Genome resources for three modern cotton lines guide future breeding efforts.

Cotton (Gossypium hirsutum L.) is the key renewable fibre crop worldwide, yet its yield and fibre quality show high variability due to genotype-specific traits and complex interactions among cultivars, management practices and environmental factors. Modern breeding practices may limit future yield gains due to a narrow founding gene pool. Precision breeding and biotechnological approaches offer potential solutions, contingent on accurate cultivar-specific data. Here we address this need by generating high-quality reference genomes for three modern cotton cultivars ('UGA230', 'UA48' and 'CSX8308') and updating the 'TM-1' cotton genetic standard reference. Despite hypothesized genetic uniformity, considerable sequence and structural variation was observed among the four genomes, which overlap with ancient and ongoing genomic introgressions from 'Pima' cotton, gene regulatory mechanisms and phenotypic trait divergence. Differentially expressed genes across fibre development correlate with fibre production, potentially contributing to the distinctive fibre quality traits observed in modern cotton cultivars. These genomes and comparative analyses provide a valuable foundation for future genetic endeavours to enhance global cotton yield and sustainability.

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.

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.

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.

Studies of Evaluation Methods for Resistance to Fusarium Wilt Race 4 (Fusarium oxysporum f. sp. vasinfectum) in Cotton: Effects of Cultivar, Planting Date, and Inoculum Density on Disease Progression.

Fusarium wilt caused by Fusarium oxysporum f. sp. vasinfectum race 4 (FOV4) is an early season disease causing root rot, seedling wilt, and death. To develop an appropriate field evaluation method for resistance to FOV4 in cotton breeding, the objectives of this study were to investigate the effects of cultivar, planting date, and inoculum density on disease progression in 2020-2021. Results showed that the usual local mid-April planting had the lowest disease severity (DSR) or mortality rate (MR) in 2020 and 2021. DSR or MR increased at the late April and early May plantings in both years and reached the highest at the early May planting in 2020, while MR in 2021 was followed by a decrease in the late May planting and reached the highest in the mid-June planting. Local daily low temperatures between mid-April and mid-June were favorable for FOV4 infections, whereas daily high temperatures at 35°C or higher suppressed wilt severity. When seedlings at the 2-true leaf stage were inoculated with 104, 105, 106, and 107 conidia ml-1 per plant in 2020, DSR was low but a linear relationship between inoculum density and DSR was observed. When a FOV4-infested soil supplemented with artificial inoculation was used, disease progression in three moderately susceptible or moderately resistant cultivars followed a linear model, while it followed a quadratic model in the highly susceptible Pima S-7 cultivar only. Among the other three cultivars, FM 2334GLT had the lowest DSR or MR except for one planting date in both years, followed by PHY 725 RF and Pima PHY 881 RF in ascending order, which were consistent with the difference in regression coefficients of the linear models. This study demonstrates that disease progression curves due to FOV4 can be used to compare responses to FOV4 infections among cotton genotypes in cotton breeding and genetic studies, regardless of planting date and inoculation method.

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.

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.

Stories from the Greenhouse-A Brief on Cotton Seed Germination.

Seed germination is the basis for the proliferation of sexual-reproducing plants, efficient crop production, and a successful crop improvement research program. Cotton (Gossypium spp.), the subject of this review, can be often sensitive to germination conditions. The hardness of the cotton seed coat, storage, extreme temperatures, and dormancy are some of the factors that can influence cotton seed germination. Research programs conducting studies on exotic and wild cotton species are especially affected by those hurdles. Here, we briefly review the challenges of cotton seed germination and some of the approaches our cotton breeding program explored throughout the years.

A nonproteinaceous Fusarium cell wall extract triggers receptor-like protein-dependent immune responses in Arabidopsis and cotton.

