Alternative splicing (AS) represents a pivotal post-transcriptional regulatory mechanism, profoundly expanding proteomic diversity and functional complexity by enabling single genes to generate multiple mRNA isoforms. In plants, AS serves as a survival toolkit, dynamically modulating stress-responsive signaling pathways, transcriptional networks, and protein functional specialization to optimize environmental fitness. Recent advances in high-throughput sequencing technologies and computational tools have significantly deepened our understanding of AS regulation in plants. Notably, breakthroughs such as long-read transcriptome sequencing and single-cell RNA analysis have revolutionized the resolution at which we can characterize AS landscapes. These developments have collectively illuminated the critical role of AS in mediating plant responses to diverse abiotic stresses, including drought, salinity, and extreme temperatures. The resulting discoveries have opened transformative avenues for crop improvement through precise manipulation of splicing patterns. Innovative strategies such as CRISPR-Cas9-based splice editing and engineered splicing factors now provide powerful platforms for developing climate-resilient, high-yielding crop varieties with enhanced stress tolerance and nutritional quality. Here, we systematically examine the molecular mechanisms underlying AS-mediated plant stress responses, and cutting-edge applications of AS engineering in precision agriculture. By synthesizing fundamental insights with biotechnological innovations, we highlight the transformative potential of AS manipulation in addressing the pressing global agricultural challenges.

Flax (Linum usitatissimum) is a valuable oilseed and cash crop with multiple applications as food, animal feed, and medicine, in addition to its industrial applications. Thousand-seed weight (TSW) is one of the most critical components of seed yield in flax, and breeding efforts have been directed toward obtaining flax varieties with high TSW. However, the genes associated with TSW in flax remain understudied. This study investigated the major locus and genes linked to TSW in flax through a comprehensive analysis, encompassing bulked segregant analysis, fine mapping, qRT-PCR, and functional annotations. Herein, we performed a genetic analysis of the F2 and F2:3 populations from our previous study, which indicated that TSW is controlled by multiple genes. Bulked segregant analysis revealed a major quantitative trait locus linked to TSW, namely TSW3.1, which is located on Chromosome 3. Subsequently, TSW3.1 was fine-mapped to a 23.38-kb interval comprising 5 genes through the linkage mapping analysis. Four of the five genes exhibited variations in either their coding regions or their upstream/downstream regulatory regions. Based on the qRT-PCR analysis and gene annotation, all five genes were identified as the candidates for TSW. This study offers fresh insights into the genetic mechanisms of TSW regulation in flax, and the findings may serve as reference for breeding new varieties with high TSW.

The increasing frequency and intensity of heatwaves due to climate change pose significant challenges to sturgeon aquaculture. This study investigated the effects of gradual heat stress (1 °C every 8 h) on two reciprocal hybrid sturgeon strains (Acipenser baerii ♀ × A. schrenckii ♂, (BS hybrid); A. schrenckii ♀ × A. baerii ♂, (SB hybrid)), focusing on their antioxidant defense mechanisms, heat shock protein (HSP) expression, and liver and gill tissue histology. When water temperature raised to 34.3 °C (about 104 h), LOE (loss of equilibrium) individuals appeared. Twenty-four hours after sampling, fifteen BS hybrid sturgeon remained alive, whereas no SB hybrid sturgeon survived. In this study, the slow heat stress significantly elevated the expression of HSP-related genes (hsc70, hsp70, hsp90) in both the liver of BS hybrid sturgeon and the gills of SB hybrid sturgeon. However, in the gills of BS hybrid sturgeon and the liver of SB hybrid sturgeon, the expression of hsp family genes in the experimental groups was either lower than or comparable to the control group. Significant liver damage, including cellular vacuolization and necrosis, was observed in BS hybrids, while SB hybrid sturgeon exhibited more pronounced gill tissue damage. Among the four antioxidant enzymes-superoxide dismutase (SOD), lactate dehydrogenase (LDH), catalase (CAT) glutathione peroxidase (GPx)-only LDH activity was elevated in the hepatic tissue of BS hybrid sturgeon, corresponding to increased serum lactate levels, while gill LDH activity was higher in SB hybrid sturgeon. In both hybrids, LDH activity exhibited an increasing trend in the kidney. However, total antioxidant capacity (T-AOC) remained unchanged across all three tissues. Both plasma cortisol and lactate were substantially affected by thermal stress. MDA remained at a relatively stable level after heat stress and recovery. These results demonstrate differential tissue-specific responses to heat stress in the reciprocal hybrids. More importantly, the BS hybrid sturgeon exhibited significantly higher thermal tolerance and post-stress survival compared to the SB hybrid sturgeon. These findings reveal that the choice of maternal parent is a critical factor influencing heat resistance in these hybrids, providing a key basis for selective breeding programs and optimizing aquaculture management.

