Characterization of centromeric DNA of Gossypium anomalum reveals sequence-independent enrichment dynamics of centromeric repeats.

Centromeres in eukaryotes are composed of highly repetitive DNAs, which evolve rapidly and are thought to achieve a favorable structure in mature centromeres. However, how the centromeric repeat evolves into an adaptive structure is largely unknown. We characterized the centromeric sequences of Gossypium anomalum through chromatin immunoprecipitation against CENH3 antibodies. We revealed that the G. anomalum centromeres contained only retrotransposon-like repeats but were depleted in long arrays of satellites. These retrotransposon-like centromeric repeats were present in the African-Asian and Australian lineage species, suggesting that they might have arisen in the common ancestor of these diploid species. Intriguingly, we observed a substantial increase and decrease in copy numbers among African-Asian and Australian lineages, respectively, for the retrotransposon-derived centromeric repeats without apparent structure or sequence variation in cotton. This result indicates that the sequence content is not a decisive aspect of the adaptive evolution of centromeric repeats or at least retrotransposon-like centromeric repeats. In addition, two active genes with potential roles in gametogenesis or flowering were identified in CENH3 nucleosome-binding regions. Our results provide new insights into the constitution of centromeric repetitive DNA and the adaptive evolution of centromeric repeats in plants.

A recessive LRR-RLK gene causes hybrid breakdown in cotton.

KEY MESSAGE: An LRR-RLK gene causing interspecific hybrid breakdown between Gossypium. anomalum and G. hirsutum was identified by deploying a map-based cloning strategy. The self-destructing symptoms of hybrid incompatibility in most cases are attributed to autoimmunity. The cloning of genes responsible for hybrid incompatibility in cotton is helpful to clarify the mechanisms underlying hybrid incompatibility and can break the barriers in distant hybridization. In this study, a temperature-dependent lethality was identified in CSSL11-9 (chromosome segment substitution line) with Gossypium anomalum chromosome segment on chromosome A11. Transcriptome analysis showed the differentially expressed genes related to autoimmune responses were highly enriched, suggesting that expression of CSSL11-9 plant lethal gene activated autoimmunity in the absence of any pathogen or external stimulus, inducing programmed cell death (PCD) and causing a lethal phenotype. The lethal phenotype was controlled by a pair of recessive genes and then fine mapped between JAAS3191-JAAS3050 interval, which covered 63.87 kb in G. hirsutum genome and 98.66 kb in G. anomalum. We demonstrated that an LRR-RLK gene designated as hybrid breakdown 1 (GoanoHBD1) was the causal gene underlying this locus for interspecific hybrid incompatibility between G. anomalum and G. hirsutum. Silencing this LRR-RLK gene could restore CSSL11-9 plants from a lethal to a normal phenotype. Our findings provide new insights into reproductive isolation and may benefit cotton breeding.

Genome-wide association analysis reveals genetic variations and candidate genes associated with salt tolerance related traits in Gossypium hirsutum.

BACKGROUND: Cotton is more resistant to salt and drought stresses as compared to other field crops, which makes itself as a pioneer industrial crop in saline-alkali lands. However, abiotic stresses still negatively affect its growth and development significantly. It is therefore important to breed salt tolerance varieties which can help accelerate the improvement of cotton production. The development of molecular markers linked to causal genes has provided an effective and efficient approach for improving salt tolerance. RESULTS: In this study, a genome-wide association study (GWAS) of salt tolerance related traits at seedling stage was performed based on 2 years of phenotype identification for 217 representative upland cotton cultivars by genotyping-by-sequencing (GBS) platform. A total of 51,060 single nucleotide polymorphisms (SNPs) unevenly distributed among 26 chromosomes were screened across the cotton cultivars, and 25 associations with 27 SNPs scattered over 12 chromosomes were detected significantly (-log10p > 4) associated with three salt tolerance related traits in 2016 and 2017. Among these, the associations on chromosome A13 and D08 for relative plant height (RPH), A07 for relative shoot fresh matter weight (RSFW), A08 and A13 for relative shoot dry matter weight (RSDW) were expressed in both environments, indicating that they were likely to be stable quantitative trait loci (QTLs). A total of 12 salt-induced candidate genes were identified differentially expressed by the combination of GWAS and transcriptome analysis. Three promising genes were selected for preliminary function verification of salt tolerance. The increase of GH_A13G0171-silenced plants in salt related traits under salt stress indicated its negative function in regulating the salt stress response. CONCLUSIONS: These results provided important genetic variations and candidate genes for accelerating the improvement of salt tolerance in cotton.

