Multi-omics analysis of pigmentation related to proanthocyanidin biosynthesis in brown cotton (Gossypium hirsutum L.).

Naturally-colored brown cotton (NBC) fiber is an environmentally friendly raw source of fiber for textile applications. The fiber of some NBC cultivars exhibits flame-retardant properties, which can be used in textiles that require flame resistance. Proanthocyanidins or their derivatives are responsible for the brown pigment in NBC; however, how flame retardancy is related to pigmentation in NBC is poorly understood. To gain insight into brown pigment biosynthesis, we conducted comparative transcripts and metabolites profiling analysis of developing cotton fibers between the brown (MC-BL) and white (MC-WL) cotton near-isogenic lines (NILs), genetically different only in the Lc1 locus. In this study, mass spectrometry was used to detect metabolites in BL and WL developing fibers at 8, 12, 16, 20, 24, 36, and 40 days post anthesis (DPA) and mature fibers. Transcripts analysis was performed at two critical fiber developmental points, 8 DPA (fiber elongation) and 20 DPA (secondary cell wall deposition). We found 5836 (ESI MS positive mode) and 4541 (ESI MS negative mode) metabolites significantly different accumulated between BL and WL. Among them, 142 were known non-redundant metabolites, including organic acids, amino acids, and derivatives of the phenylpropanoid pathway. Transcript analysis determined 1691 (8 DPA) and 5073 (20 DPA) differentially expressed genes (DEGs) between BL and WL, with the majority of DEGs down-regulated at 20 DPA. Organic acids of the citric acid cycle were induced, while most of the detected amino acids were reduced in the MC-BL line. Both cis- and trans-stereoisomers of flavan-3-ols were detected in developing MC-WL and MC-BL fibers; however, the gallocatechin and catechin accumulated multiple times higher. Gas chromatography-mass spectrometry (GC-MS) analysis of fatty acids determined that palmitic acid long-chain alcohols were the main constituents of waxes of mature fibers. Energy-dispersive X-ray spectrometry (EDS) analysis of mature fibers revealed that potassium accumulated three times greater in MC-BL than in MC-WL mature fibers. This study provides novel insights into the biosynthesis of pigments and its association with flame retardancy in NBC fibers.

Virulence of Two Isolates of Meloidogyne enterolobii (Guava Root-Knot Nematode) from North Carolina on Cotton Lines Resistant to Southern Root-Knot Nematode (M. incognita) and Reniform Nematode (Rotylenchulus reniformis).

Meloidogyne enterolobii [the guava root-knot nematode (RKN)] is an emerging plant-parasitic nematode that poses a threat to Upland cotton (Gossypium hirsutum) production in the southeastern United States. Like other RKN spp., M. enterolobii has a wide host range and proven ability to overcome resistance sources that have helped protect crops from other Meloidogyne spp., including the southern RKN (Meloidogyne incognita). In this study we evaluated the virulence of two North Carolina M. enterolobii isolates on Upland cotton germplasm lines having resistance quantitative trait loci (QTL) to RKN (M240 RNR, MRk-Rn-1) and/or reniform nematode (Rotylenchulus reniformis) (M713 Ren1, MRk-Rn-1) in comparison to their susceptible recurrent parents (DPL61, SG747). Multiple assays using eggs or J2 as inoculum demonstrated that both isolates reproduced equally well on all germplasm lines, producing reproductive factor (RF) values ≥ 6 on the otherwise nematode-resistant lines. Measurements of seedling growth in control and inoculated containers suggested that existing nematode-resistance QTL may offer a level of tolerance to M. enterolobii infection that should be further explored in greenhouse and field environments. Meloidogyne enterolobii infection of SG747 and MRk-Rn-1 showed nearly identical stages of symptom and nematode development over a time-course of 24 days. These data demonstrate that existing RKN and RN resistance QTL available in elite cotton varieties to producers are most likely insufficient in preventing yield loss due to M. enterolobii and that future research should focus on (i) understanding the M. enterolobii-cotton interaction at the molecular level, and (ii) screening novel germplasm collections to identify resistance loci.