In resource-constrained industrial scene, the application of neural networks is a challenge due to the requirement for powerful high-performance computing devices to handle large amounts of floating-point data. The paper proposes a quantified stochastic configuration network model called Stochastic Configuration Networks with Incremental Encoding (SCN-IE), aiming to improve the operating efficiency of the model. To quantize the model, a novel feature encoding is developed to convert the input data into bit vectors. The characteristic of this model is that its hidden layer inputs and weights are represented in the form of bit vectors. We use basic bit logic operations to effectively calculate the output of the hidden layer, achieving lightweight computation. In addition, the stochastic configuration algorithm is used to solve the approximation problem of the model. The results demonstrate that SCN-IE exhibits powerful real-time reasoning capabilities compared to SCN and IRVFLN, and it holds great potential for application on resource-constrained devices.

The global yield of cottonseed could meet the annual protein requirements of approximately half a billion people if gossypol were absent from the seeds. Here, we characterize the molecular mechanism by which the Gl2e mutation exerts a dominant-negative effect on gland development, providing a mechanistic basis for engineering seed-specific gossypol-free (SSGF) cotton. We show that Gl2/Gl3 form multimers-likely tetramers-that function as master regulators within the transcriptional network controlling gossypol gland development. Further analyses demonstrate that Gl2e, a dominant mutant allele of Gl2, induces a glandless phenotype through its dominant-negative effect. In addition, multimers composed of Gl2e and Gl2/Gl3 retain E-box binding activity but lack transcriptional activation capacity, thereby inhibiting gland organogenesis. Guided by these insights, we engineered SSGF cotton by driving Gl2e expression specifically during seed development, effectively suppressing gossypol gland formation in seeds. Multi-year, multi-location field trials of the SSGF cotton confirmed the stable production of gossypol-free seeds without compromising fiber yield or other key agronomic traits. Notably, completely gossypol-free oil and flour can be produced directly from SSGF seeds without the need for degossypolization. This work establishes a mechanistic foundation for understanding gland development and offers a sustainable path toward enhancing global plant-derived protein and oil resources.

In our study, we identified the gene Gohir.A08G240900 as a potential target for regulating cotton plant height, providing a genetic basis for enhancing cotton morphology. Arabinogalactan peptides are a class of hydroxyproline-rich proteins widely distributed in plants that participate in many life processes, including growth and development, cell division and even plant reproductive development. In this study, we identified 122 members of the AGP gene family via genome-wide identification in six cotton species. Through phylogenetic tree analysis, the AGP family was divided into six different subgroups. A core yet variable region composed of proline, hydroxyproline, serine, threonine, and alanine (PAST) was identified among these members. Furthermore, Ka/Ks analysis revealed that the AGP gene family underwent multiple fragment duplication events. Additionally, we analyzed the 1.5 kb upstream cis-acting elements of all upland cotton family members and identified numerous functional elements associated with growth and development, suggesting a close relationship among the family members. The results of RT‒qPCR analysis revealed that the expression level of Gohir.A08G240900 was significantly different among the four upland cotton varieties, with significant differences in plant height. Virus-induced gene silencing (VIGS) experiments revealed that the height of Gohir.A08G240900 gene-silenced plants significantly decreased. The results revealed that Gohir.A08G240900 may affect plant growth and development and may be a potential functional gene regulating cotton plant height.

Abstract Background Aldehyde dehydrogenases (ALDHs) are a family of enzymes that catalyze the oxidation of aldehyde molecules into the corresponding carboxylic acid, regulate the balance of aldehydes and protect plants from the poisoning caused by excessive accumulation of aldehydes; however, this gene family has rarely been studied in cotton. Results In the present study, genome-wide identification was performed, and a total of 114 ALDH family members were found in three cotton species, Gossypium hirsutum, Gossypium arboreum and Gossypium raimondii. The ALDH genes were divided into six subgroups by evolutionary analysis. ALDH genes in the same subgroup showed similar gene structures and conserved motifs, but some genes showed significant differences, which may result in functional differences. Chromosomal location analysis and selective pressure analysis revealed that the ALDH gene family had experienced many fragment duplication events. Cis-acting element analysis revealed that this gene family may be involved in the response to various biotic and abiotic stresses. The RT‒qPCR results showed that the expression levels of some members of this gene family were significantly increased under salt stress conditions. Gohir.A11G040800 and Gohir.D06G046200 were subjected to virus-induced gene silencing (VIGS) experiments, and the sensitivity of the silenced plants to salt stress was significantly greater than that of the negative control plants, suggesting that Gohir.A11G040800 and Gohir.D06G046200 may be involved in the response of cotton to salt stress. Conclusions In total, 114 ALDH genes were identified in three Gossypium species by a series of bioinformatics analysis. Gene silencing of the ALDH genes of G. hirsutum revealed that ALDH plays an important role in the response of cotton to salt stress.

