Fine mapping and candidate gene analysis of qFL-A12-5: a fiber length-related QTL introgressed from Gossypium barbadense into Gossypium hirsutum.

KEY MESSAGE: The fiber length-related qFL-A12-5 identified in CSSLs introgressed from Gossypium barbadense into Gossypium hirsutum was fine-mapped to an 18.8 kb region on chromosome A12, leading to the identification of the GhTPR gene as a potential regulator of cotton fiber length. Fiber length is a key determinant of fiber quality in cotton, and it is a key target of artificial selection for breeding and domestication. Although many fiber length-related quantitative trait loci have been identified, there are few reports on their fine mapping or candidate gene validation, thus hampering efforts to understand the mechanistic basis of cotton fiber development. Our previous study identified the qFL-A12-5 associated with superior fiber quality on chromosome A12 in the chromosome segment substitution line (CSSL) MBI7747 (BC4F3:5). A single segment substitution line (CSSL-106) screened from BC6F2 was backcrossed to construct a larger segregation population with its recurrent parent CCRI45, thus enabling the fine mapping of 2852 BC7F2 individuals using denser simple sequence repeat markers to narrow the qFL-A12-5 to an 18.8 kb region of the genome, in which six annotated genes were identified in Gossypium hirsutum. Quantitative real-time PCR and comparative analyses led to the identification of GH_A12G2192 (GhTPR) encoding a tetratricopeptide repeat-like superfamily protein as a promising candidate gene for qFL-A12-5. A comparative analysis of the protein-coding regions of GhTPR among Hai1, MBI7747, and CCRI45 revealed two non-synonymous mutations. The overexpression of GhTPR resulted in longer roots in Arabidopsis, suggesting that GhTPR may regulate cotton fiber development. These results provide a foundation for future efforts to improve cotton fiber length.

Genome-Wide Analysis and Functional Characterization of LACS Gene Family Associated with Lipid Synthesis in Cotton (Gossypium spp.).

Cotton (Gossypium spp.) is the fifth largest oil crop in the world, and cottonseed provides abundant vegetable oil resources and industrial bioenergy fuels for people; therefore, it is of practical significance to increase the oil content of cotton seeds for improving the oil yield and economic benefits of planting cotton. Long-chain acyl-coenzyme A (CoA) synthetase (LACS) capable of catalyzing the formation of acyl-CoAs from free fatty acids has been proven to significantly participate in lipid metabolism, of which whole-genome identification and functional characterization of the gene family have not yet been comprehensively analyzed in cotton. In this study, a total of sixty-five LACS genes were confirmed in two diploid and two tetraploid Gossypium species, which were divided into six subgroups based on phylogenetic relationships with twenty-one other plants. An analysis of protein motif and genomic organizations displayed structural and functional conservation within the same group but diverged among the different group. Gene duplication relationship analysis illustrates the LACS gene family in large scale expansion through WGDs/segmental duplications. The overall Ka/Ks ratio indicated the intense purifying selection of LACS genes in four cotton species during evolution. The LACS genes promoter elements contain numerous light response cis-elements associated with fatty acids synthesis and catabolism. In addition, the expression of almost all GhLACS genes in high seed oil were higher compared to those in low seed oil. We proposed LACS gene models and shed light on their functional roles in lipid metabolism, demonstrating their engineering potential for modulating TAG synthesis in cotton, and the genetic engineering of cottonseed oil provides a theoretical basis.

QTL mapping and candidate gene prediction for fiber yield and quality traits in a high-generation cotton chromosome substitution line with Gossypium barbadense segments.

Gossypium provides the foremost natural fiber for supporting the rapid development of the textile industry. Quantitative trait locus (QTL) mapping of fiber yield and quality traits is, thus, of great significance for providing a foundation for the genetic improvement of key target traits in cotton production. In this study, a superior chromosome segment substitution line (CSSL), MBI8255, with high yield and premium fiber quality characteristics was cultivated from the BC5F3:5 lineage derived from G. barbadense Hai1 and G. hirsutum CCRI36, and was chosen to construct a segregation population containing 123 F2 individuals with CCRI36. A total of 71 polymorphic SSR (simple sequence repeat) markers were identified based on a previous high-density linkage map, and 17 QTLs distributed on five chromosomes were detected, of which 10 QTLs for cotton yield explained 0.26-15.41% of phenotypic variations, while 7 QTLs for fiber quality explained 0.84-9.38% of phenotypic variations, separately containing four and one stable QTLs detected from over two environments. Among three identified QTL clusters, only the Chr19 QTL cluster harbored two stable and one unstable QTL for three different traits, and hence this significant region, which included 1546 genes, was subjected to functional enrichment and transcriptome expression analyses, ultimately screening eight candidate genes relevant to fiber development. This study not only provides useful information for the further fine-mapping and functional verification of candidate genes, but also offers a solid foundation for revealing the molecular mechanisms of fiber formation.

