Quantitative Trait Locus Mapping for Plant Height and Branch Number in CCRI70 Recombinant Inbred Line Population of Upland Cotton (Gossypium hirsutum).

Upland cotton accounts for a high percentage (95%) of the world's cotton production. Plant height (PH) and branch number (BN) are two important agronomic traits that have an impact on improving the level of cotton mechanical harvesting and cotton yield. In this research, a recombinant inbred line (RIL) population with 250 lines developed from the variety CCRI70 was used for constructing a high-density genetic map and identification of quantitative trait locus (QTL). The results showed that the map harbored 8298 single nucleotide polymorphism (SNP) markers, spanning a total distance of 4876.70 centimorgans (cMs). A total of 69 QTLs for PH (9 stable) and 63 for BN (11 stable) were identified and only one for PH was reported in previous studies. The QTLs for PH and BN harbored 495 and 446 genes, respectively. Combining the annotation information, expression patterns and previous studies of these genes, six genes could be considered as potential candidate genes for PH and BN. The results could be helpful for cotton researchers to better understand the genetic mechanism of PH and BN development, as well as provide valuable genetic resources for cotton breeders to manipulate cotton plant architecture to meet future demands.

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.

Genome-wide characterization of trichome birefringence-like genes provides insights into fiber yield improvement.

Cotton is an important fiber crop. The cotton fiber is an extremely long trichome that develops from the epidermis of an ovule. The trichome is a general and multi-function plant organ, and trichome birefringence-like (TBL) genes are related to trichome development. At the genome-wide scale, we identified TBLs in four cotton species, comprising two cultivated tetraploids (Gossypium hirsutum and G. barbadense) and two ancestral diploids (G. arboreum and G. raimondii). Phylogenetic analysis showed that the TBL genes clustered into six groups. We focused on GH_D02G1759 in group IV because it was located in a lint percentage-related quantitative trait locus. In addition, we used transcriptome profiling to characterize the role of TBLs in group IV in fiber development. The overexpression of GH_D02G1759 in Arabidopsis thaliana resulted in more trichomes on the stems, thereby confirming its function in fiber development. Moreover, the potential interaction network was constructed based on the co-expression network, and it was found that GH_D02G1759 may interact with several genes to regulate fiber development. These findings expand our knowledge of TBL family members and provide new insights for cotton molecular breeding.

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 artificial introgressions of Gossypium barbadense into G. hirsutum reveal superior loci for simultaneous improvement of cotton fiber quality and yield traits.

INTRODUCTION: The simultaneous improvement of fiber quality and yield for cotton is strongly limited by the narrow genetic backgrounds of Gossypium hirsutum (Gh) and the negative genetic correlations among traits. An effective way to overcome the bottlenecks is to introgress the favorable alleles of Gossypium barbadense (Gb) for fiber quality into Gh with high yield. OBJECTIVES: This study was to identify superior loci for the improvement of fiber quality and yield. METHODS: Two sets of chromosome segment substitution lines (CSSLs) were generated by crossing Hai1 (Gb, donor-parent) with cultivar CCRI36 (Gh) and CCRI45 (Gh) as genetic backgrounds, and cultivated in 6 and 8 environments, respectively. The kmer genotyping strategy was improved and applied to the population genetic analysis of 743 genomic sequencing data. A progeny segregating population was constructed to validate genetic effects of the candidate loci. RESULTS: A total of 68,912 and 83,352 genome-wide introgressed kmers were identified in the CCRI36 and CCRI45 populations, respectively. Over 90 % introgressions were homologous exchanges and about 21 % were reverse insertions. In total, 291 major introgressed segments were identified with stable genetic effects, of which 66(22.98 %), 64(21.99 %), 35(12.03 %), 31(10.65 %) and 18(6.19 %) were beneficial for the improvement of fiber length (FL), strength (FS), micronaire, lint-percentage (LP) and boll-weight, respectively. Thirty-nine introgression segments were detected with stable favorable additive effects for simultaneous improvement of 2 or more traits in Gh genetic background, including 6 could increase FL/FS and LP. The pyramiding effects of 3 pleiotropic segments (A07:C45Clu-081, D06:C45Clu-218, D02:C45Clu-193) were further validated in the segregating population. CONCLUSION: The combining of genome-wide introgressions and kmer genotyping strategy showed significant advantages in exploring genetic resources. Through the genome-wide comprehensive mining, a total of 11 clusters (segments) were discovered for the stable simultaneous improvement of FL/FS and LP, which should be paid more attention in the future.

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.

Linkage and association analyses reveal that hub genes in energy-flow and lipid biosynthesis pathways form a cluster in upland cotton.