Flame resistant cotton lines generated by synergistic epistasis in a MAGIC population.

Textiles made from cotton fibers are flammable and thus often include flame retardant additives for consumer safety. Transgressive segregation in multi-parent populations facilitates new combinations of alleles of genes and can result in traits that are superior to those of any of the parents. A screen of 257 recombinant inbred lines from a multi-parent advanced generation intercross (MAGIC) population for naturally enhance flame retardance (FR) was conducted. All eleven parents, like all conventional white fiber cotton cultivars produce flammable fabric. MAGIC recombinant inbred lines (RILs) that produced fibers with significantly lower heat release capacities (HRC) as measured by microscale combustion calorimetry (MCC) were identified and the stability of the phenotypes of the outliers were confirmed when the RILs were grown at an additional location. Of the textiles fabricated from the five superior RILs, four exhibited the novel characteristic of inherent flame resistance. When exposed to open flame by standard 45° incline flammability testing, these four fabrics self-extinguished. To determine the genetic architecture of this novel trait, linkage, epistatic and multi-locus genome wide association studies (GWAS) were conducted with 473k SNPs identified by whole genome sequencing (WGS). Transcriptomes of developing fiber cells from select RILs were sequenced (RNAseq). Together, these data provide insight into the genetic mechanism of the unexpected emergence of flame-resistant cotton by transgressive segregation in a breeding program. The incorporation of this trait into global cotton germplasm by breeding has the potential to greatly reduce the costs and impacts of flame-retardant chemicals.

Genomic confirmation of Gossypium barbadense introgression into G. hirsutum and a subsequent MAGIC population.

Introgression of superior fiber traits from Pima cotton (Gossypium barbadense, GB) into high yield Upland cotton (G. hirsutum) has been a breeding objective for many years in a few breeding programs in the world. However, progress has been very slow due to introgression barriers resulting from whole genome hybridization between the two species. To minimize such barriers, chromosome substitution lines (CS-B) from Pima cotton 3-79 in an Upland cotton cultivar TM-1 were developed. A multiparent advanced generation inter-cross (MAGIC) population consisting of 180 recombinant inbred lines (RILs) was subsequently made using the 18 CS-B lines and three Upland cotton cultivars as parents. In this research, we sequenced the whole genomes of the 21 parents and 180 RILs to examine the G. barbadense introgression. Of the 18 CS-B lines, 11 contained the target GB chromosome or chromosome segment, two contained more than two GB chromosomes, and five did not have the expected introgression. Residual introgression in non-target chromosomes was prevalent in all CS-B lines. A clear structure existed in the MAGIC population and the 180 RILs were distributed into three groups, i.e., high, moderate, and low GB introgression. Large blocks of GB chromosome introgression were still present in some RILs after five cycles of random-mating, an indication of recombination suppression or other unknown reasons present in the population. Identity by descent analysis revealed that the MAGIC RILs contained less introgression than expected. This research presents an insight on understanding the complex problems of introgression between cotton species.

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.

Genomic interrogation of a MAGIC population highlights genetic factors controlling fiber quality traits in cotton.

Cotton (Gossypium hirsutum L.) fiber is the most important resource of natural and renewable fiber for the textile industry. However, the understanding of genetic components and their genome-wide interactions controlling fiber quality remains fragmentary. Here, we sequenced a multiple-parent advanced-generation inter-cross (MAGIC) population, consisting of 550 individuals created by inter-crossing 11 founders, and established a mosaic genome map through tracing the origin of haplotypes that share identity-by-descent (IBD). We performed two complementary GWAS methods-SNP-based GWAS (sGWAS) and IBD-based haplotype GWAS (hGWAS). A total of 25 sQTLs and 14 hQTLs related to cotton fiber quality were identified, of which 26 were novel QTLs. Two major QTLs detected by both GWAS methods were responsible for fiber strength and length. The gene Ghir_D11G020400 (GhZF14) encoding the MATE efflux family protein was identified as a novel candidate gene for fiber length. Beyond the additive QTLs, we detected prevalent epistatic interactions that contributed to the genetics of fiber quality, pinpointing another layer for trait variance. This study provides new targets for future molecular design breeding of superior fiber quality.