Identification and analysis of oil candidate genes reveals the molecular basis of cottonseed oil accumulation in Gossypium hirsutum L.

KEY MESSAGE: Based on the integration of QTL-mapping and regulatory network analyses, five high-confidence stable QTL regions, six candidate genes and two microRNAs that potentially affect the cottonseed oil content were discovered. Cottonseed oil is increasingly becoming a promising target for edible oil with its high content of unsaturated fatty acids. In this study, a recombinant inbred line (RIL) cotton population was constructed to detect quantitative trait loci (QTLs) for the cottonseed oil content. A total of 39 QTLs were detected across eight different environments, of which five QTLs were stable. Forty-three candidate genes potentially involved in carbon metabolism, fatty acid synthesis and triacylglycerol biosynthesis processes were further obtained in the stable QTL regions. Transcriptome analysis showed that nineteen of these candidate genes expressed during  the developing cottonseed ovules and may affect the cottonseed oil content. Besides, transcription factor (TF) and microRNA (miRNA) co-regulatory network analyses based on the nineteen candidate genes suggested that six genes, two core miRNAs (ghr-miR2949b and ghr-miR2949c), and one TF GhHSL1 were considered to be closely associated with the cottonseed oil content. Moreover, four vital genes were validated by quantitative real-time PCR (qRT-PCR). These results provide insights into the oil accumulation mechanism in developing cottonseed ovules through the construction of a detailed oil accumulation model.

AAQSP increases mapping resolution of stable QTLs through applying NGS-BSA in multiple genetic backgrounds.

KEY MESSAGE: In this study, we present AAQSP as an extension of existing NGS-BSA applications for identifying stable QTLs at high resolution. GhPAP16 and GhIQD14 fine mapped on chromosome D09 of upland cotton are identified as important candidate genes for lint percentage (LP). Bulked segregant analysis combined with next generation sequencing (NGS-BSA) allows rapid identification of genome sequence differences responsible for phenotypic variation. The NGS-BSA approach applied to crops mainly depends on comparing two bulked DNA samples of individuals from an F2 population. Since some F2 individuals still maintain high heterozygosity, heterosis will exert complications in pursuing NGS-BSA in such populations. In addition, the genetic background influences the stability of gene expression in crops, so some QTLs mapped in one segregating population may not be widely applied in crop improvement. The AAQSP (Association Analysis of QTL-seq on Semi-homologous Populations) reported in our study combines the optimized scheme of constructing BSA bulks with NGS-BSA analysis in two (or more) different parental genetic backgrounds for isolating the stable QTLs. With application of AAQSP strategy and construction of a high-density linkage map, we have successfully identified a QTL significantly related to lint percentage (LP) in cultivated upland cotton, followed by map-based cloning to dissect two candidate genes, GhPAP16 and GhIQD14. This study demonstrated that AAQSP can efficiently identify stable QTLs for complex traits of interest, and thus accelerate the genetic improvement of upland cotton and other crop plants.

Identification of hub genes through co-expression network of major QTLs of fiber length and strength traits in multiple RIL populations of cotton.

The present study demonstrated a de novo correlation among fiber quality genes in multiple RIL populations including sGK9708 × 0-153, LMY22 × LY343 and Lumianyan28 × Xinluzao24. The current study was conducted to identify the major common QTLs including fiber length and strength, and to identify the co-expression networks of fiber length and strength QTLs harbored genes to target the hub genes. The RNA-seq data of sGK9708 × 0-153 population highlighted 50 and 48 candidate genes of fiber length and fiber strength QTLs. A total of 29 and 21 hub genes were identified in fiber length and strength co-expression network modules. The absolute values of correlation coefficient close to 1 resulted highly positive correlation among hub genes. Results also suggested that the gene correlation significantly influence the gene expression at different fiber development stages. These results might provide useful reference for further experiments in multiple RIL populations and suggest potential candidate genes for functional studies in cotton.