A complete set of monosomic alien addition lines developed from Gossypium anomalum in a Gossypium hirsutum background: genotypic and phenotypic characterization.

Gossypium anomalum (B1B1) is a valuable wild resource for the genetic improvement of G. hirsutum (A1A1D1D1) in terms of fiber quality and disease and pest resistance, but the inherent difficulties in distant hybridization hinder its utilization in breeding programs. Monosomic alien addition lines (MAALs) are powerful tools for interspecific gene transfer. First, to access useful genes from G. anomalum, a fertile hexaploid from G. hirsutum × G. anomalum was obtained and then additional chromosomes were selected using SSR markers in successive backcrosses and self-crossing from BC2F1 to BC4F4. Finally, a complete set of 13 MAALs were developed. All the MAALs were confirmed by chromosome-specific anchored SSRs and genome-wide resequencing. The MAALs demonstrated abundant variation in morphological, agronomic, yield-related, and fiber quality traits. MAAL_3B had excellent fiber strength and fineness, indicating that the transmitted chromosome may carry desirable genes for the observed phenotypes. This complete set of MAALs will provide important genetic bridge material for the identification and introgression of favorable genes from G. anomalum and lay an important foundation for the genetic improvement of cotton.

Transcription Factor GarWRKY5 Is Involved in Salt Stress Response in Diploid Cotton Species (Gossypium aridum L.).

Cotton is one of the most economically important crops in the world, and it is exposed to various abiotic stresses during its lifecycle, especially salt stress. However, the molecular mechanisms underlying cotton tolerance to salt stress are still not fully understood due to the complex nature of salt response. Therefore, identification of salt stress tolerance-related functional genes will help us understand key components involved in stress response and provide valuable genes for improving salt stress tolerance via genetic engineering in cotton. In the present study, virus-induced gene silencing of GhWRKY5 in cotton showed enhanced salt sensitivity compared to wild-type plants under salt stress. Overexpression of GarWRKY5 in Arabidopsis positively regulated salt tolerance at the stages of seed germination and vegetative growth. Additionally, GarWRKY5-overexpressing plants exhibited higher activities of superoxide dismutase (SOD) and peroxidase (POD) under salt stress. The transcriptome sequencing analysis of transgenic Arabidopsis plants and wild-type plants revealed that there was enriched coexpression of genes involved in reactive oxygen species (ROS) scavenging (including glutamine S-transferases (GSTs) and SODs) and altered response to jasmonic acid and salicylic acid in the GarWRKY5-OE lines. GarWRKY5 is involved in salt stress response by the jasmonic acid- or salicylic acid-mediated signaling pathway based on overexpression of GarWRKY5 in Arabidopsis and virus-induced gene silencing of GarWRKY5 in cotton.

Fine mapping and candidate gene analysis of qFL-chr1, a fiber length QTL in cotton.

KEY MESSAGE: A fiber length QTL, qFL-chr1, was fine mapped to a 0.9 cM interval of cotton chromosome 1. Two positional candidate genes showed positive correlation between gene expression level and fiber length. Prior analysis of a backcross-self mapping population derived from a cross between Gossypium hirsutum L. and G. barbadense L. revealed a QTL on chromosome 1 associated with increased fiber length (qFL-chr1), which was confirmed in three independent populations of near-isogenic introgression lines (NIILs). Here, a single NIIL, R01-40-08, was used to develop a large population segregating for the target region. Twenty-two PCR-based polymorphic markers used to genotype 1672 BC4F2 plants identified 432 recombinants containing breakpoints in the target region. Substitution mapping using 141 informative recombinants narrowed the position of qFL-chr1 to a 1.0-cM interval between SSR markers MUSS084 and CIR018. To exclude possible effects of non-target introgressions on fiber length, different heterozygous BC4F3 plants introgressed between SSR markers NAU3384 and CGR5144 were selected to develop sub-NILs. The qFL-chr1 was further mapped at 0.9-cM interval between MUSS422 and CIR018 by comparisons of sub-NIL phenotype, and increased fiber length by ~1 mm. The 2.38-Mb region between MUSS422 and CIR018 in G. barbadense contained 19 annotated genes. Expression levels of two of these genes, GOBAR07705 (encoding 1-aminocyclopropane-1-carboxylate synthase) and GOBAR25992 (encoding amino acid permease), were positively correlated with fiber length in a small F2 population, supporting these genes as candidates for qFL-chr1.