Fusarium head blight (FHB) threatens wheat production worldwide. Utilization of FHB resistant varieties is the most effective solution for disease control. Owing to the limited sources of FHB resistance, mining of novel resistance genes is crucial. Here, we report an FHB resistance gene from a wild wheat relative species, Roegneria ciliaris and developed FHB resistant germplasm containing this gene. Wheat-R. ciliaris disomic addition line DA3Sc showed enhanced type II FHB resistance compared to its sister line 3Sc-Null without chromosome 3Sc, indicating that the resistance was contributed by the addition of 3Sc. The resistance gene on 3Sc was validated using F2 and F2:3 populations derived from the cross between DA3Sc and susceptible Aikang 58 (a susceptible cultivar), demonstrating that the lines with 3Sc had significantly enhanced FHB resistance compared to the individuals without 3Sc. This was the second resistance gene identified in R. ciliaris, designated FhbRc2. To transfer FhbRc2 to common wheat, we produced a double-monosomic chromosome population by crossing DA3Sc with the Chinese Spring nulli-tetrasomic line N3DT3B. Eight alien chromosome lines containing 3Sc were identified using genomic/fluorescence in situ hybridization and 3Sc-specific marker analysis. Only the lines carrying the long arm of 3Sc conferred FHB resistance, further locating FhbRc2 on 3ScL. A compensating wheat-R. ciliaris Robertsonian translocation line T3DS·3ScL harboring FhbRc2 is developed and provides a potential genetic resource in wheat breeding for enhanced FHB resistance.

BACKGROUND:As the world's leading fiber crop and a major oil-producing crop, cotton fiber yield and fiber quality are affected by environmental stresses, especially heat, drought and salinity. The LAZ1 (Lazarus 1) family genes are responsive to abscisic acid, drought, and salt treatments. Currently, mining and functional analyses of LAZ1 family genes in cotton have not been reported.METHODS AND RESULTS:In this study, 20 GhLAZ1 genes, designated GhLAZ1-1 - GhLAZ1-20, were identified in the genome of Gossypium hirsutum through the construction of an HMM model, and their molecular properties, chromosomal localization, phylogeny, gene structure, evolutionary selection pressure, promoter cis elements and gene expression under salt stress were analyzed. With the exception of GhLAZ1-17 and GhLAZ1-20, the remaining 18 GhLAZ1 genes were unevenly localized on 13 chromosomes in G. hirsutum; evolutionary analysis showed that these genes could be divided into three subfamilies; and evolutionary selection pressure analysis demonstrated that the GhLAZ1 genes were all under purifying selection. Many elements related to light responses, hormone responses, and abiotic stresses were predicted on the GhLAZ1 family gene promoters, and real-time quantitative PCR results showed that GhLAZ1-2, GhLAZ1-8, and GhLAZ1-18 were upregulated significantly in salt-treated cotton leaves.CONCLUSIONS:Our results suggested that GhLAZ1 genes were involved in the salt tolerance mechanism in G. hirsutum and provided a reference for further exploring the function and molecular mechanism of LAZ1 genes.