Multi-responses of O-methyltransferase genes to salt stress and fiber development of Gossypium species.

BACKGROUND: O-methyltransferases (OMTs) are an important group of enzymes that catalyze the transfer of a methyl group from S-adenosyl-L-methionine to their acceptor substrates. OMTs are divided into several groups according to their structural features. In Gossypium species, they are involved in phenolics and flavonoid pathways. Phenolics defend the cellulose fiber from dreadful external conditions of biotic and abiotic stresses, promoting strength and growth of plant cell wall. RESULTS: An OMT gene family, containing a total of 192 members, has been identified and characterized in three main Gossypium species, G. hirsutum, G. arboreum and G. raimondii. Cis-regulatory elements analysis suggested important roles of OMT genes in growth, development, and defense against stresses. Transcriptome data of different fiber developmental stages in Chromosome Substitution Segment Lines (CSSLs), Recombination Inbred Lines (RILs) with excellent fiber quality, and standard genetic cotton cultivar TM-1 demonstrate that up-regulation of OMT genes at different fiber developmental stages, and abiotic stress treatments have some significant correlations with fiber quality formation, and with salt stress response. Quantitative RT-PCR results revealed that GhOMT10_Dt and GhOMT70_At genes had a specific expression in response to salt stress while GhOMT49_At, GhOMT49_Dt, and GhOMT48_At in fiber elongation and secondary cell wall stages. CONCLUSIONS: Our results indicate that O-methyltransferase genes have multi-responses to salt stress and fiber development in Gossypium species and that they may contribute to salt tolerance or fiber quality formation in Gossypium.

Disequilibrium evolution of the Fructose-1,6-bisphosphatase gene family leads to their functional biodiversity in Gossypium species.

BACKGROUND: Fructose-1,6-bisphosphatase (FBP) is a key enzyme in the plant sucrose synthesis pathway, in the Calvin cycle, and plays an important role in photosynthesis regulation in green plants. However, no systemic analysis of FBPs has been reported in Gossypium species. RESULTS: A total of 41 FBP genes from four Gossypium species were identified and analyzed. These FBP genes were sorted into two groups and seven subgroups. Results revealed that FBP family genes were under purifying selection pressure that rendered FBP family members as being conserved evolutionarily, and there was no tandem or fragmental DNA duplication in FBP family genes. Collinearity analysis revealed that a FBP gene was located in a translocated DNA fragment and the whole FBP gene family was under disequilibrium evolution that led to a faster evolutionary progress of the members in G. barbadense and in At subgenome than those in other Gossypium species and in the Dt subgenome, respectively, in this study. Through RNA-seq analyses and qRT-PCR verification, different FBP genes had diversified biological functions in cotton fiber development (two genes in 0 DPA and 1DPA ovules and four genes in 20-25 DPA fibers), in plant responses to Verticillium wilt onset (two genes) and to salt stress (eight genes). CONCLUSION: The FBP gene family displayed a disequilibrium evolution pattern in Gossypium species, which led to diversified functions affecting not only fiber development, but also responses to Verticillium wilt and salt stress. All of these findings provide the foundation for further study of the function of FBP genes in cotton fiber development and in environmental adaptability.

Quantification of Trace Organophosphorus Pesticides in Environmental Water via Enrichment by Magnetic-Zirconia Nanocomposites and Online Extractive Electrospray Ionization Mass Spectrometry.

A total of 15 representative organophosphorus pesticides (OPPs), a class of water pollutants causing serious global concerns, have been sensitively quantified by internal extractive electrospray ionization mass spectrometry (iEESI-MS) after enrichment with Fe3O4-ZrO2 innovatively synthesized in our lab by a one step coprecipitation method. For the premium enrichment, the amounts of Fe3O4-ZrO2, pH value, adsorption time, type and volume of desorption solvent, and shaking time of desorption were systematically optimized. Under optimized conditions, the proposed method provided low limits of detection (LODs) of 0.14-16.39 ng L-1 with relative standard deviations (RSDs) of less than 8.7%. A wide linear response range of about 4 orders of magnitude was achieved with linear coefficients (R2) of 0.9921-0.9999 for all the analytes tested. The present method also provided good recoveries (85.4-105.9%) with acceptable precision (RSDs < 7.2%) in spiked environmental water samples. Furthermore, multiple analytes including dimethoate, omethoate, etc. were simultaneously detected in a single sample run, which was accomplished within 1 min, resulting in significantly improved analytical throughput for quantitative analysis of bulk amounts of samples. The experimental results demonstrated that Fe3O4-ZrO2-iEESI-MS provided advantages, including high sensitivity, high speed, and reasonable selectivity for the detection of OPPs, showing potential applications in environmental water sample analysis and environmental science.