Fusarium wilt caused by the ascomycete fungus Fusarium oxysporum is a devastating disease of many economically important crops. The mechanisms underlying plant responses to F. oxysporum infections remain largely unknown. We demonstrate here that a water-soluble, heat-resistant and nonproteinaceous F. oxysporum cell wall extract (FoCWE) component from multiple F. oxysporum isolates functions as a race-nonspecific elicitor, also termed pathogen-associated molecular pattern (PAMP). FoCWE triggers several demonstrated immune responses, including mitogen-activated protein (MAP) kinase phosphorylation, reactive oxygen species (ROS) burst, ethylene production, and stomatal closure, in cotton and Arabidopsis. Pretreated FoCWE protects cotton seeds against infections by virulent F. oxysporum f. sp. vasinfectum (Fov), and Arabidopsis plants against the virulent bacterium, Pseudomonas syringae, suggesting the potential application of FoCWEs in crop protection. Host-mediated responses to FoCWE do not appear to require LYKs/CERK1, BAK1 or SOBIR1, which are commonly involved in PAMP perception and/or signalling. However, FoCWE responses and Fusarium resistance in cotton partially require two receptor-like proteins, GhRLP20 and GhRLP31. Transcriptome analysis suggests that FoCWE preferentially activates cell wall-mediated defence, and Fov has evolved virulence mechanisms to suppress FoCWE-induced defence. These findings suggest that FoCWE is a classical PAMP that is potentially recognised by a novel pattern-recognition receptor to regulate cotton resistance to Fusarium infections.

Responses of Upland Cotton (Gossypium hirsutum L.) Lines to Irrigated and Rainfed Conditions of Texas High Plains.

Understanding drought stress responses and the identification of phenotypic traits associated with drought are key factors in breeding for sustainable cotton production in limited irrigation water of semi-arid environments. The objective of this study was to evaluate the responses of upland cotton lines to rainfed and irrigated conditions. We compared selected agronomic traits over time, final yield and fiber quality of cotton lines grown in irrigated and rainfed trials. Under rainfed conditions, the average number of squares per plant sharply declined during weeks 10 to 14 while the average number of bolls per plant significantly reduced during weeks 13 to 15 after planting. Therefore, weeks 10 to 14 and weeks 13 to 15 are critical plant growth stages to differentiate among upland cotton lines for square and boll set, respectively, under drought stress. Variation in square and boll set during this stage may translate into variable lint percent, lint yield and fiber properties under water-limited conditions. Lint yield and fiber quality were markedly affected under rainfed conditions in all cotton lines tested. Despite significantly reduced lint yield in rainfed trials, some cotton lines including 11-21-703S, 06-46-153P, CS 50, L23, FM 989 and DP 491 performed relatively well under stress compared to other cotton lines. The results also reveal that cotton lines show variable responses for fiber properties under irrigated and rainfed trials. Breeding line 12-8-103S produced long, uniform and strong fibers under both irrigated and rainfed conditions. The significant variation observed among cotton genotypes for agronomic characteristics, yield and fiber quality under rainfed conditions indicate potential to breed cotton for improved drought tolerance.

The relationship between commercial cotton cultivars with varying Meloidogyne incognita resistance genes and yield.