Effects of Interspecific Chromosome Substitution in Upland Cotton on Cottonseed Macronutrients.

Nutrients, including macronutrients such as Ca, P, K, and Mg, are essential for crop production and seed quality, and for human and animal nutrition and health. Macronutrient deficiencies in soil lead to poor crop nutritional qualities and a low level of macronutrients in cottonseed meal-based products, leading to malnutrition. Therefore, the discovery of novel germplasm with a high level of macronutrients or significant variability in the macronutrient content of crop seeds is critical. To our knowledge, there is no information available on the effects of chromosome or chromosome arm substitution on cottonseed macronutrient content. The objective of this study was to evaluate the effects of chromosome or chromosome arm substitution on the variability and content of the cottonseed macronutrients Ca, K, Mg, N, P, and S in chromosome substitution lines (CS). Nine chromosome substitution lines were grown in two-field experiments at two locations in 2013 in South Carolina, USA, and in 2014 in Mississippi, USA. The controls used were TM-1, the recurrent parent of the CS line, and the cultivar AM UA48. The results showed major variability in macronutrients among CS lines and between CS lines and controls. For example, in South Carolina, the mean values showed that five CS lines (CS-T02, CS-T04, CS-T08sh, CS-B02, and CS-B04) had higher Ca level in seed than controls. Ca levels in these CS lines varied from 1.88 to 2.63 g kg-1 compared with 1.81 and 1.72 g kg-1 for TM-1 and AMUA48, respectively, with CS-T04 having the highest Ca concentration. CS-M08sh exhibited the highest K concentration (14.50 g kg-1), an increase of 29% and 49% over TM-1 and AM UA48, respectively. Other CS lines had higher Mg, P, and S than the controls. A similar trend was found at the MS location. This research demonstrated that chromosome substitution resulted in higher seed macronutrients in some CS lines, and these CS lines with a higher content of macronutrients can be used as a genetic tool towards the identification of desired seed nutrition traits. Also, the CS lines with higher desired macronutrients can be used as parents to breed for improved nutritional quality in Upland cotton, Gossypium hirsutum L., through improvement by the interspecific introgression of desired seed nutrient traits such as Ca, K, P, S, and N. The positive and significant (p ≤ 0.0001) correlation of P with Ca, P with Mg, S with P, and S with N will aid in understanding the relationships between nutrients to improve the fertilizer management program and maintain higher cottonseed nutrient content.

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.

Effects of Interspecific Chromosome Substitution in Upland Cotton on Cottonseed Micronutrients.

Micronutrients are essential for plant growth and development, and important for human health nutrition and livestock feed. Therefore, the discovery of novel germplasm with significant variability or higher micronutrients content in crop seeds is critical. Currently, there is no information available on the effects of chromosome or chromosome arm substitution in cotton on cottonseed micronutrients. Thus, the objective of this study was to evaluate the effects of chromosome or chromosome arm substitution on the variability and levels of micronutrients B, Fe, Cu, Zn, Mn, and Ni in cottonseed from chromosome substitution (CS) cotton lines. Our hypothesis was that interspecific chromosome substitution in cotton can affect cottonseed micronutrients content, resulting in significant differences and variabilities of these nutrients among CS lines and between CS lines and the controls. Nine CS lines were grown in two-field experiments at two locations (in 2013 in South Carolina, USA; and in 2014 in Mississippi, USA). TM-1 (the recurrent parent of the CS line) and AM UA48 (cultivar) were used as control. The results showed significant variability among CS lines compared to the controls AM UA48 and TM-1. For example, in South Carolina (SC), B concentration in cottonseed ranged from 10.35 mg kg-1 in CS-M02 to 13.67 mg kg-1 in CS-T04. The concentration of Cu ranged from 4.81 mg kg-1 in CS-B08sh to 7.65 mg kg-1 in CS-T02, and CS-T02 was higher than both controls. The concentration of Fe ranged from 36.09 mg kg-1 to 56.69 mg kg-1 (an increase up to 57%), and six CS lines (CS-B02, CS-B08sh, CS-M02, CS-M04, CS-T02, and CS-T04) had higher concentration than both controls in 2013. In 2014 at the Mississippi location (MS), similar observation was found with CS lines for micronutrients content. The CS lines with higher concentrations of these micronutrients can be used as a genetic tool toward QTL identification for desired seed traits because these lines are genetically similar with TM-1, except the substituted chromosome or chromosome segment pairs from the alien species. Chromosome substitution provides an effective means for upland cotton improvement by targeted interspecific introgression, yielding CS lines that facilitate trait discovery, such as seed micronutritional qualities, due to increased isogenicity and markedly reduced complexity from epistatic interactions with non-target alien chromosomes. The positive correlation between B, Cu, and Fe at both locations, between Ni and Mn, between Zn and Cu, and between Zn and Ni at both locations signify the importance of a good agricultural and fertilizer management of these nutrients to maintain higher cottonseed nutrient content.