Identification of candidate genes for key fibre-related QTLs and derivation of favourable alleles in Gossypium hirsutum recombinant inbred lines with G. barbadense introgressions.

Fine mapping QTLs and identifying candidate genes for cotton fibre-quality and yield traits would be beneficial to cotton breeding. Here, we constructed a high-density genetic map by specific-locus amplified fragment sequencing (SLAF-seq) to identify QTLs associated with fibre-quality and yield traits using 239 recombinant inbred lines (RILs), which was developed from LMY22 (a high-yield Gossypium hirsutumL. cultivar) × LY343 (a superior fibre-quality germplasm with G. barbadenseL. introgressions). The genetic map spanned 3426.57 cM, including 3556 SLAF-based SNPs and 199 SSR marker loci. A total of 104 QTLs, including 67 QTLs for fibre quality and 37 QTLs for yield traits, were identified with phenotypic data collected from 7 environments. Among these, 66 QTLs were co-located in 19 QTL clusters on 12 chromosomes, and 24 QTLs were detected in three or more environments and determined to be stable. We also investigated the genomic components of LY343 and their contributions to fibre-related traits by deep sequencing the whole genome of LY343, and we found that genomic components from G. hirsutum races (which entered LY343 via its G. barbadense parent) contributed more favourable alleles than those from G. barbadense. We further identified six putative candidate genes for stable QTLs, including Gh_A03G1147 (GhPEL6), Gh_D07G1598 (GhCSLC6) and Gh_D13G1921 (GhTBL5) for fibre-length QTLs and Gh_D03G0919 (GhCOBL4), Gh_D09G1659 (GhMYB4) and Gh_D09G1690 (GhMYB85) for lint-percentage QTLs. Our results provide comprehensive insight into the genetic basis of the formation of fibre-related traits and would be helpful for cloning fibre-development-related genes as well as for marker-assisted genetic improvement in cotton.

Genome-wide quantitative trait loci reveal the genetic basis of cotton fibre quality and yield-related traits in a Gossypium hirsutum recombinant inbred line population.

Cotton is widely cultivated globally because it provides natural fibre for the textile industry and human use. To identify quantitative trait loci (QTLs)/genes associated with fibre quality and yield, a recombinant inbred line (RIL) population was developed in upland cotton. A consensus map covering the whole genome was constructed with three types of markers (8295 markers, 5197.17 centimorgans (cM)). Six fibre yield and quality traits were evaluated in 17 environments, and 983 QTLs were identified, 198 of which were stable and mainly distributed on chromosomes 4, 6, 7, 13, 21 and 25. Thirty-seven QTL clusters were identified, in which 92.8% of paired traits with significant medium or high positive correlations had the same QTL additive effect directions, and all of the paired traits with significant medium or high negative correlations had opposite additive effect directions. In total, 1297 genes were discovered in the QTL clusters, 414 of which were expressed in two RNA-Seq data sets. Many genes were discovered, 23 of which were promising candidates. Six important QTL clusters that included both fibre quality and yield traits were identified with opposite additive effect directions, and those on chromosome 13 (qClu-chr13-2) could increase fibre quality but reduce yield; this result was validated in a natural population using three markers. These data could provide information about the genetic basis of cotton fibre quality and yield and help cotton breeders to improve fibre quality and yield simultaneously.

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

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

Selective detection of phospholipids in human blood plasma and single cells for cancer differentiation using dispersed solid-phase microextraction combined with extractive electrospray ionization mass spectrometry.