To investigate the genetic mechanisms underlying the reproductive traits (time to flowering and maturity) in wheat and identify candidate genes associated, a phenotypic analysis was conducted on 239 wheat accessions (lines) from around the world. A genome-wide association study (GWAS) of wheat heading and maturity phases was performed using the MLM (Q+K) model in the TASSLE software, combined with the Wheat 55K SNP array. The results revealed significant phenotypic variation in heading and maturity among the wheat accessions across different years, with coefficients of variation ranging from 0.96% to 1.97%. The phenotypic data from different years exhibited excellent correlation, with a genome-wide linkage disequilibrium (LD) attenuation distance of 3 Mb. Population structure analysis, evolutionary tree analysis, and principal component analysis indicated that the 239 wheat accessions formed a relatively homogeneous natural population, which could be divided into three subgroups. The GWAS results identified a total of 293 SNP marker loci that were significantly associated with wheat heading and maturity stages (P ≤ 0.001) in different environments. Among them, nine stable SNP marker loci were consistently detected in multiple environments. These marker loci were distributed on wheat chromosomes 1A、1B、2D、3A、5B、6D and 7A. Each individual locus explained 4.03%-16.06% of the phenotypic variation. Furthermore, through careful analysis of the associated loci with large phenotypic effect values and stable inheritance, a total of nine candidate genes related to wheat heading and maturity stages were identified. These findings have implications for molecular marker-assisted selection breeding programs targeting specific wheat traits at the heading and maturity stages. In summary, this study conducted a comprehensive GWAS of wheat heading and maturity phases, revealing significant associations between genetic markers and key developmental stages in wheat. The identification of candidate genes and marker loci provides valuable information for further studies on wheat breeding and genetic improvement targeted at enhancing heading and maturity traits.

BACKGROUND:Drought stress has significantly hampered agricultural productivity worldwide and can also result in modifications to DNA methylation levels. However, the dynamics of DNA methylation and its association with the changes in gene transcription and alternative splicing (AS) under drought stress are unknown in linseed, which is frequently cultivated in arid and semiarid regions.RESULTS:We analysed AS events and DNA methylation patterns in drought-tolerant (Z141) and drought-sensitive (NY-17) linseed under drought stress (DS) and repeated drought stress (RD) treatments. We found that the number of intron-retention (IR) and alternative 3' splice site (Alt3'SS) events were significantly higher in Z141 and NY-17 under drought stress. We found that the linseed response to the DS treatment was mainly regulated by transcription, while the response to the RD treatment was coregulated by transcription and AS. Whole genome-wide DNA methylation analysis revealed that drought stress caused an increase in the overall methylation level of linseed. Although we did not observe any correlation between differentially methylated genes (DMGs) and differentially spliced genes (DSGs) in this study, we found that the DSGs whose gene body region was hypermethylated in Z141 and hypomethylated in NY-17 were enriched in abiotic stress response Gene Ontology (GO) terms. This finding implies that gene body methylation plays an important role in AS regulation in some specific genes.CONCLUSION:Our study is the first comprehensive genome-wide analysis of the relationship between linseed methylation changes and AS under drought and repeated drought stress. Our study revealed different interaction patterns between differentially expressed genes (DEGs) and DSGs under DS and RD treatments and differences between methylation and AS regulation in drought-tolerant and drought-sensitive linseed varieties. The findings will probably be of interest in the future. Our results provide interesting insights into the association between gene expression, AS, and DNA methylation in linseed under drought stress. Differences in these associations may account for the differences in linseed drought tolerance.

Cotton is not only the most important fiber crop but also the fifth most important oilseed crop in the world because of its oil-rich seeds as a byproduct of fiber production. By comparative transcriptome analysis between two germplasms with diverse oil accumulation, we reveal pieces of the gene expression network involved in the process of oil synthesis in cottonseeds. Approximately, 197.16 Gb of raw data from 30 RNA sequencing samples with 3 biological replicates were generated. Comparison of the high-oil and low-oil transcriptomes enabled the identification of 7682 differentially expressed genes (DEGs). Based on gene expression profiles relevant to triacylglycerol (TAG) biosynthesis, we proposed that the Kennedy pathway (diacylglycerol acyltransferase-catalyzed diacylglycerol to TAG) is the main pathway for oil production, rather than the phospholipid diacylglycerol acyltransferase-mediated pathway. Using weighted gene co-expression network analysis, 5312 DEGs were obtained and classified into 14 co-expression modules, including the MEblack module containing 10 genes involved in lipid metabolism. Among the DEGs in the MEblack module, GhCYSD1 was identified as a potential key player in oil biosynthesis. The overexpression of GhCYSD1 in yeast resulted in increased oil content and altered fatty acid composition. This study may not only shed more light on the underlying molecular mechanism of oil accumulation in cottonseed oil, but also provide a set of new gene for potential enhancement of oil content in cottonseeds.