Fine mapping and identification of candidate genes for a QTL affecting Meloidogyne incognita reproduction in Upland cotton.

BACKGROUND: The southern root-knot nematode (Meloidogyne incognita; RKN) is one of the most important economic pests of Upland cotton (Gossypium hirsutum L.). Host plant resistance, the ability of a plant to suppress nematode reproduction, is the most economical, practical, and environmentally sound method to provide protection against this subterranean pest. The resistant line Auburn 623RNR and a number of elite breeding lines derived from it remain the most important source of root-knot nematode (RKN) resistance. Prior genetic analysis has identified two epistatically interacting RKN resistance QTLs, qMi-C11 and qMi-C14, affecting gall formation and RKN reproduction, respectively. RESULTS: We developed a genetic population segregating only for the qMi-C14 locus and evaluated the genetic effects of this QTL on RKN resistance in the absence of the qMi-C11 locus. The qMi-C14 locus had a LOD score of 12 and accounted for 24.5 % of total phenotypic variation for egg production. In addition to not being significantly associated with gall formation, this locus had a lower main effect on RKN reproduction than found in our previous study, which lends further support to evidence of epistasis with qMi-C11 in imparting RKN resistance in the Auburn 623RNR source. The locus qMi-C14 was fine-mapped with the addition of 16 newly developed markers. By using the reference genome sequence of G. raimondii, we identified 20 candidate genes encoding disease resistance protein homologs in the newly defined 2.3 Mb region flanked by two SSR markers. Resequencing of an RKN resistant and susceptible G. hirsutum germplasm revealed non-synonymous mutations in only four of the coding regions of candidate genes, and these four genes are consequently of high interest. CONCLUSIONS: Our mapping results validated the effects of the qMi-C14 resistance locus, delimiting the QTL to a smaller region, and identified tightly linked SSR markers to improve the efficiency of marker-assisted selection. The candidate genes identified warrant functional studies that will help in identifying and characterizing the actual qMi-C14 defense gene(s) against root-knot nematodes.

Development of Gossypium anomalum-derived microsatellite markers and their use for genome-wide identification of recombination between the G. anomalum and G. hirsutum genomes.

KEY MESSAGE: We reported the first development of Gossypium anomalum -derived microsatellite markers and identification of recombination between sexually incompatible species by a synthesized hexaploid on genome level. To continue to develop improved cotton varieties, it is essential to transfer desired characters from diploid wild cotton species such as Gossypium anomalum to cultivated allotetraploid cotton species. However, interspecific reproductive barriers limit gene transfer between species. In a previous study, we used colchicine treatment to produce a synthesized hexaploid derived from an interspecific hybrid between Gossypium hirsutum and G. anomalum and demonstrated its hybridity and doubled status using morphological, cytological and molecular marker methods. In the current study, to effectively monitor G. anomalum genome components in the G. hirsutum background, we developed 5974 non-redundant G. anomalum-derived SSR primer pairs using RNA-Seq technology, which were combined with a publicly available physical map. Based on this combined map and segregation data from the BC2F1 population, we identified a set of 230 informative G. anomalum-specific SSR markers distributed on the chromosomes, which cover 95.72% of the cotton genome. After analyzing BC2F1 segregation data, 50 recombination types from 357 recombination events were identified, which cover 81.48% of the corresponding G. anomalum genome. A total of 203 recombination events occurred on chromosome 11, accounting for 56.86% of the recombination events on all chromosomes. Recombination hotspots were observed at marker intervals JAAS1148-NAU5100 on chromosome 1 and JAAS0426-NAU998 on chromosome 2. Therefore, all G. anomalum chromosomes are capable of recombining with At chromosomes in G. hirsutum. This study represents an important step towards introgressing desirable traits into cultivated cotton from the wild cotton species G. anomalum.