Small plot cotton cultivar trials (12 trials) were conducted from 2016 to 2019 in fields infested with Meloidogyne incognita. Entries in these trials included commercial cultivars with partial and high resistance to M. incognita, as well as cultivars with no known resistance. Different resistant groups were created based on different cotton seed companies and their descriptions of the M. incognita resistant cultivars. Groups were none (susceptible); partial resistance found in Stoneville or Fibermax cultivars (PR-FM/ST); partial resistance found in PhytoGen cultivars (PR-PHY); resistance (unknown gene(s)) in Deltapine cultivars (NR-DP); and highly resistant cultivars homozygous for RK1 and RK2 resistant genes in PhytoGen cultivars (HR-PHY). The highest lint yields using a mixed model analysis were found in the PR-FM/ST (1,396 kg lint/ha), HR-PHY (1,327 kg lint/ha), and PR-PHY (1,314 kg lint/ha) groups. Yield for NR-DP (1,234 kg lint/ha) was not different (p > 0.05) than yield for susceptible cultivars (1,243 kg lint/ha). If the older resistant cultivars from Deltapine and PhytoGen (those with only Roundup Ready® herbicide technology) were removed from the analysis, then HR-PHY yields increased by 133 kg of lint/ha to 1,460 kg lint/ha and NR-DP yields remained approximately unchanged (1,227 kg lint/ha). Newer HR-PHY had much improved yield over the first HR-PHY cultivars. Newer HR-PHY averaged 17% higher yield than the susceptible group. LOG10 (M. incognita eggs/500 cm3 soil + 1) were highest for the susceptible cultivars (3.2), followed by PR-FM/ST (2.6), NR-DP (2.4), PR-PHY (2.1), and lowest with HR-PHY (1.4). The newer HR-PHY cultivars (those with ENLIST® herbicide technology) combine excellent yields (17% higher than susceptible cultivars) with high (96%) suppression of M. incognita.Small plot cotton cultivar trials (12 trials) were conducted from 2016 to 2019 in fields infested with Meloidogyne incognita. Entries in these trials included commercial cultivars with partial and high resistance to M. incognita, as well as cultivars with no known resistance. Different resistant groups were created based on different cotton seed companies and their descriptions of the M. incognita resistant cultivars. Groups were none (susceptible); partial resistance found in Stoneville or Fibermax cultivars (PR-FM/ST); partial resistance found in PhytoGen cultivars (PR-PHY); resistance (unknown gene(s)) in Deltapine cultivars (NR-DP); and highly resistant cultivars homozygous for RK1 and RK2 resistant genes in PhytoGen cultivars (HR-PHY). The highest lint yields using a mixed model analysis were found in the PR-FM/ST (1,396 kg lint/ha), HR-PHY (1,327 kg lint/ha), and PR-PHY (1,314 kg lint/ha) groups. Yield for NR-DP (1,234 kg lint/ha) was not different (p > 0.05) than yield for susceptible cultivars (1,243 kg lint/ha). If the older resistant cultivars from Deltapine and PhytoGen (those with only Roundup Ready® herbicide technology) were removed from the analysis, then HR-PHY yields increased by 133 kg of lint/ha to 1,460 kg lint/ha and NR-DP yields remained approximately unchanged (1,227 kg lint/ha). Newer HR-PHY had much improved yield over the first HR-PHY cultivars. Newer HR-PHY averaged 17% higher yield than the susceptible group. LOG10 (M. incognita eggs/500 cm3 soil + 1) were highest for the susceptible cultivars (3.2), followed by PR-FM/ST (2.6), NR-DP (2.4), PR-PHY (2.1), and lowest with HR-PHY (1.4). The newer HR-PHY cultivars (those with ENLIST® herbicide technology) combine excellent yields (17% higher than susceptible cultivars) with high (96%) suppression of M. incognita.

Impacts of Verticillium Wilt on Photosynthesis Rate, Lint Production, and Fiber Quality of Greenhouse-Grown Cotton (Gossypium hirsutum).

