Background: Cotton, with a large genome, is an important crop throughout the world. A high-density genetic linkage map is the prerequisite for cotton genetics and breeding. A genetic map based on simple polymerase chain reaction markers will be efficient for marker-assisted breeding in cotton, and markers from transcribed sequences have more chance to target genes related to traits. To construct a genome-wide, functional marker-based genetic linkage map in cotton, we isolated and mapped expressed sequence tag-simple sequence repeats (EST-SSRs) from cotton ESTs derived from the A₁, D₅, (AD)₁, and (AD)₂ genome. Results: A total of 3177 new EST-SSRs developed in our laboratory and other newly released SSRs were used to enrich our interspecific BC₁ genetic linkage map. A total of 547 loci and 911 loci were obtained from our EST-SSRs and the newly released SSRs, respectively. The 1458 loci together with our previously published data were used to construct an updated genetic linkage map. The final map included 2316 loci on the 26 cotton chromosomes, 4418.9 cM in total length and 1.91 cM in average distance between adjacent markers. To our knowledge, this map is one of the three most dense linkage maps in cotton. Twenty-one segregation distortion regions (SDRs) were found in this map; three segregation distorted chromosomes, Chr02, Chr16, and Chr18, were identified with 99.9% of distorted markers segregating toward the heterozygous allele. Functional analysis of SSR sequences showed that 1633 loci of this map (70.6%) were transcribed loci and 1332 loci (57.5%) were translated loci. Conclusions: This map lays groundwork for further genetic analyses of important quantitative traits, marker-assisted selection, and genome organization architecture in cotton as well as for comparative genomics between cotton and other species. The segregation distorted chromosomes can be a guide to identify segregation distortion loci in cotton. The annotation of SSR sequences identified frequent and rare gene ontology items on each chromosome, which is helpful to discover functions of cotton chromosomes.

Background: The low genetic diversity of Upland cotton limits the potential for genetic improvement. Making full use of the genetic resources of Sea-island cotton will facilitate genetic improvement of widely cultivated Upland cotton varieties. The chromosome segments substitution lines (CSSLs) provide an ideal strategy for mapping quantitative trait loci (QTL) in interspecific hybridization. Results: In this study, a CSSL population was developed by PCR-based markers assisted selection (MAS), derived from the crossing and backcrossing of Gossypium hirsutum (Gh) and G. barbadense (Gb), firstly. Then, by whole genome re-sequencing, 11,653,661 high-quality single nucleotide polymorphisms (SNPs) were identified which ultimately constructed 1211 recombination chromosome introgression segments from Gb. The sequencing-based physical map provided more accurate introgressions than the PCR-based markers. By exploiting CSSLs with mutant morphological traits, the genes responding for leaf shape and fuzz-less mutation in the Gb were identified. Based on a high-resolution recombination bin map to uncover genetic loci determining the phenotypic variance between Gh and Gb, 64 QTLs were identified for 14 agronomic traits with an interval length of 158 kb to 27 Mb. Surprisingly, multiple alleles of Gb showed extremely high value in enhancing cottonseed oil content (SOC). Conclusions: This study provides guidance for studying interspecific inheritance, especially breeding researchers, for future studies using the traditional PCR-based molecular markers and high-throughput re-sequencing technology in the study of CSSLs. Available resources include candidate position for controlling cotton quality and quantitative traits, and excellent breeding materials. Collectively, our results provide insights into the genetic effects of Gb alleles on the Gh, and provide guidance for the utilization of Gb alleles in interspecific breeding.

Availability of molecular markers has proven to be an efficient tool in facilitating progress in plant breeding, which is particularly important in the case of less researched crops such as cotton. Considering the obvious advantages of single nucleotide polymorphisms (SNPs) and insertion-deletion polymorphisms (InDels), expressed sequence tags (ESTs) were analyzed in silico to identify SNPs and InDels in this study, aiming to develop more molecular markers in cotton. A total of 1,349 EST-based SNP and InDel markers were developed by comparing ESTs between Gossypium hirsutum and G. barbadense, mining G. hirsutum unigenes, and analyzing 3′ untranslated region (3′UTR) sequences. The marker polymorphisms were investigated using the two parents of the mapping population based on the single-strand conformation polymorphism (SSCP) analysis. Of all the markers, 137 (10.16%) were polymorphic, and revealed 142 loci. Linkage analysis using a BC1 population mapped 133 loci on the 26 chromosomes. Statistical analysis of base variations in SNPs showed that base transitions accounted for 55.78% of the total base variations and gene ontology indicated that cotton genes varied greatly in harboring SNPs ranging from 1.00 to 24.00 SNPs per gene. Sanger sequencing of three randomly selected SNP markers revealed discrepancy between the in silico predicted sequences and the actual sequencing results. In silico analysis is a double-edged blade to develop EST-SNP/InDel markers. On the one hand, the designed markers can be well used in tetraploid cotton genetic mapping. And it plays a certain role in revealing transition preference and SNP frequency of cotton genes. On the other hand, the developmental efficiency of markers and polymorphism of designed primers are comparatively low.