Re-evaluation of the inheritance for root-knot nematode resistance in the Upland cotton germplasm line M-120 RNR revealed two epistatic QTLs conferring resistance.

KEY MESSAGE: We report a second major QTL for root-knot nematode resistance in the highly resistant Upland cotton line M-120RNR and show epistasis between two resistant QTLs with different mechanisms conferring resistance. In an earlier study, we identified a major QTL on Chromosome 11 associated with resistance to root-knot nematode in the M-120 RNR Upland cotton line (Gossypium hirsutum L.) of the Auburn 623 RNR source. Herein, we re-evaluated the genetics of the resistance to root-knot nematode in the M-120 RNR × Pima S-6 population by linkage mapping using recently published SSR markers. The QTL analysis detected two regions significantly associated with the resistance phenotype. In addition to the QTL previously identified on Chromosome 11 (qMi-C11), a major QTL was identified on Chromosome 14 (qMi-C14). The resistance locus on qMi-C11 originated from the Clevewilt parent, while the qMi-C14 locus originated from the other resistant parent, Mexico Wild Jack Jones. The qMi-C14 locus had logarithms of odds score of 17 and accounted for 45 % of the total phenotype variation in egg production. It was also associated with galling index, but the percent variation explained was only 6 %, suggesting that the qMi-C11 locus had a much stronger effect on root gall suppression than egg production, while the qMi-C14 locus had a stronger effect on egg production than galling. The results also suggest that the transgressive segregation observed in the development of Auburn 623 RNR was due to the pyramiding of at least two main effect QTLs as well as an additive-by-additive epistatic effects between the two resistant loci. The SSRs markers tightly linked to the qMi-C11 and qMi-C14 loci will greatly facilitate the improvement of RKN resistance in cotton via marker-assisted breeding.

Transcriptome Expression Profiling Reveals the Molecular Response to Salt Stress in Gossypium anomalum Seedlings.

Some wild cotton species are remarkably tolerant to salt stress, and hence represent valuable resources for improving salt tolerance of the domesticated allotetraploid species Gossypium hirsutum L. Here, we first detected salt-induced stress changes in physiological and biochemical indexes of G. anomalum, a wild African diploid cotton species. Under 350 mmol/L NaCl treatment, the photosynthetic parameters declined significantly, whereas hydrogen peroxide (H2O2) and malondialdehyde (MDA) contents increased. Catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) activity and proline (PRO) content also significantly increased, reaching peak values at different stages of salt stress. We used RNA-Seq to characterize 15,476 differentially expressed genes in G. anomalum roots after 6, 12, 24, 72, and 144 h of salt stress. Gene Ontology enrichment analysis revealed these genes to be related to sequence-specific DNA and iron ion binding and oxidoreductase, peroxidase, antioxidant, and transferase activity; meanwhile, the top enriched pathways from the Kyoto Encyclopedia of Genes and Genomes database were plant hormone signal transduction, phenylpropanoid biosynthesis, fatty acid degradation, carotenoid biosynthesis, zeatin biosynthesis, starch and sucrose metabolism, and MAPK signaling. A total of 1231 transcription factors were found to be expressed in response to salt stress, representing ERF, MYB, WRKY, NAC, C2H2, bZIP, and HD-ZIP families. Nine candidate genes were validated by quantitative real-time PCR and their expression patterns were found to be consistent with the RNA-Seq data. These data promise to significantly advance our understanding of the molecular response to salt stress in Gossypium spp., with potential value for breeding applications.

Transcriptome Profiling of Gossypium anomalum Seedlings Reveals Key Regulators and Metabolic Pathways in Response to Drought Stress.