N6-methyladenosine (m6A) is a ubiquitous reversible epigenetic RNA modification which plays a significant role in regulation of gene expression and cell functions. However, m6A abundance, dynamics and topology remain unexplored in cotton. Here, we performed transcriptome-wide m6A profiling and comparative analysis of cotton root tissues under normal and salt stress treatments. Totally, 3360 and 3771 high confidence m6A peaks, involved in 3219 and 3566 expressed genes under normal and salt stress, were identified, respectively. Of them, 1311 mRNA transcripts from salt treatment and 964 in control were specifically detected m6A peaks. Functional analysis indicated that the 1311 methylated genes specifically from salt stress were mainly involved in zeatin biosynthesis, taurine and hypotaurine metabolism, ABC transporters, and anthocyanin biosynthesis, while 964 in control were involved in ribosome and proteasome processes. Furthermore, mRNA m6A sites were enriched around the stop codons and coding regions, and m6A methylation exhibited dynamic changes after salt treatment, with 434 m6A peaks in 430 genes up-regulated and 290 m6A peaks in 287 genes downregulated. Combined with RNA-seq analysis, we found that the m6A deposition specifically in normal or salt treatment is positively correlated with their corresponding gene transcripts abundance. We confirmed that cotton YTH (YT512-B homology) domain gene GhECT6 (evolutionarily conserved C-terminal region 6), a deduced m6A reader, played an important role against salt stress. GhECT6 was significantly induced expression after salt treatment, and silencing GhECT6 caused a decrease in salt tolerance. This first high-throughput epitranscriptomic data provide new insights into understanding the critical mRNA modification in response to salt stress in cotton.

Background: Oil flax (linseed, Linum usitatissimum L.) is one of the most important oil crops., However, the increases in drought resulting from climate change have dramatically reduces linseed yield and quality, but very little is known about how linseed coordinates the expression of drought resistance gene in response to different level of drought stress (DS) on the genome-wide level.Results: To explore the linseed transcriptional response of DS and repeated drought (RD) stress, we determined the drought tolerance of different linseed varieties. Then we performed full-length transcriptome sequencing of drought-resistant variety (Z141) and drought-sensitive variety (NY-17) under DS and RD stress at the seedling stage using single-molecule real-time sequencing and RNA-sequencing. Gene Ontology (GO) and reduce and visualize GO (REVIGO) enrichment analysis showed that upregulated genes of Z141 were enriched in more functional pathways related to plant drought tolerance than those of NY-17 were under DS. In addition, 4436 linseed transcription factors were identified, and 1190 were responsive to stress treatments. Moreover, protein-protein interaction (PPI) network analysis showed that the proline biosynthesis pathway interacts with stress response genes through RAD50 (DNA repair protein 50) interacting protein 1 (RIN-1). Finally, proline biosynthesis and DNA repair structural gene expression patterns were verified by RT- PCR.Conclusions: The drought tolerance of Z141 may be related to its upregulation of drought tolerance genes under DS. Proline may play an important role in linseed drought tolerance by maintaining cell osmotic and protecting DNA from ROS damage. In summary, this study provides a new perspective to understand the drought adaptability of linseed.

BACKGROUND:Numerous quantitative trait loci (QTLs) and candidate genes associated with yield-related traits have been identified in cotton by genome-wide association study (GWAS) analysis. However, most of the phenotypic data were from a single or few environments, and the stable loci remained to be validated under multiple field environments.RESULTS:Here, 242 upland cotton accessions collected from different origins were continuously investigated for phenotypic data of four main yield components, including boll weight (BW) and lint percentage (LP) under 13 field environments, and boll number per plant (BN) and seed index (SI) under 11 environments. Correlation analysis revealed a positive correlation between BN and LP, BW and SI, while SI had a negative correlation with LP and BN. Genetic analysis indicated that LP had the highest heritability estimates of 94.97%, followed by 92.08% for SI, 86.09% for BW, and 72.92% for BN, indicating LP and SI were more suitable traits for genetic improvement. Based on 56,010 high-quality single nucleotide polymorphisms (SNPs) and GWAS analysis, a total of 95 non-redundant QTLs were identified, including 12 of BN, 23 of BW, 45 of LP, and 33 of SI, respectively. Of them, 10 pairs of homologous QTLs were detected between A and D sub-genomes. We also found that 15 co-located QTLs with more than two traits and 12 high-confidence QTLs were detected under more than six environments, respectively. Further, two NET genes (GH_A08G0716 and GH_A08G0783), located in a novel QTL hotspot (qtl24, qtl25 and qlt26) were predominately expressed in early fiber development stages, exhibited significant correlation with LP and SI. The GH_A07G1389 in the stable qtl19 region encoded a tetratricopeptide repeat (TPR)-like superfamily protein and was a homologous gene involved in short fiber mutant ligon lintless-y (Liy), implying important roles in cotton yield.CONCLUSIONS:The present study provides a foundation for understanding the regulatory mechanisms of yield components and may enhance yield improvement through molecular breeding in cotton.