Analysis of Cotton Chromosome 11 and 14 Root-Knot Nematode Resistance Quantitative Trait Loci Effects on Root-Knot Nematode Postinfection Development, Egg Mass Formation, and Fecundity.

Cotton (Gossypium hirsutum) resistance to root-knot nematode (RKN) (Meloidogyne incognita) is controlled by quantitative trait loci (QTLs) on chromosomes 11 (CHR11) and 14 (CHR14). The individual contributions of these QTLs to resistance are not completely understood. We developed near isogenic lines susceptible at both loci (null), having CHR11 or CHR14 alone, and having both QTLs (CHR11/CHR14). RKN reproduction, postinfection development, egg mass formation, and adult female fecundity were evaluated. Total RKN reproduction was reduced more in CHR14 versus CHR11 but not as greatly as in CHR11/CHR14. Second-stage juvenile (J2) development to the J3 and J4 (J3+J4) life stages was delayed in CHR11, whereas the J2 transition to J3+J4 in CHR14 followed a similar track as in null plants. Development of J3+J4 nematodes to adult females was inhibited in CHR14 at 21 days after inoculation (DAI). Adult female numbers were decreased in CHR11 and CHR14 at 21 and 28 DAI, with CHR11/CHR14 showing an even greater reduction by 28 DAI. The number of egg masses per gram of root at 21, 28, and 35 DAI formed on CHR11 and CHR14 followed a similar track as numbers of adult females. RKN adult female fecundity (eggs/egg mass) was reduced for CHR11 and CHR14 compared with the null at 21 DAI; however, CHR11 eggs/egg mass was only slightly reduced versus the null by 28 DAI. In contrast, CHR14 eggs/egg mass was like CHR11/CHR14, showing a 4-fold decrease compared with CHR11 and the null.

Evaluation of genomic selection methods for predicting fiber quality traits in Upland cotton.

The use of genomic selection (GS) has stimulated a new way to utilize molecular markers in breeding for complex traits in the absence of phenotypic data. GS can potentially decrease breeding cycle by selecting the progeny in the early stages. The objective of this study was to experimentally evaluate the potential value of genomic selection in Upland cotton breeding. Six fiber quality traits were obtained in 3 years of replicated field trials in Starkville, MS. Genotyping-by-sequencing-based genotyping was performed using 550 recombinant inbred lines of the multi-parent advanced generation inter-cross population, and 6292 molecular markers were used for the GS analysis. Several methods were compared including genomic BLUP (GBLUP), ridge regression BLUP (rrBLUP), BayesB, Bayesian LASSO, and reproducing kernel hilbert spaces (RKHS). The average heritability (h2) ranged from 0.38 to 0.88 for all tested traits across the 3 years evaluated. BayesB predicted the highest accuracies among the five GS methods tested. The prediction ability (PA) and prediction accuracy (PACC) varied widely across 3 years for all tested traits and the highest PA and PACC were 0.65, and 0.69, respectively, in 2010 for fiber elongation. Marker density and training population size appeared to be very important factors for PA and PACC in GS. Results indicated that BayesB-based GS method could predict genomic estimated breeding value efficiently in Upland cotton fiber quality attributes and has great potential utility in breeding by reducing cost and time.