Drought stress is a key limiting factor for cotton (Gossypium spp.) growth, production, development, and production worldwide. Some wild diploid cotton species are remarkably tolerant of water deficit and constitute an important reservoir for understanding the molecular mechanisms of Gossypium spp. drought tolerance and improving cultivated upland cotton. Here, we utilized RNA-Seq technology to characterize the leaf transcriptomes of a wild African diploid cotton species, Gossypium anomalum, under drought stress. A total of 12,322 differentially expressed genes (DEGs) were identified after mapping valid clean reads to the reference genome of G. anomalum, of which 1243 were commonly differentially expressed at all stages of drought stress. These genes were significantly enriched for molecular functions Gene Ontology terms related to cytoskeleton, hydrolase activity, cellular redox, and binding. Additionally, a substantial proportion of enriched biological process terms concerned cell or subcellular processes, while most in the cellular components category concerned membrane function and photosynthesis. An enrichment analysis against the Kyoto Encyclopedia of Genes and Genomes showed the top significantly enriched pathways to be photosynthesis-antenna proteins, amino sugar and nucleotide sugar metabolism, starch and sucrose metabolism, MAPK signaling pathway, glutathione metabolism, and plant hormone signal transduction. The DEGs also exhibited interestingly significant enrichments for drought stress-induced tandemly repeated genes involved in iron ion binding, oxidoreductase activity, heme binding, and other biological processes. A large number of genes encoding transcription factors, such as MYB, bHLH, ERF, NAC, WRKY, and bZIP, were identified as playing key roles in acclimatizing to drought stress. These results will provide deeper insights into the molecular mechanisms of drought stress adaptation in Gossypium spp.

Genome sequence of Gossypium anomalum facilitates interspecific introgression breeding.

Crop wild relatives are an important reservoir of natural biodiversity. However, incorporating wild genetic diversity into breeding programs is often hampered by reproductive barriers and a lack of accurate genomic information. We assembled a high-quality, accurately centromere-anchored genome of Gossypium anomalum, a stress-tolerant wild cotton species. We provided a strategy to discover and transfer agronomically valuable genes from wild diploid species to tetraploid cotton cultivars. With a (Gossypium hirsutum × G. anomalum)2 hexaploid as a bridge parent, we developed a set of 74 diploid chromosome segment substitution lines (CSSLs) of the wild cotton species G. anomalum in the G. hirsutum background. This set of CSSLs included 70 homozygous substitutions and four heterozygous substitutions, and it collectively contained about 72.22% of the G. anomalum genome. Twenty-four quantitative trait loci associated with plant height, yield, and fiber qualities were detected on 15 substitution segments. Integrating the reference genome with agronomic trait evaluation of the CSSLs enabled location and cloning of two G. anomalum genes that encode peroxiredoxin and putative callose synthase 8, respectively, conferring drought tolerance and improving fiber strength. We have demonstrated the power of a high-quality wild-species reference genome for identifying agronomically valuable alleles to facilitate interspecific introgression breeding in crops.

Fine mapping QMi-C11 a major QTL controlling root-knot nematodes resistance in Upland cotton.

The identification and utilization of a high-level of host plant resistance is the most effective and economical approach to control root-knot nematode (Meloidogyne incognita). In an earlier study, we identified a major quantitative trait locus (QTL) for resistance to root-knot nematode in the M-120 RNR Upland cotton line (Gossypium hirsutum L.) of the Auburn 623 RNR source. The QTL is located in a 12.9-cM interval flanked by the two SSR markers CIR069 and CIR316 on the distal segment of chromosome 11. To construct a fine map around the target region, a bulked segregation analysis was performed using two DNA pools consisting of five individuals, with each being homozygous for the two parental alleles. From a survey of 1,152 AFLP primer combinations, 9 AFLP markers closely linked to the target region were identified. By screening an additional 1,221 F(2) individuals developed from the initial mapping population, the Mi-C11 locus was delimited to a 3.6-cM interval flanked by the SSR marker CIR069 and the AFLP marker E14M27-375. These results further elucidate the genetic fine structure of the Mi-C11 locus and provide the basis for map-based isolation of the nematode resistance gene in M-120 RNR.

GrTEdb: the first web-based database of transposable elements in cotton (Gossypium raimondii).

Although several diploid and tetroploid Gossypium species genomes have been sequenced, the well annotated web-based transposable elements (TEs) database is lacking. To better understand the roles of TEs in structural, functional and evolutionary dynamics of the cotton genome, a comprehensive, specific, and user-friendly web-based database, Gossypium raimondii transposable elements database (GrTEdb), was constructed. A total of 14 332 TEs were structurally annotated and clearly categorized in G. raimondii genome, and these elements have been classified into seven distinct superfamilies based on the order of protein-coding domains, structures and/or sequence similarity, including 2929 Copia-like elements, 10 368 Gypsy-like elements, 299 L1 , 12 Mutators , 435 PIF-Harbingers , 275 CACTAs and 14 Helitrons . Meanwhile, the web-based sequence browsing, searching, downloading and blast tool were implemented to help users easily and effectively to annotate the TEs or TE fragments in genomic sequences from G. raimondii and other closely related Gossypium species. GrTEdb provides resources and information related with TEs in G. raimondii , and will facilitate gene and genome analyses within or across Gossypium species, evaluating the impact of TEs on their host genomes, and investigating the potential interaction between TEs and protein-coding genes in Gossypium species. Database URL: http://www.grtedb.org/.

