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.

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.

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.

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 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.

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.

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.

Dissection of complicate genetic architecture and breeding perspective of cottonseed traits by genome-wide association study.

BACKGROUND: Cottonseed is one of the most important raw materials for plant protein, oil and alternative biofuel for diesel engines. Understanding the complex genetic basis of cottonseed traits is requisite for achieving efficient genetic improvement of the traits. However, it is not yet clear about their genetic architecture in genomic level. GWAS has been an effective way to explore genetic basis of quantitative traits in human and many crops. This study aims to dissect genetic mechanism seven cottonseed traits by a GWAS for genetic improvement. RESULTS: A genome-wide association study (GWAS) based on a full gene model with gene effects as fixed and gene-environment interaction as random, was conducted for protein, oil and 5 fatty acids using 316 accessions and ~ 390 K SNPs. Totally, 124 significant quantitative trait SNPs (QTSs), consisting of 16, 21, 87 for protein, oil and fatty acids (palmitic, linoleic, oleic, myristic, stearic), respectively, were identified and the broad-sense heritability was estimated from 71.62 to 93.43%; no QTS-environment interaction was detected for the protein, the palmitic and the oleic contents; the protein content was predominantly controlled by epistatic effects accounting for 65.18% of the total variation, but the oil content and the fatty acids except the palmitic were mainly determined by gene main effects and no epistasis was detected for the myristic and the stearic. Prediction of superior pure line and hybrid revealed the potential of the QTSs in the improvement of cottonseed traits, and the hybrid could achieve higher or lower genetic values compared with pure lines. CONCLUSIONS: This study revealed complex genetic architecture of seven cottonseed traits at whole genome-wide by mixed linear model approach; the identified genetic variants and estimated genetic component effects of gene, gene-gene and gene-environment interaction provide cotton geneticist or breeders new knowledge on the genetic mechanism of the traits and the potential molecular breeding design strategy.

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.

Broiler Litter × Industrial By-Products Reduce Nutrients and Microbial Losses in Surface Runoff When Applied to Forages.

The inability to incorporate broiler litter (BL) into permanent hayfields and pastures leads to nutrient accumulation near the soil surface and increases the potential transport of nutrients in runoff. This study was conducted on Marietta silt loam soil to determine the effect of flue gas desulfurization (FGD) gypsum and lignite on P, N, C, and microbial concentrations in runoff. Treatments were (i) control (unfertilized) and (ii) BL at 13.4 Mg ha alone or (iii) treated with either FGD gypsum or lignite applied at 20% (w/w) (2.68 Mg ha). Rainfall simulators were used to produce a 5.6 cm h storm event sufficient in duration to cause 15 min of continuous runoff. Repeated rains were applied at 3-d intervals to determine how long FGD gypsum and lignite are effective in reducing loss of litter-derived N, P, and C from soil. Application of BL increased N, P, and C concentrations in runoff as compared to the control. Addition of FGD gypsum reduced ( < 0.05) water-soluble P and dissolved organic C concentrations in runoff by 39 and 16%, respectively, as compared to BL alone. Lignite reduced runoff total N and NH-N concentrations by 38 and 70%, respectively, as compared to BL alone. Addition of FGD gypsum or lignite failed to significantly reduce microbial loads in runoff, although both treatments reduced microbial concentration by >20%. Thus, BL treated with FGD and lignite can be considered as cost-effective management practices in the mitigation of P, N, and C and possibly microbial concentration in runoff.

Small RNA sequencing and degradome analysis of developing fibers of short fiber mutants Ligon-lintles-1 (Li 1 ) and -2 (Li 2 ) revealed a role for miRNAs and their targets in cotton fiber elongation.