Fiber strength is a critical determinant of fiber quality, with stronger fibers being highly preferred in the cotton textile industry. However, the genetic basis and the specific regulatory mechanism underlying the formation of cotton fiber strength remain largely unknown. To explore fiber strength-related genes, QTL mapping, map-based cloning, and gene function verification were conducted in a backcross inbred line BS41 derived from interspecific hybridization between upland cotton and sea-island cotton. Upland cotton Emian22 (E22) and an interspecific backcross inbred line (BIL) BS41 were used as parents to construct secondary segregation populations for BSA and QTL mapping of fiber strength. The candidate gene GbNTL9 was identified through map-based cloning and expression analysis. The function of NTL9 was determined through transgenic experiments and cytological observations. The regulatory mechanisms of NTL9 were explored using RNA-seq, RT-qPCR, yeast two-hybrid, bimolecular fluorescence complementation, and yeast one-hybrid. A major QTL for fiber strength, qFS-A11-1, was mapped to a 14.6-kb genomic region using segregating populations from E22 × BS41. GbNTL9, which encodes a NAC transcription factor, was identified as the candidate gene. Overexpression of both upland cotton genotype NTL9^E22 and sea-island genotype NTL9^BS41 in upland cotton enhanced fiber strength by facilitating the dense accumulation and orderly organization of cellulose microfibrils within the cell wall. Transcriptomic analysis revealed that NTL9 inhibited the expression of genes involved in secondary wall synthesis, such as CESA4, CESA7, and CESA8, thereby delaying cell wall cellulose deposition and altering the microfibril deposition pattern. NTL9 interacted with MYB6 and functioned as a downstream gene in the ethylene signaling pathway. Additionally, an effective gene marker NTL9-24 was developed to distinguish haplotypes from G. barbadense and G. hirsutum for fiber quality breeding program. Our findings demonstrate that GbNTL9 positively regulates fiber strength through altering the microfibril deposition pattern, and provide a new insight into the molecular mechanism underlying fiber strength.

Root architectural traits at the seedling stage have been demonstrated to be crucial for the efficient uptake of nutrients and drought tolerance in wheat. To dissect the genetic basis of these traits from the D genome, 182 recombinant inbred lines (RILs) derived from the common wheat TAA10 crossed with resynthesized allohexaploid wheat XX329 possessed similar AABB genomes were used for QTL mapping of five root traits in hydroponic-cultured seedlings, including lateral root number (LRN), seminal root number (SRN), root hair length (RHL), root diameter (RD), and total root volume (TRV). A total of seven QTLs were identified for the five root traits, with six possible novel QTLs for LRN, RHL, RD and TRV, accounting for 4.98–12.17% of phenotypic variation. One QTL (QLrn.qau-5D.2), controlling lateral root number, was fine mapped an approximate 5.0-Mb interval harboring 80 annotated genes, including five auxin-related genes. We further validated that QLrn.qau-5D.2 in NIL^TAA10 significantly enhanced yield-related traits, such as plant height, spike length, spike compactness, tiller number per plant and grain yield per plant, as comparison with NIL^XX329. Collectively, these results provide vital insights for fine-mapping QTLs associated with LRN, SRN, RHL, RD and TRV and facilitate the root morphologic designs for enhancing yield performance.

Drought stress is a devastating natural stress that threatens crop productivity and quality. Mitigating the adverse effects of drought stress on wheat is a key object in agriculture. C-repeat binding transcription factor/DROUGHT RESPONSE ELEMENT BINDING FACTOR 1 (CBF/DREB1) transcription factors are well known for their role in cold acclimation. However, the involvement of CBF genes in drought stress and the mechanisms underlying their function remain poorly understood. In this study, 81 CBFs were identified in wheat, which were further clustered into four distinct lineages based on phylogenetic analysis. Chromosomal localization indicated that most CBF genes were dispersed across chromosome 5. We identified three homoeologous genes (TaCBF14A, TaCBF14B, and TaCBF14D) that were simultaneously upregulated under drought stress based on RNA-seq analysis. According to the high expression after drought stress, TaCBF14B was selected for further functional analysis. Subcellular localization and transcriptional activation activity analysis indicated that TaCBF14B likely functions as a transcription factor involved in drought stress tolerance. Overexpression of TaCBF14B in Arabidopsis enhanced the primary root growth by 13.49% (OE1), 12.56% (OE2), and 19.53% (OE3) under 200 mM mannitol treatment, and 21.65% (OE1), 16.63% (OE2), and 28.13% (OE3) under 250 mM mannitol treatment compared to WT. Meanwhile, the water loss rate of transgenic lines was 56% in WT leaves, but only 44%, 50%, and 40% in OE1, OE2, and OE3 lines, respectively. Compared to the wild type, POD activities of OE1, OE2, and OE3 were significantly increased by 42.94%, 29.41%, and 62.52%, respectively. And the Pro activities in OE1, OE2, and OE3 were significantly increased by 16.33%, 5.18%, and 29.09%, respectively, compared to the wild type. Additionally, the MDA content in OE1, OE2, and OE3 was significantly reduced by 40.53%, 15.81%, and 54.36%, respectively. Further analysis showed that the transgenic lines were hypersensitive to abscisic acid (ABA), and exhibited increased expression of AtABI3. We speculate that TaCBF14B plays an important role in enhancing drought tolerance. In summary, our findings provide new insights into the functional roles of CBF genes in drought stress tolerance.