Genetic and transcriptomic dissection of the fiber length trait from a cotton (Gossypium hirsutum L.) MAGIC population.

BACKGROUND: Improving cotton fiber length without reducing yield is one of the major goals of cotton breeding. However, genetic improvement of cotton fiber length by breeding has been a challenge due to the narrow genetic diversity of modern cotton cultivars and negative correlations between fiber quality and yield traits. A multi-parent advanced generation inter-cross (MAGIC) population developed through random mating provides an excellent genetic resource that allows quantitative trait loci (QTL) and causal genes to be identified. RESULTS: An Upland cotton MAGIC population, consisting of 550 recombinant inbred lines (RILs) derived from eleven different cultivars, was used to identify fiber length QTLs and potential genes that contribute to longer fibers. A genome wide association study (GWAS) identified a cluster of single nucleotide polymorphisms (SNPs) on chromosome (Chr.) D11 that is significantly associated with fiber length. Further evaluation of the Chr. D11 genomic region among lines of the MAGIC population detected that 90% of RILs have a D11 haplotype similar to the reference TM-1 genome (D11-ref), whereas 10% of RILs inherited an alternative haplotype from one of the parents (D11-alt). The average length of fibers of D11-alt RILs was significantly shorter compared to D11-ref RILs, suggesting that alleles in the D11-alt haplotype contributed to the inferior fiber quality. RNAseq analysis of the longest and shortest fiber length RILs from D11-ref and D11-alt populations identified 949 significantly differentially expressed genes (DEGs). Gene set enrichment analysis revealed that different functional categories of genes were over-represented during fiber elongation between the four selected RILs. We found 12 genes possessing non-synonymous SNPs (nsSNPs) significantly associated with the fiber length, and three that were highly significant and were clustered at D11:24-Mb, including D11G1928, D11G1929 and D11G1931. CONCLUSION: The results of this study provide insights into molecular aspects of genetic variation in fiber length and suggests candidate genes for genetic manipulation for cotton improvement.

A novel variant of Gh_D02G0276 is required for root-knot nematode resistance on chromosome 14 (D02) in Upland cotton.

KEY MESSAGE: MAGIC population sequencing and virus-induced gene silencing identify Gh_D02G0276 as a novel root-knot nematode resistance gene on chromosome 14 in Upland cotton. The southern root-knot nematode [RKN; Meloidogyne incognita (Kofoid & White)] remains the primary yield-limiting biotic stress to Upland cotton (Gossypium hirsutum L.) throughout the southeastern USA. While useful genetic markers have been developed for two major RKN resistance loci on chromosomes 11 (A11) and 14 (D02), these markers are not completely effective because the causative genes have not been identified. Here, we sequenced 550 recombinant inbred lines (RILs) from a multi-parent advanced generation intercross (MAGIC) population to identify five RILs that had informative recombinations near the D02-RKN resistance locus. The RKN resistance phenotypes of these five RILs narrowed the D02-RKN locus to a 30-kb region with four candidate genes. We conducted virus-induced gene silencing (VIGS) on each of these genes and found that Gh_D02G0276 was required for suppression of RKN egg production conferred by the Chr. D02 resistance gene. The resistant lines all possessed an allele of Gh_D02G0276 that showed non-synonymous mutations and was prematurely truncated. Furthermore, a Gh_D02G0276-specific marker for the resistance allele variant was able to identify RKN-resistant germplasm from a collection of 367 cotton accessions. The Gh_D02G0276 peptide shares similarity with domesticated hAT-like transposases with additional novel N- and C-terminal domains that resemble the target of known RKN effector molecules and a prokaryotic motif, respectively. The truncation in the resistance allele results in a loss of a plant nuclear gene-specific C-terminal motif, potentially rendering this domain antigenic due to its high homology with bacterial proteins. The conclusive identification of this RKN resistance gene opens new avenues for understanding plant resistance mechanisms to RKN as well as opportunities to develop more efficient marker-assisted selection in cotton breeding programs.

Whole genome sequencing of a MAGIC population identified genomic loci and candidate genes for major fiber quality traits in upland cotton (Gossypium hirsutum L.).