BACKGROUND: The length of cotton fiber is an important agronomic trait that directly affects the quality of yarn and fabric. Understanding the molecular basis of fiber elongation would provide a means for improvement of fiber length. Ligon-lintless-1 (Li 1 ) and -2 (Li 2 ) are monogenic and dominant mutations that result in an extreme reduction in the length of lint fiber on mature seeds. In a near-isogenic state with wild type cotton these two short fiber mutants provide an effective model system to study the mechanisms of fiber elongation. Plant miRNAs regulate many aspects of growth and development. However, the mechanism underlying the miRNA-mediated regulation of fiber development is largely unknown. RESULTS: Small RNA libraries constructed from developing fiber cells of the short fiber mutants Li 1 and Li 2 and their near-isogenic wild type lines were sequenced. We identified 24 conservative and 147 novel miRNA families with targets that were detected through degradome sequencing. The distribution of the target genes into functional categories revealed the largest set of genes were transcription factors. Expression profiles of 20 miRNAs were examined across a fiber developmental time course in wild type and short fiber mutations. We conducted correlation analysis between miRNA transcript abundance and the length of fiber for 11 diverse Upland cotton lines. The expression patterns of 4 miRNAs revealed significant negative correlation with fiber lengths of 11 cotton lines. CONCLUSIONS: Our results suggested that the mutations have changed the regulation of miRNAs expression during fiber development. Further investigations of differentially expressed miRNAs in the Li 1 and Li 2 mutants will contribute to better understanding of the regulatory mechanisms of cotton fiber development. Four miRNAs negatively correlated with fiber length are good candidates for further investigations of miRNA regulation of important genotype dependent fiber traits. Thus, our results will contribute to further studies on the role of miRNAs in cotton fiber development and will provide a tool for fiber improvement through molecular breeding.

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.

Comparative assessment of genetic diversity in cytoplasmic and nuclear genome of upland cotton.

The importance of the cytoplasmic genome for many economically important traits is well documented in several crop species, including cotton. There is no report on application of cotton chloroplast specific SSR markers as a diagnostic tool to study genetic diversity among improved Upland cotton lines. The complete plastome sequence information in GenBank provided us an opportunity to report on 17 chloroplast specific SSR markers using a cost-effective data mining strategy. Here we report the comparative analysis of genetic diversity among a set of 42 improved Upland cotton lines using SSR markers specific to chloroplast and nuclear genome, respectively. Our results revealed that low to moderate level of genetic diversity existed in both nuclear and cytoplasm genome among this set of cotton lines. However, the specific estimation suggested that genetic diversity is lower in cytoplasmic genome compared to the nuclear genome among this set of Upland cotton lines. In summary, this research is important from several perspectives. We detected a set of cytoplasm genome specific SSR primer pairs by using a cost-effective data mining strategy. We reported for the first time the genetic diversity in the cytoplasmic genome within a set of improved Upland cotton accessions. Results revealed that the genetic diversity in cytoplasmic genome is narrow, compared to the nuclear genome within this set of Upland cotton accessions. Our results suggested that most of these polymorphic chloroplast SSRs would be a valuable complementary tool in addition to the nuclear SSR in the study of evolution, gene flow and genetic diversity in Upland cotton.

Coupling of MIC-3 overexpression with the chromosomes 11 and 14 root-knot nematode (RKN) (Meloidogyne incognita) resistance QTLs provides insights into the regulation of the RKN resistance response in Upland cotton (Gossypium hirsutum).

KEY MESSAGE: Genetic analysis of MIC-3 transgene with RKN resistance QTLs provides insight into the resistance regulatory mechanism and provides a framework for testing additional hypotheses. Resistance to root-knot nematode (RKN) (Meloidogyne incognita) in Upland cotton (Gossypium hirsutum) is mediated by two major quantitative trait loci (QTL) located on chromosomes 11 and 14. The MIC-3 (Meloidogyne Induced Cotton3) protein accumulates specifically within the immature galls of RKN-resistant plants that possess these QTLs. Recently, we showed that MIC-3 overexpression in an RKN-susceptible cotton genotype suppressed RKN egg production but not RKN-induced root galling. In this study, the MIC-3 overexpression construct T-DNA in the single-copy transgenic line '14-7-1' was converted into a codominant molecular marker that allowed the marker assisted selection of F2:3 cotton lines, derived from a cross between 14-7-1 and M-240 RNR, having all possible combinations of the chromosomes 11 and 14 QTLs with and without the MIC-3 overexpression construct. Root-knot nematode reproduction (eggs g(-1) root) and severity of RKN-induced root galling were assessed in these lines. We discovered that the addition of MIC-3 overexpression suppressed RKN reproduction in lines lacking both resistance QTLs and in lines having only the chromosome 14 QTL, suggesting an additive effect of the MIC-3 construct with this QTL. In contrast, MIC-3 overexpression did not improve resistance in lines having the single chromosome 11 QTL or in lines having both resistance QTLs, suggesting an epistatic interaction between the chromosome 11 QTL and the MIC-3 construct. Overexpression of MIC-3 did not affect the severity of RKN-induced root galling regardless of QTL genotype. These data provide new insights into the relative order of action of the chromosomes 11 and 14 QTLs and their potential roles in regulating MIC-3 expression as part of the RKN resistance response.