[Molecular cloning and expression analysis of a SUPERMAN-like zinc finger protein gene in upland cotton].

The zinc finger proteins belong to the largest family of regulatory transcription factors, which play an important role in growth and development in animal and plant systems. SUPERMAN-like zinc finger protein gene has only one "finger like" motif. A pair of degenerate primers was designed according to the conserved regions, and 3 kinds of EST of this family were isolated from cotton through RT-PCR. The full length of one SUPERMAN-like zinc finger protein also has been acquired. The entire coding region is 744 bp and encodes a polypeptide of 248 amino acids with 40% homology to RBE protein of Arabidopsis deposited in the GenBank. This gene was designated as GZFP. It has the conserved zinc finger domain and the leucine rich region at the carboxyl terminus but no intron in the coding region. GZFP also has the plant nuclear localization signal. GZFP shows a more expression pattern in floral buds, ovaries, petals and roots than in phloem, xylem, fibers, leaves and seeds of cotton by RT-PCR, although it has a very low detection level and there is not any homologous ESTs found in the GenBank. Analysis of the 5' flanking sequence shows there are several regulatory elements responsible for pollen and root expression, four core sites required for binding of Dof proteins and four light-regulated elements.

Molecular marker assisted selection and pyramiding of two QTLs for fiber strength in upland cotton.

Based on two major QTLs that control high fiber strength which originated from an elite fiber germ-plasm line 7235 (Gossypium hiusutum L.), the efficiency of molecular marker-assisted selection (MAS) was investigated using two populations from pedigree selection and modified backcrossing pyramiding developed for the breeding purpose. Simian 3 (SM3), a widely planted variety in the Yangtze River Valley, and 7235 were used as parents to develop the two populations. In the two major QTLs for fiber strength from 7235, QTLfs-1 could explain more than 30% of the phenotypic variation (PV) in the (7235 x TM-1) F2 population. QTLfs-2 was at first identified in another super quality fiber line HS427-10 from (HS427-10 x TM-1) F2 population with 12.5% of PV explanation,which was further also identified in 7235 line but was non-allelic with QTLfs-1. The result of molecular marker-assisted selection for fiber strength showed that the genetic effect of the QTLfs-1 was stable under different environmental conditions, and its molecular marker-assisted selection showed significant selective efficiency among breeding populations with different genetic backgrounds. QTLfs-2 also showed high selective efficiency in advanced generation populations though its effect was a little lower than the former. When QTLfs-1 was selected simultaneously with 2 molecular markers with known genetic distance, the selection efficiency for the fiber strength was greatly increased. The pyramiding for two QTLs that control high fiber strength by MAS greatly improved the selection efficiency for cotton fiber strength. This report provides a successful example of MAS pyramiding for QTL for favorable traits in breeding programs.

Transcriptome-wide identification of salt-responsive members of the WRKY gene family in Gossypium aridum.

WRKY transcription factors are plant-specific, zinc finger-type transcription factors. The WRKY superfamily is involved in abiotic stress responses in many crops including cotton, a major fiber crop that is widely cultivated and consumed throughout the world. Salinity is an important abiotic stress that results in considerable yield losses. In this study, we identified 109 WRKY genes (GarWRKYs) in a salt-tolerant wild cotton species Gossypium aridum from transcriptome sequencing data to elucidate the roles of these factors in cotton salt tolerance. According to their structural features, the predicted members were divided into three groups (Groups I-III), as previously described for Arabidopsis. Furthermore, 28 salt-responsive GarWRKY genes were identified from digital gene expression data and subjected to real-time quantitative RT-PCR analysis. The expression patterns of most GarWRKY genes revealed by this analysis are in good agreement with those revealed by RNA-Seq analysis. RT-PCR analysis revealed that 27 GarWRKY genes were expressed in roots and one was exclusively expressed in roots. Analysis of gene orthology and motif compositions indicated that WRKY members from Arabidopsis, rice and soybean generally shared the similar motifs within the same subgroup, suggesting they have the similar function. Overexpression-GarWRKY17 and -GarWRKY104 in Arabidopsis revealed that they could positively regulate salt tolerance of transgenic Arabidopsis during different development stages. The comprehensive data generated in this study provide a platform for elucidating the functions of WRKY transcription factors in salt tolerance of G. aridum. In addition, GarWRKYs related to salt tolerance identified in this study will be potential candidates for genetic improvement of cultivated cotton salt stress tolerance.