KEY MESSAGE: Significant associations between candidate genes and six major cotton fiber quality traits were identified in a MAGIC population using GWAS and whole genome sequencing. Upland cotton (Gossypium hirsutum L.) is the world's major renewable source of fibers for textiles. To identify causative genetic variants that influence the major agronomic measures of cotton fiber quality, which are used to set discount or premium prices on each bale of cotton in the USA, we measured six fiber phenotypes from twelve environments, across three locations and 7 years. Our 550 recombinant inbred lines were derived from a multi-parent advanced generation intercross population and were whole-genome-sequenced at 3× coverage, along with the eleven parental cultivars at 20× coverage. The segregation of 473,517 single nucleotide polymorphisms (SNPs) in this population, including 7506 non-synonymous mutations, was combined with phenotypic data to identify seven highly significant fiber quality loci. At these loci, we found fourteen genes with non-synonymous SNPs. Among these loci, some had simple additive effects, while others were only important in a subset of the population. We observed additive effects for elongation and micronaire, when the three most significant loci for each trait were examined. In an informative subset where the major multi-trait locus on chromosome A07:72-Mb was fixed, we unmasked the identity of another significant fiber strength locus in gene Gh_D13G1792 on chromosome D13. The micronaire phenotype only revealed one highly significant genetic locus at one environmental location, demonstrating a significant genetic by environment component. These loci and candidate causative variant alleles will be useful to cotton breeders for marker-assisted selection with minimal linkage drag and potential biotechnological applications.

The P450 gene CYP749A16 is required for tolerance to the sulfonylurea herbicide trifloxysulfuron sodium in cotton (Gossypium hirsutum L.).

BACKGROUND: Weed management is critical to global crop production and is complicated by rapidly evolving herbicide resistance in weeds. New sources of herbicide resistance are needed for crop plants so that applied herbicides can be rotated or combined to thwart the evolution of resistant weeds. The diverse family of cytochrome P450 proteins has been suggested to be a source of detoxifying herbicide metabolism in both weed and crop plants, and greater understanding of these genes will offer avenues for crop improvement and novel weed management practices. RESULTS: Here, we report the identification of CYP749A16 (Gh_D10G1401) which is responsible for the natural tolerance exhibited by most cotton, Gossypium hirsutum L., cultivars to the herbicide trifloxysulfuron sodium (TFS, CGA 362622, commercial formulation Envoke). A 1-bp frameshift insertion in the third exon of CYP749A16 results in the loss of tolerance to TFS. The DNA marker designed from this insertion perfectly co-segregated with the phenotype in 2145 F2 progeny of a cross between the sensitive cultivar Paymaster HS26 and tolerant cultivar Stoneville 474, and in 550 recombinant inbred lines of a multi-parent advanced generation inter-cross population. Marker analysis of 382 additional cotton cultivars identified twelve cultivars containing the 1-bp frameshift insertion. The marker genotypes matched perfectly with phenotypes in 188 plants from the selected twelve cultivars. Virus-induced gene silencing of CYP749A16 generated sensitivity in the tolerant cotton cultivar Stoneville 474. CONCLUSIONS: CYP749A16 located on chromosome D10 is required for TFS herbicide tolerance in cotton. This finding should add to the repertoire of tools available to farmers and breeders for the advancement of agricultural productivity.

A High-Throughput Standard PCR-Based Genotyping Method for Determining Transgene Zygosity in Segregating Plant Populations.