Phospholipids in microvolume biofluid samples (≤0.5 µL), including human plasma and single cells, were selectively captured by dispersed magnetic Fe3O4@TiO2 nanocomposite particles (40 µg). A suspension containing Fe3O4@TiO2 nanoparticles was loaded into a glass capillary (i.d. 0.75 mm) by capillary force. The supernatant solution was discarded, while the Fe3O4@TiO2 particles were retained inside the capillary by using an external magnetic field (ca. 1.3 T). The phospholipids on the surface of Fe3O4@TiO2 nanoparticles were directly analyzed using internal extractive electrospray ionization mass spectrometry (iEESI-MS) by pumping ≤1 µL of extraction solution of methanol containing 1.5% ammonia (w/w) through the capillary tube toward the ESI tip. A single sample analysis was accomplished within 4 min. Phospholipids in blood plasma samples from 59 patients with ovarian cancer and 43 healthy controls, and 28 patients with pancreatic cancer and 23 healthy controls were studied. Based on the orthogonal partial least squares discriminant analysis (OPLS-DA), the cancer patients were confidently discriminated from the healthy controls. Phospholipids in single human cells (MV4-11 and NB4) were determined, showing the sensitivity for single cell analysis. Therefore the results demonstrated that rapid cancer differentiation is achieved using this approach through the detection of trace phospholipids in microvolume blood and cell samples with high sensitivity, high specificity, low sample consumption, and high throughput.

Generating Supercharged Protein Ions for Breath Analysis by Extractive Electrospray Ionization Mass Spectrometry.

Supercharged protein ions produced by electrospray ionization are extremely efficient proton donors for secondary ionization. Here, by electrospraying the protein solutions containing 5% 1,2-butylene carbonate, the supercharged protein ions with unusually high proton density were produced as the primary ions for the ionization of exhaled breath samples in the extractive electrospray ionization mass spectrometry (EESI-MS), which resulted in the enhanced ionization efficiency for the breath analytes even with relatively low gas phase basicity. Moreover, the total number of metabolites detected in breath increased by about 260% in the mass range of 200-500 Da, owing to the substantial signal enhancement for breath metabolites, providing complementary and additional information to conventional SESI.

Transcriptomic and biochemical analysis of upland cotton (Gossypium hirsutum) and a chromosome segment substitution line from G. hirsutum × G. barbadense in response to Verticillium dahliae infection.

BACKGROUND: Verticillium wilt (VW), also known as "cotton cancer," is one of the most destructive diseases in global cotton production that seriously impacts fiber yield and quality. Despite numerous attempts, little significant progress has been made in improving the VW resistance of upland cotton. The development of chromosome segment substitution lines (CSSLs) from Gossypium hirsutum × G. barbadense has emerged as a means of simultaneously developing new cotton varieties with high-yield, superior fiber, and resistance to VW. RESULTS: In this study, VW-resistant investigations were first conducted in an artificial greenhouse, a natural field, and diseased nursery conditions, resulting in the identification of one stably VW-resistant CSSL, MBI8255, and one VW-susceptible G. hirsutum, CCRI36, which were subsequently subjected to biochemical tests and transcriptome sequencing during V991 infection (0, 1, and 2 days after inoculation). Eighteen root samples with three replications were collected to perform multiple comparisons of enzyme activity and biochemical substance contents. The findings indicated that VW resistance was positively correlated with peroxidase and polyphenol oxidase activity, but negatively correlated with malondialdehyde content. Additionally, RNA sequencing was used for the same root samples, resulting in a total of 77,412 genes, of which 23,180 differentially expressed genes were identified from multiple comparisons between samples. After Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis on the expression profiles identified using Short Time-series Expression Miner, we found that the metabolic process in the biological process, as well as the pathways of phenylpropanoid biosynthesis and plant hormone signal transduction, participated significantly in the response to VW. Gene functional annotation and expression quantity analysis indicated the important roles of the phenylpropanoid metabolic pathway and oxidation-reduction process in response to VW, which also provided plenty of candidate genes related to plant resistance. CONCLUSIONS: This study concentrates on the preliminary response to V991 infection by comparing the VW-resistant CSSL and its VW-susceptible recurrent parent. Not only do our findings facilitate the culturing of new resistant varieties with high yield and superior performance, but they also broaden our understanding of the mechanisms of cotton resistance to VW.

QTL mapping and genetic effect of chromosome segment substitution lines with excellent fiber quality from Gossypium hirsutum × Gossypium barbadense.