De novo transcriptome sequencing and comparative analysis of differentially expressed genes in Gossypium aridum under salt stress.

Salinity stress is one of the most serious factors that impede the growth and development of various crops. Wild Gossypium species, which are remarkably tolerant to salt water immersion, are valuable resources for understanding salt tolerance mechanisms of Gossypium and improving salinity resistance in upland cotton. To generate a broad survey of genes with altered expression during various stages of salt stress, a mixed RNA sample was prepared from the roots and leaves of Gossypium aridum plants subjected to salt stress. The transcripts were sequenced using the Illumina sequencing platform. After cleaning and quality checks, approximately 41.5 million clean reads were obtained. Finally, these reads were eventually assembled into 98,989 unigenes with a mean size of 452 bp. All unigenes were compared to known cluster of orthologous groups (COG) sequences to predict and classify the possible functions of these genes, which were classified into at least 25 molecular families. Variations in gene expression were then examined after exposing the plants to 200 mM NaCl for 3, 12, 72 or 144 h. Sequencing depths of approximately six million raw tags were achieved for each of the five stages of salt stress. There were 2634 (1513 up-regulated/1121 down-regulated), 2449 (1586 up-regulated/863 down-regulated), 2271 (946 up-regulated/1325 down-regulated) and 3352 (933 up-regulated/2419 down-regulated) genes that were differentially expressed after exposure to NaCl for 3, 12, 72 and 144 h, respectively. Digital gene expression analysis indicated that pathways involved in "transport", "response to hormone stimulus" and "signaling" play important roles during salt stress, while genes involved in "protein kinase activity" and "transporter activity" undergo major changes in expression during early and later stages of salt stress, respectively.

QTL mapping for resistance to root-knot nematodes in the M-120 RNR Upland cotton line (Gossypium hirsutum L.) of the Auburn 623 RNR source.

Root-knot nematodes Meloidogyne incognita (Kofoid and White) can cause severe yield loss in cotton (Gossypium hirsutum L.). The objectives of this study were to determine the inheritance and genomic location of genes conferring root-knot nematode resistance in M-120 RNR, a highly resistant G. hirsutum line with the Auburn 623 RNR source of resistance. Utilizing two interspecific F(2) populations developed from the same M-120 RNR by Gossypium barbadense (cv. Pima S-6) cross, genome-wide scanning with RFLP markers revealed a marker on Chromosome 7 and two on Chromosome 11 showing significant association with the resistant phenotype. The association was confirmed using SSR markers with the detection of a minor and a major dominant QTL on Chromosome 7 and 11, respectively. Combined across the two populations, the major QTL on Chromosome 11 Mi-C11 had a LOD score of 19.21 (9.69 and 9.61 for Pop1 and Pop2, respectively) and accounted for 63.7% (52.6 and 65.56% for Pop1 and Pop2, respectively) of the total phenotypic variation. The minor QTL locus on Chromosome 7 Mi ( 1 ) -C07 had a LOD score of 3.48 and accounted for 7.7% of the total phenotypic variation in the combined dataset but was detected in only one population. The allele from the M-120 RNR parent contributed to increased resistance in the Mi-C11 locus, but surprisingly, the Pima S-6 allele contributed to increased resistance in the Mi-C07 locus. The M-120 RNR allele in the Mi-C11 locus, derived from the Auburn 623 RNR, is likely to have originated from the Clevewilt 6 cultivar. Results from this study indicated that the SSR marker CIR316 may replace the laborious greenhouse screening in breeding programs to identify genotypes resistant to M. incognita.