Background Virescent mutation broadly exists in plants and is an ideal experimental material to investigate regulatory mechanisms underlying chlorophyll synthesis, photosynthesis and plant growth. Up to date, the molecular mechanisms in two virescent mutations have been clarified in cottons ( Gossypiuma hirsutum ). A virescent mutation has been found in the cotton strain Sumian 22, and the underlying molecular mechanisms have been studied. Methods The virescent mutant and wild type (WT) of Sumian 22 were cross-bred, and the F 1 population were self-pollinated to calculate the segregation ratio. Green and yellow leaves from F 2 populations were subjected to genome sequencing and bulked-segregant analysis was performed to screen mutations. Real-time quantitative PCR (RT-qPCR) were performed to identify genes in relations to chlorophyll synthesis. Intermediate products for chlorophyll synthesis were determined to validate the RT-qPCR results. Results The segregation ratio of green and virescent plants in F2 population complied with 3:1. Compared with WT, a 0.34 Mb highly mutated interval was identified on the chromosome D10 in mutant, which contained 31 genes. Among them, only ABCI1 displayed significantly lower levels in mutant than in WT. Meanwhile, the contents of Mg-protoporphyrin IX, protochlorophyllide, chlorophyll a and b were all significantly lower in mutant than in WT, which were consistent with the inhibited levels of ABCI1 . In addition, a mutation from A to T at the -317 bp position from the start codon of ABCI1 was observed in the genome sequence of mutant. Conclusions Inhibited transcription of ABCI1 might be the mechanism causing virescent mutation in Sumian 22 cotton, which reduced the transportation of protoporphyrin IX to plastid, and then inhibited Mg-protoporphyrin IX, Protochlorophyllide and finally chlorophyll synthesis. These results provided novel insights into the molecular mechanisms underlying virescent mutation in cotton.

Cotton (Gossypium L.) is the most important fiber crop worldwide. Here, transcriptome analysis was conducted on developing fibers of a G. mustelinum introgression line, IL9, and its recurrent parent, PD94042, at 17 and 21 days post-anthesis (dpa). Differentially expressed genes (DEGs) of PD94042 and IL9 were identified. Gene Ontology (GO) enrichment analysis showed that the annotated DEGs were rich in two main biological processes and two main molecular functions. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis likewise showed that the annotated DEGs were mainly enriched in metabolic pathways and biosynthesis of secondary metabolites. In total, 52 DEGs were selected as candidate genes based on comparison of the DEGs and GO function annotation information. Quantitative real-time PCR (RT-qPCR) analysis results for 12 randomly selected DEGs were consistent with transcriptome analysis. SNP identification based on G. mustelinum chromatin segment introgression showed that 394 SNPs were identified in 268 DEGs, and two genes with known functions were identified within fiber strength quantitative trait loci (QTL) regions or near the confidence intervals. We identified 52 key genes potentially related to high fiber strength in a G. mustelinum introgression line and provided significant insights into the study of cotton fiber quality improvement.

Gene introgression from wild species has been shown to be a feasible approach for fiber quality improvement in Upland cotton. Previously, we developed an interspecific G. mustelinum × G. hirsutum advanced-backcross population and mapped over one hundred QTL for fiber quality traits. In the current study, a trait-based selective genotyping approach was utilized to prioritize a small subset of introgression lines with high phenotypic values for different fiber quality traits, to simultaneously validate multiple fiber quality QTL in a single experiment. A total of 75 QTL were detected by CIM and/or single-marker analysis, including 11 significant marker-trait associations (P < 0.001) and three putative associations (P < 0.005) also reported in earlier studies. The QTL that have been validated include three each for fiber length, micronaire, and elongation, and one each for fiber strength and uniformity. Collectively, about 10% of the QTL previously reported have been validated here, indicating that selective genotyping has the potential to validate multiple marker-trait associations for different traits, especially those with a moderate to large-effect detected simultaneously in one experimental population. The G. mustelinum alleles contributed to improved fiber quality for all validated loci. The results from this study will lay the foundation for further fine mapping, marker-assisted selection and map-based gene cloning.