Development, genetic mapping and QTL association of cotton PHYA, PHYB, and HY5-specific CAPS and dCAPS markers.

BACKGROUND: Among SNP markers that become increasingly valuable in molecular breeding of crop plants are the CAPS and dCAPS markers derived from the genes of interest. To date, the number of such gene-based markers is small in polyploid crop plants such as allotetraploid cotton that has A- and D-sub-genomes. The objective of this study was to develop and map new CAPS and dCAPS markers for cotton developmental-regulatory genes that are important in plant breeding programs. RESULTS: Gossypium hirsutum and G. barbadense, are the two cultivated allotetraploid cotton species. These have distinct fiber quality and other agronomic traits. Using comparative sequence analysis of characterized GSTs of the PHYA1, PHYB, and HY5 genes of G. hirsutum and G. barbadense one PHYA1-specific Mbo I/Dpn II CAPS, one PHYB-specific Alu I dCAPS, and one HY5-specific Hinf I dCAPS cotton markers were developed. These markers have successfully differentiated the two allotetraploid genomes (AD1 and AD2) when tested in parental genotypes of 'Texas Marker-1' ('TM-1'), 'Pima 3-79' and their F1 hybrids. The genetic mapping and chromosome substitution line-based deletion analyses revealed that PHYA1 gene is located in A-sub-genome chromosome 11, PHYB gene is in A-sub-genome chromosome 10, and HY5 gene is in D-sub-genome chromosome 24, on the reference 'TM-1' x 'Pima 3-79' RIL genetic map. Further, it was found that genetic linkage map regions containing phytochrome and HY5-specific markers were associated with major fiber quality and flowering time traits in previously published QTL mapping studies. CONCLUSION: This study detailed the genome mapping of three cotton phytochrome genes with newly developed CAPS and dCAPS markers. The proximity of these loci to fiber quality and other cotton QTL was demonstrated in two A-subgenome and one D-subgenome chromosomes. These candidate gene markers will be valuable for marker-assisted selection (MAS) programs to rapidly introgress G. barbadense phytochromes and/or HY5 gene (s) into G. hirsutum cotton genotypes or vice versa.

Overexpression of MIC-3 indicates a direct role for the MIC gene family in mediating Upland cotton (Gossypium hirsutum) resistance to root-knot nematode (Meloidogyne incognita).

KEY MESSAGE: Transgene-based analysis of the MIC-3 gene provides the first report of a cotton gene having a direct role in mediating cotton resistance to root-knot nematode. Major quantitative trait loci have been mapped to Upland cotton (Gossypium hirsutum L.) chromosomes 11 and 14 that govern the highly resistant phenotype in response to infection by root-knot nematode (RKN; Meloidogyne incognita); however, nearly nothing is known regarding the underlying molecular determinants of this RKN-resistant phenotype. Multiple lines of circumstantial evidence have strongly suggested that the MIC (Meloidogyne Induced Cotton) gene family plays an integral role in mediating cotton resistance to RKN. In this report, we demonstrate that overexpression of MIC-3 in the RKN-susceptible genetic background Coker 312 reduces RKN egg production by ca. 60-75 % compared to non-transgenic controls and transgene-null sibling lines. MIC-3 transcript and protein overexpression were confirmed in root tissues of multiple independent transgenic lines with each line showing a similar level of increased resistance to RKN. In contrast to RKN fecundity, transgenic lines showed RKN-induced root galling similar to the susceptible controls. In addition, we determined that this effect of MIC-3 overexpression was specific to RKN as no effect was observed on reniform nematode (Rotylenchulus reniformis) reproduction. Transgenic lines did not show obvious alterations in growth, morphology, flowering, or fiber quality traits. Gene expression analyses showed that MIC-3 transcript levels in uninfected transgenic roots exceeded levels observed in RKN-infected roots of naturally resistant plants and that overexpression did not alter the regulation of native MIC genes in the genome. These results are the first report describing a direct role for a specific gene family in mediating cotton resistance to a plant-parasitic nematode.

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.

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.