Upland cotton is an important allotetraploid crop that provides both natural fiber for the textile industry and edible vegetable oil for the food or feed industry. To better understand the genetic mechanism that regulates the biosynthesis of storage oil in cottonseed, we identified the genes harbored in the major quantitative trait loci/nucleotides (QTLs/QTNs) of kernel oil content (KOC) in cottonseed via both multiple linkage analyses and genome-wide association studies (GWAS). In 'CCRI70' RILs, six stable QTLs were simultaneously identified by linkage analysis of CHIP and SLAF-seq strategies. In '0-153' RILs, eight stable QTLs were detected by consensus linkage analysis integrating multiple strategies. In the natural panel, thirteen and eight loci were associated across multiple environments with two algorithms of GWAS. Within the confidence interval of a major common QTL on chromosome 3, six genes were identified as participating in the interaction network highly correlated with cottonseed KOC. Further observations of gene differential expression showed that four of the genes, LtnD, PGK, LPLAT1, and PAH2, formed hub genes and two of them, FER and RAV1, formed the key genes in the interaction network. Sequence variations in the coding regions of LtnD, FER, PGK, LPLAT1, and PAH2 genes may support their regulatory effects on oil accumulation in mature cottonseed. Taken together, clustering of the hub genes in the lipid biosynthesis interaction network provides new insights to understanding the mechanism of fatty acid biosynthesis and TAG assembly and to further genetic improvement projects for the KOC in cottonseeds.

Multi-environment Evaluations Across Ecological Regions Reveal That the Kernel Oil Content of Cottonseed Is Equally Determined by Genotype and Environment.

Cotton is the fifth-largest oil crop in the world. A high kernel oil content (KOC) and high stability are important cottonseed attributes for food security. In this study, the phenotype of KOC and the genotype-by-environment interaction factors were collectively dissected using 250 recombinant inbred lines, their parental cultivars sGK156 and 901-001, and CCRI70 across multi-environments. ANOVA and correlation analysis showed that both genotype and environment contributed significantly to KOC accumulation. Analyses of additive main effect multiplicative interaction and genotype-by-environment interaction biplot models presented the effects of genotype, environment, and genotype by environment on KOC performance and the stability of the experimental materials. Interaction network analysis revealed that meteorological and geographical factors explained 38% of the total KOC variance, with average daily rainfall contributing the largest positive impact and cumulative rainfall having the largest negative impact on KOC accumulation. This study provides insight into KOC accumulation and could direct selection strategies for improved KOC and field management of cottonseed in the future.

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.

Transformation and Overexpression of Primary Cell Wall Synthesis-Related Zinc Finger Gene Gh_A07G1537 to Improve Fiber Length in Cotton.

Molecular interventions have helped to explore the genes involved in fiber length, fiber strength, and other quality parameters with improved characteristics, particularly in cotton. The current study is an extension and functional validation of previous findings that Gh_A07G1537 influences fiber length in cotton using a chromosomal segment substitution line MBI7747 through RNA-seq data. The recombinant Gh_A07G1537 derived from the MBI7747 line was over-expressed in CCRI24, a genotype with a low profile of fiber quality parameters. Putative transformants were selected on MS medium containing hygromycin (25mg/ml), acclimatized, and shifted to a greenhouse for further growth and proliferation. Transgene integration was validated through PCR and Southern Blot analysis. Stable integration of the transgene (ΔGh_A07G1537) was validated by tracking its expression in different generations (T0, T1, and T2) of transformed cotton plants. It was found to be 2.97-, 2.86-, and 2.92-folds higher expression in T0, T1, and T2 plants, respectively, of transgenic compared with non-transgenic cotton plants. Fiber quality parameters were also observed to be improved in the engineered cotton line. Genetic modifications of Gh_A07G1537 support the improvement in fiber quality parameters and should be appreciated for the textile industry.

Genome-wide quantitative trait loci mapping on Verticillium wilt resistance in 300 chromosome segment substitution lines from Gossypium hirsutum × Gossypium barbadense.

Cotton Verticillium wilt (VW) is a devastating disease seriously affecting fiber yield and quality, and the most effective and economical prevention measure at present is selection and extension of Gossypium varieties harboring high resistance to VW. However, multiple attempts to improve the VW resistance of the most widely cultivated upland cottons have made little significant progress. The introduction of chromosome segment substitution lines (CSSLs) provide the practical solutions for merging the superior genes related with high yield and wide adaptation from Gossypium hirsutum and VW resistance and the excellent fiber quality from Gossypium barbadense. In this study, 300 CSSLs were chosen from the developed BC5F3:5 CSSLs constructed from CCRI36 (G. hirsutum) and Hai1 (G. barbadense) to conduct quantitative trait locus (QTL) mapping of VW resistance, and a total of 40 QTL relevant to VW disease index (DI) were identified. Phenotypic data were obtained from a 2-year investigation in two fields with two replications per year. All the QTL were distributed on 21 chromosomes, with phenotypic variation of 1.05%-10.52%, and 21 stable QTL were consistent in at least two environments. Based on a meta-analysis, 34 novel QTL were identified, while 6 loci were consistent with previously identified QTL. Meanwhile, 70 QTL hotspot regions were detected, including 44 novel regions. This study concentrates on QTL identification and screening for hotspot regions related with VW in the 300 CSSLs, and the results lay a solid foundation not only for revealing the genetic and molecular mechanisms of VW resistance but also for further fine mapping, gene cloning and molecular designing in breeding programs for resistant cotton varieties.

