results indicate that genetic background more strongly impacts on fiber quality traits than environmental factors. Twenty-three clusters with BI- and/or SE-QTLs were identified, 19 of which harbored favorable alleles from G. barbadense for two or three fiber quality traits. This study is the first report using two sets of introgression lines to identify fiber quality QTLs across environments in cotton, providing insights into the effect of genetic backgrounds and environments on the QTL expression of fiber quality and important information for the genetic basis underlying fiber quality traits toward QTL cloning and molecular breeding.

Genome-Wide Characterization and Analysis of CIPK Gene Family in Two Cultivated Allopolyploid Cotton Species: Sequence Variation, Association with Seed Oil Content, and the Role of GhCIPK6.

Calcineurin B-like protein-interacting protein kinases (CIPKs), as key regulators, play an important role in plant growth and development and the response to various stresses. In the present study, we identified 80 and 78 CIPK genes in the Gossypium hirsutum and G. barbadense, respectively. The phylogenetic and gene structure analysis divided the cotton CIPK genes into five groups which were classified into an exon-rich clade and an exon-poor clade. A synteny analysis showed that segmental duplication contributed to the expansion of Gossypium CIPK gene family, and purifying selection played a major role in the evolution of the gene family in cotton. Analyses of expression profiles showed that GhCIPK genes had temporal and spatial specificity and could be induced by various abiotic stresses. Fourteen GhCIPK genes were found to contain 17 non-synonymous single nucleotide polymorphisms (SNPs) and co-localized with oil or protein content quantitative trait loci (QTLs). Additionally, five SNPs from four GhCIPKs were found to be significantly associated with oil content in one of the three field tests. Although most GhCIPK genes were not associated with natural variations in cotton oil content, the overexpression of the GhCIPK6 gene reduced the oil content and increased C18:1 and C18:1+C18:1d6 in transgenic cotton as compared to wild-type plants. In addition, we predicted the potential molecular regulatory mechanisms of the GhCIPK genes. In brief, these results enhance our understanding of the roles of CIPK genes in oil synthesis and stress responses.