In crop research programs that implement transgene-based strategies for trait improvement it is necessary to distinguish between transgene homozygous and hemizygous individuals in segregating populations. Direct methods for determining transgene zygosity are technically challenging, expensive, and require specialized equipment. In this report, we describe a standard PCR-based protocol coupled with capillary electrophoresis that can identify transgene homozygous and hemizygous individuals in a segregating population without knowledge of transgene insertion site. PCR primers were designed to amplify conserved T-DNA segments of the 35S promoter, OCS terminator, and NPTII kanamycin resistance gene in the pHellsgate-8 RNAi construct for the Gossypium hirsutum phytochrome A1 gene. Using an optimized multiplexed reaction mixture and an amplification program of only 10 cycles we could discriminate between transgene homozygous and hemizygous cotton control DNA samples based on PCR product peak characteristics gathered by capillary electrophoresis. The protocol was refined by evaluating segregating transgenic progeny from nine BC1S1 populations derived from crosses between the transgenic cotton parent 'E-1-7-6' and other cotton cultivars. OCS PCR product peak height and peak area, normalized by amplification of the native cotton gene GhUBC1, revealed clear bimodal distributions of OCS product characteristics for each BC1S1 population indicating the presence of homozygous and hemizygous clusters which was further confirmed via K-means clustering. BC1S1 plants identified as homozygous or hemizygous were self-fertilized to produce BC1S2 progeny. For the homozygous class, 19/20 BC1S2 families confirmed the homozygous BC1S1 prediction while 21/21 BC1S2 families confirmed the hemizygous prediction of the original parent. This relatively simple protocol provides a reliable, rapid, and high-throughput way of evaluating segregating transgenic populations using methods and equipment common to crop molecular breeding labs.

A MAGIC population-based genome-wide association study reveals functional association of GhRBB1_A07 gene with superior fiber quality in cotton.

BACKGROUND: Cotton supplies a great majority of natural fiber for the global textile industry. The negative correlation between yield and fiber quality has hindered breeders' ability to improve these traits simultaneously. A multi-parent advanced generation inter-cross (MAGIC) population developed through random-mating of multiple diverse parents has the ability to break this negative correlation. Genotyping-by-sequencing (GBS) is a method that can rapidly identify and genotype a large number of single nucleotide polymorphisms (SNP). Genotyping a MAGIC population using GBS technologies will enable us to identify marker-trait associations with high resolution. RESULTS: An Upland cotton MAGIC population was developed through random-mating of 11 diverse cultivars for five generations. In this study, fiber quality data obtained from four environments and 6071 SNP markers generated via GBS and 223 microsatellite markers of 547 recombinant inbred lines (RILs) of the MAGIC population were used to conduct a genome wide association study (GWAS). By employing a mixed linear model, GWAS enabled us to identify markers significantly associated with fiber quantitative trait loci (QTL). We identified and validated one QTL cluster associated with four fiber quality traits [short fiber content (SFC), strength (STR), length (UHM) and uniformity (UI)] on chromosome A07. We further identified candidate genes related to fiber quality attributes in this region. Gene expression and amino acid substitution analysis suggested that a regeneration of bulb biogenesis 1 (GhRBB1_A07) gene is a candidate for superior fiber quality in Upland cotton. The DNA marker CFBid0004 designed from an 18 bp deletion in the coding sequence of GhRBB1_A07 in Acala Ultima is associated with the improved fiber quality in the MAGIC RILs and 105 additional commercial Upland cotton cultivars. CONCLUSION: Using GBS and a MAGIC population enabled more precise fiber QTL mapping in Upland cotton. The fiber QTL and associated markers identified in this study can be used to improve fiber quality through marker assisted selection or genomic selection in a cotton breeding program. Target manipulation of the GhRBB1_A07 gene through biotechnology or gene editing may potentially improve cotton fiber quality.

Quantitative trait loci analysis of fiber quality traits using a random-mated recombinant inbred population in Upland cotton (Gossypium hirsutum L.).