Chromosome segment substitution lines (CSSLs) are ideal materials for identifying genetic effects. In this study, CSSL MBI7561 with excellent fiber quality that was selected from BC4F3:5 of CCRI45 (Gossypium hirsutum) × Hai1 (Gossypium barbadense) was used to construct 3 secondary segregating populations with 2 generations (BC5F2 and BC5F2:3). Eighty-one polymorphic markers related to 33 chromosome introgressive segments on 18 chromosomes were finally screened using 2292 SSR markers which covered the whole tetraploid cotton genome. A total of 129 quantitative trait loci (QTL) associated with fiber quality (103) and yield-related traits (26) were detected on 17 chromosomes, explaining 0.85-30.35% of the phenotypic variation; 39 were stable (30.2%), 53 were common (41.1%), 76 were new (58.9%), and 86 had favorable effects on the related traits. More QTL were distributed in the Dt subgenome than in the At subgenome. Twenty-five stable QTL clusters (with stable or common QTL) were detected on 22 chromosome introgressed segments. Finally, the 6 important chromosome introgressed segments (Seg-A02-1, Seg-A06-1, Seg-A07-2, Seg-A07-3, Seg-D07-3, and Seg-D06-2) were identified as candidate chromosome regions for fiber quality, which should be given more attention in future QTL fine mapping, gene cloning, and marker-assisted selection (MAS) breeding.

Dissecting the genetic basis of fiber quality and yield traits in interspecific backcross populations of Gossypium hirsutum × Gossypium barbadense.

Fiber quality and yield are important traits of cotton. Quantitative trait locus (QTL) mapping is a prerequisite for marker-assisted selection (MAS) in cotton breeding. To identify QTLs for fiber quality and yield traits, 4 backcross-generation populations (BC1F1, BC1S1, BC2F1, and BC3F0) were developed from an interspecific cross between CCRI36 (Gossypium hirsutum L.) and Hai1 (G. barbadense L.). A total of 153 QTLs for fiber quality and yield traits were identified based on data from the BC1F1, BC1S1, BC2F1 and BC3F0 populations in the field and from the BC2F1 population in an artificial disease nursery using a high-density genetic linkage map with 2292 marker loci covering 5115.16 centimorgans (cM) from the BC1F1 population. These QTLs were located on 24 chromosomes, and each could explain 4.98-19.80% of the observed phenotypic variations. Among the 153 QTLs, 30 were consistent with those identified previously. Specifically, 23 QTLs were stably detected in 2 or 3 environments or generations, 6 of which were consistent with those identified previously and the other 17 of which were stable and novel. Ten QTL clusters for different traits were found and 9 of them were novel, which explained the significant correlations among some phenotypic traits in the populations. The results including these stable or consensus QTLs provide valuable information for marker-assisted selection (MAS) in cotton breeding and will help better understand the genetic basis of fiber quality and yield traits, which can then be used in QTL cloning.

Coupling of micro-solid-phase extraction and internal extractive electrospray ionization mass spectrometry for ultra-sensitive detection of 1-hydroxypyrene and papaverine in human urine samples.

Quantification of ultra-trace analytes in complex biological samples using micro-solid-phase extraction followed by direct detection with internal extractive electrospray ionization mass spectrometry (µSPE-iEESI-MS) was demonstrated. 1-Hydroxypyrene (1-OHP) and papaverine at attomole levels in human raw urine samples were analyzed under negative and positive ion detection mode, respectively. The µSPE was simply prepared by packing a disposable syringe filter with octadecyl carbon chain (C18)-bonded micro silica particles, which were then treated as the "bulk sample" after the analytes were efficiently enriched by the C18 particles. Under the optimized experimental conditions, the analytes were readily eluted by isopropanol/water (80/20, V/V) at a high voltage of ± 4.0 kV, producing analyte ions under ambient conditions. The limit of detection (LOD) was 0.02 pg/L (9.2 amol) for 1-hydroxypyrene and 0.02 pg/L (5.9 amol) for papaverine. The acceptable linearity (R2 > 0.99), signal stability (RSD ≤ 10.7%), spike recoveries (91-95%), and comparable results for real urine samples were also achieved, opening up possibilities for quantitative analysis of trace compounds (at attomole levels) in complex bio-samples. Graphical abstract.

Comparative transcriptome analysis of cotton fiber development of Upland cotton (Gossypium hirsutum) and Chromosome Segment Substitution Lines from G. hirsutum × G. barbadense.