Verticillium wilt, caused by Verticillium dahliae Kleb., leads to significant losses in cotton yield and fiber quality worldwide. To investigate Verticillium wilt impact on photosynthesis rate, yield, and fiber quality, six upland cotton genotypes, namely Verticillium susceptible (DP 1612 B2XF) and partially resistant (FM 2484B2F) commercial cultivars and four breeding lines, were grown to maturity under greenhouse conditions in soil either infested or not infested with V. dahliae microsclerotia. Photosynthetic rate, lint, and seed yield were all higher (p < 0.05) for FM 2484B2F than DP 1612 B2XF when infected with V. dahliae. When comparing healthy (H) to Verticillium wilt (VW) affected plants, fiber properties were greatly impacted. Micronaire decreased from 5.0 (H) to 3.6 (VW) with DP 1612 B2XF and 4.4 (H) to 4.1 (VW) with FM 2484B2F. The maturity ratio decreased from 0.90 (H) to 0.83 (VW) for DP 1612 B2XF and was unchanged for FM 2484B2F (0.90). Fiber properties such as short fiber content, nep count, fineness, and immature fiber content were also significantly affected under Verticillium wilt pressure. With Verticillium wilt affected plants, lines 16-13-601V and 17-17-206V performed similarly to FM 2484B2F for photosynthetic rate, yield, and all fiber properties measured. When selecting for improved cultivars in the presence of Verticillium wilt, it is important to select for relatively unchanged fiber properties under disease pressure in addition to reduced disease severity and increased yield.

Regulation of cotton (Gossypium hirsutum) drought responses by mitogen-activated protein (MAP) kinase cascade-mediated phosphorylation of GhWRKY59.

Drought is a key limiting factor for cotton (Gossypium spp.) production, as more than half of the global cotton supply is grown in regions with high water shortage. However, the underlying mechanism of the response of cotton to drought stress remains elusive. By combining genome-wide transcriptome profiling and a loss-of-function screen using virus-induced gene silencing, we identified Gossypium hirsutum GhWRKY59 as an important transcription factor that regulates the drought stress response in cotton. Biochemical and genetic analyses revealed a drought stress-activated mitogen-activated protein (MAP) kinase cascade consisting of GhMAP3K15-Mitogen-activated Protein Kinase Kinase 4 (GhMKK4)-Mitogen-activated Protein Kinase 6 (GhMPK6) that directly phosphorylates GhWRKY59 at residue serine 221. Interestingly, GhWRKY59 is required for dehydration-induced expression of GhMAPK3K15, constituting a positive feedback loop of GhWRKY59-regulated MAP kinase activation in response to drought stress. Moreover, GhWRKY59 directly binds to the W-boxes of DEHYDRATION-RESPONSIVE ELEMENT-BINDING PROTEIN 2 (GhDREB2), which encodes a dehydration-inducible transcription factor regulating the plant hormone abscisic acid (ABA)-independent drought response. Our study identified a complete MAP kinase cascade that phosphorylates and activates a key WRKY transcription factor, and elucidated a regulatory module, consisting of GhMAP3K15-GhMKK4-GhMPK6-GhWRKY59-GhDREB2, that is involved in controlling the cotton drought response.

Cotton GhBAK1 mediates Verticillium wilt resistance and cell death.

Virus-induced gene silencing (VIGS) offers a powerful approach for functional analysis of individual genes by knocking down their expression. We have adopted this approach to dissect gene functions in cotton resistant to Verticillium wilt, one of the most devastating diseases worldwide. We showed here that highly efficient VIGS was obtained in a cotton breeding line (CA4002) with partial resistance to Verticillium wilt, and GhMKK2 and GhVe1 are required for its resistance to Verticillium wilt. Arabidopsis AtBAK1/SERK3, a central regulator in plant disease resistance, belongs to a subfamily of somatic embryogenesis receptor kinases (SERKs) with five members, AtSERK1 to AtSERK5. Two BAK1 orthologs and one SERK1 ortholog were identified in the cotton genome. Importantly, GhBAK1 is required for CA4002 resistance to Verticillium wilt. Surprisingly, silencing of GhBAK1 is sufficient to trigger cell death accompanied with production of reactive oxygen species in cotton. This result is distinct from Arabidopsis in which AtBAK1 and AtSERK4 play redundant functions in cell death control. Apparently, cotton has only evolved SERK1 and BAK1 whereas AtSERK4/5 are newly evolved genes in Arabidopsis. Our studies indicate the functional importance of BAK1 in Verticillium wilt resistance and suggest the dynamic evolution of SERK family members in different plant species.