BACKGROUND: Upland cotton (Gossypium hirsutum L.) accounts for about 95% of world cotton production. Improving Upland cotton cultivars has been the focus of world-wide cotton breeding programs. Negative correlation between yield and fiber quality is an obstacle for cotton improvement. Random-mating provides a potential methodology to break this correlation. The suite of fiber quality traits that affect the yarn quality includes the length, strength, maturity, fineness, elongation, uniformity and color. Identification of stable fiber quantitative trait loci (QTL) in Upland cotton is essential in order to improve cotton cultivars with superior quality using marker-assisted selection (MAS) strategy. RESULTS: Using 11 diverse Upland cotton cultivars as parents, a random-mated recombinant inbred (RI) population consisting of 550 RI lines was developed after 6 cycles of random-mating and 6 generations of self-pollination. The 550 RILs were planted in triplicates for two years in Mississippi State, MS, USA to obtain fiber quality data. After screening 15538 simple sequence repeat (SSR) markers, 2132 were polymorphic among the 11 parents. One thousand five hundred eighty-two markers covering 83% of cotton genome were used to genotype 275 RILs (Set 1). The marker-trait associations were analyzed using the software program TASSEL. At p < 0.01, 131 fiber QTLs and 37 QTL clusters were identified. These QTLs were responsible for the combined phenotypic variance ranging from 62.3% for short fiber content to 82.8% for elongation. The other 275 RILs (Set 2) were analyzed using a subset of 270 SSR markers, and the QTLs were confirmed. Two major QTL clusters were observed on chromosomes 7 and 16. Comparison of these 131 QTLs with the previously published QTLs indicated that 77 were identified before, and 54 appeared novel. CONCLUSIONS: The 11 parents used in this study represent a diverse genetic pool of the US cultivated cotton, and 10 of them were elite commercial cultivars. The fiber QTLs, especially QTL clusters reported herein can be readily implemented in a cotton breeding program to improve fiber quality via MAS strategy. The consensus QTL regions warrant further investigation to better understand the genetics and molecular mechanisms underlying fiber development.

Comparisons of four approximation algorithms for large-scale linkage map construction.

Efficient construction of large-scale linkage maps is highly desired in current gene mapping projects. To evaluate the performance of available approaches in the literature, four published methods, the insertion (IN), seriation (SER), neighbor mapping (NM), and unidirectional growth (UG) were compared on the basis of simulated F(2) data with various population sizes, interferences, missing genotype rates, and mis-genotyping rates. Simulation results showed that the IN method outperformed, or at least was comparable to, the other three methods. These algorithms were also applied to a real data set and results showed that the linkage order obtained by the IN algorithm was superior to the other methods. Thus, this study suggests that the IN method should be used when constructing large-scale linkage maps.

Identification of QTL regions and SSR markers associated with resistance to reniform nematode in Gossypium barbadense L. accession GB713.

The identification of molecular markers that are closely linked to gene(s) in Gossypium barbadense L. accession GB713 that confer a high level of resistance to reniform nematode (RN), Rotylenchulus reniformis Linford & Oliveira, would be very useful in cotton breeding programs. Our objectives were to determine the inheritance of RN resistance in the accession GB713, to identify SSR markers linked with RN resistance QTLs, and to map these linked markers to specific chromosomes. We grew and scored plants for RN reproduction in the P(1), P(2), F(1), F(2), BC(1)P(1), and BC(1)P(2) generations from the cross of GB713 × Acala Nem-X. The generation means analysis using the six generations indicated that one or more genes were involved in the RN resistance of GB713. The interspecific F(2) population of 300 plants was genotyped with SSR molecular markers that covered most of the chromosomes of Upland cotton (G. hirsutum L.). Results showed two QTLs on chromosome 21 and one QTL on chromosome 18. One QTL on chromosome 21 was at map position 168.6 (LOD 28.0) flanked by SSR markers, BNL 1551_162 and GH 132_199 at positions 154.2 and 177.3, respectively. A second QTL on chromosome 21 was at map position 182.7 (LOD 24.6) flanked by SSR markers BNL 4011_155 and BNL 3279_106 at positions 180.6 and 184.5, respectively. Our chromosome 21 map had 61 SSR markers covering 219 cM. One QTL with smaller genetic effects was localized to chromosome 18 at map position 39.6 (LOD 4.0) and flanked by SSR markers BNL 1721_178 and BNL 569_131 at positions 27.6 and 42.9, respectively. The two QTLs on chromosome 21 had significant additive and dominance effects, which were about equal for each QTL. The QTL on chromosome 18 showed larger additive than dominance effects. Following the precedent set by the naming of the G. longicalyx Hutchinson & Lee and G. aridum [(Rose & Standley) Skovsted] sources of resistance, we suggest the usage of Ren (barb1) and Ren (barb2) to designate these QTLs on chromosome 21 and Ren (barb3) on chromosome 18.