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.

Genome-wide identification, evolution, expression, and alternative splicing profiles of peroxiredoxin genes in cotton.

Peroxiredoxin (PRX) is a ubiquitous thioredoxin-dependent peroxidase that can eliminate excessive free radicals produced by stress and protect cells from oxidative damage. PRXs are also involved in reactive oxygen species (ROS)- and redox-dependent signaling by performing redox interactions with other proteins and modify their redox status. At present, PRX family identification, evolution and regulation research has been conducted in some plants; however, systematic research about this family is lacking in cotton. In this study, a total of 44 PRXs were identified in the cotton genome. Phylogenetic and conserved active site analyses showed that the PRXs were divided into six subfamilies according to the conserved site (PxxxTxxC…S…W/F) and conserved cysteinyl residues positions. Segmental duplication and polyploid events were the main methods for PRX family expansion, and the PRXs of diploid G. arboreum were the donors of PRXs in the D subgenomes of allotetraploid G. hirsutum and G. barbadense during the evolution of the PRX family. qRT-PCR analysis confirmed that cis-acting elements play important roles in regulating the expression of PRXs. Alternative splicing events occurred in GhPRX14-D that can increased the complexity of transcripts in G. hirsutum. Subcellular localization showed that most PRX members were located in chloroplasts, the cytoplasmic membrane and the nucleus. Our results provide systematic support for a better understanding of PRXs in cotton and a starting point for further studies of the specific functions of PRXs 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.

Quantitative Trait Loci and Transcriptome Analysis Reveal Genetic Basis of Fiber Quality Traits in CCRI70 RIL Population of Gossypium hirsutum.

Upland cotton (Gossypium hirsutum) is widely planted around the world for its natural fiber, and producing high-quality fiber is essential for the textile industry. CCRI70 is a hybrid cotton plant harboring superior yield and fiber quality, whose recombinant inbred line (RIL) population was developed from two upland cotton varieties (sGK156 and 901-001) and were used here to investigate the source of high-quality related alleles. Based on the material of the whole population, a high-density genetic map was constructed using specific locus-amplified fragment sequencing (SLAF-seq). It contained 24,425 single nucleotide polymorphism (SNP) markers, spanning a distance of 4,850.47 centimorgans (cM) over 26 chromosomes with an average marker interval of 0.20 cM. In evaluating three fiber quality traits in nine environments to detect multiple environments stable quantitative trait loci (QTLs), we found 289 QTLs, of which 36 of them were stable QTLs and 18 were novel. Based on the transcriptome analysis for two parents and two RILs, 24,941 unique differentially expressed genes (DEGs) were identified, 473 of which were promising genes. For the fiber strength (FS) QTLs, 320 DEGs were identified, suggesting that pectin synthesis, phenylpropanoid biosynthesis, and plant hormone signaling pathways could influence FS, and several transcription factors may regulate fiber development, such as GAE6, C4H, OMT1, AFR18, EIN3, bZIP44, and GAI. Notably, the marker D13_56413025 in qFS-chr18-4 provides a potential basis for enhancing fiber quality of upland cotton via marker-assisted breeding and gene cloning of important fiber quality traits.

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.

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.

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.

Enrichment of phospholipids using magnetic Fe3O4/TiO2 nanoparticles for quantitative detection at single cell levels by electrospray ionization mass spectrometry.

Quantitative detection of phospholipids at the single cell level remains in challenge. Herein, the TiO2-coated Fe3O4 nanoparticles were synthesized to selectively enrich trace phospholipids from single cell, which were then eluted using 1.5% ammonia/methanol (w/w) for sensitive detection by electrospray ionization mass spectrometry. Under the optimal experimental conditions, eighteen phospholipids in single cell samples were detected and identified by MS/MS experiments. The limit-of-detections (LODs) were 0.012 µg/L for phosphatidylcholine (PC, 34:1) and 0.014 µg/L for phosphatidylcholine (PC, 36:2) in PBS matrix, with the linear range of 0.05-50 µg/L (R2 ≥ 0.999). The recovery rates of 94.90-104.00% were obtained, with the relative standard deviations (RSDs ≤ 6.90%). Quantitative determination of PC in real unicellular samples was also achieved, with the concentration of 1.82-2.11 µg/L for PC(34:1) and 1.25-1.65 µg/L for PC(36:2) in six types of single cell, opening up possibilities for quantitative analysis of trace compounds in complex bio-samples. A set of 6 types of tumor cells were analyzed and further differentiated by the partial least squares-discriminant analysis (PLS-DA). Conclusively, a facile method for the direct quantification of phospholipids in single cell samples has been developed, showing potential applications for advanced investigation of phosphorylated substance at the single cell level.