BACKGROUND: How to develop new cotton varieties possessing high yield traits of Upland cotton and superior fiber quality traits of Sea Island cotton remains a key task for cotton breeders and researchers. While multiple attempts bring in little significant progresses, the development of Chromosome Segment Substitution Lines (CSSLs) from Gossypium barbadense in G. hirsutum background provided ideal materials for aforementioned breeding purposes in upland cotton improvement. Based on the excellent fiber performance and relatively clear chromosome substitution segments information identified by Simple Sequence Repeat (SSR) markers, two CSSLs, MBI9915 and MBI9749, together with the recurrent parent CCRI36 were chosen to conduct transcriptome sequencing during the development stages of fiber elongation and Secondary Cell Wall (SCW) synthesis (from 10DPA and 28DPA), aiming at revealing the mechanism of fiber development and the potential contribution of chromosome substitution segments from Sea Island cotton to fiber development of Upland cotton. RESULTS: In total, 15 RNA-seq libraries were constructed and sequenced separately, generating 705.433 million clean reads with mean GC content of 45.13% and average Q30 of 90.26%. Through multiple comparisons between libraries, 1801 differentially expressed genes (DEGs) were identified, of which the 902 up-regulated DEGs were mainly involved in cell wall organization and response to oxidative stress and auxin, while the 898 down-regulated ones participated in translation, regulation of transcription, DNA-templated and cytoplasmic translation based on GO annotation and KEGG enrichment analysis. Subsequently, STEM software was performed to explicate the temporal expression pattern of DEGs. Two peroxidases and four flavonoid pathway-related genes were identified in the "oxidation-reduction process", which could play a role in fiber development and quality formation. Finally, the reliability of RNA-seq data was validated by quantitative real-time PCR of randomly selected 20 genes. CONCLUSIONS: The present report focuses on the similarities and differences of transcriptome profiles between the two CSSLs and the recurrent parent CCRI36 and provides novel insights into the molecular mechanism of fiber development, and into further exploration of the feasible contribution of G. barbadense substitution segments to fiber quality formation, which will lay solid foundation for simultaneously improving fiber yield and quality of upland cotton through CSSLs.

Quantitative trait loci analysis of Verticillium wilt resistance in interspecific backcross populations of Gossypium hirsutum × Gossypium barbadense.

BACKGROUND: Verticillium wilt (VW) caused by Verticillium dahliae (Kleb) is one of the most destructive diseases of cotton. The identification of highly resistant QTLs or genes in the whole cotton genome is quite important for developing a VW-resistant variety and for further molecular design breeding. RESULTS: In the present study, BC1F1, BC1S1, and BC2F1 populations derived from an interspecific backcross between the highly resistant line Hai1 (Gossypium barbadense L.) and the susceptible variety CCRI36 (G. hirsutum L.) as the recurrent parent were constructed. Quantitative trait loci (QTL) related to VW resistance were detected in the whole cotton genome using a high-density simple sequence repeat (SSR) genetic linkage map from the BC1F1 population, with 2292 loci covering 5115.16 centiMorgan (cM) of the cotton (AD) genome, and the data concerning VW resistance that were obtained from four dates of BC2F1 in the artificial disease nursery and one date of BC1S1 and BC2F1 in the field. A total of 48 QTLs for VW resistance were identified, and 37 of these QTLs had positive additive effects, which indicated that the G. barbadense alleles increased resistance to VW and decreased the disease index (DI) by about 2.2-10.7. These QTLs were located on 19 chromosomes, in which 33 in the A subgenome and 15 QTLs in the D subgenome. The 6 QTLs were found to be stable. The 6 QTLs were consistent with those identified previously, and another 42 were new, unreported QTLs, of which 31 QTLs were from G. barbadense. By meta-analysis, 17 QTL hotspot regions were identified and 10 of them were new, unreported hotspot regions. 29 QTLs in this paper were in 12 hotspot regions and were all from G. barbadense. CONCLUSIONS: These stable or consensus QTL regions warrant further investigation to better understand the genetics and molecular mechanisms underlying VW resistance. This study provides useful information for further comparative analysis and marker-assisted selection in the breeding of disease-resistant cotton. It may also lay an important foundation for gene cloning and further molecular design breeding for the entire cotton genome.