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Segregation distortion (SD) is a genetic mechanism commonly found in segregating or stable populations. The principle behind this puzzles many researchers. The F2 generation developed from wild Gossypium darwinii and G. hirsutum CCRI12 species was used to investigate the possible transcription factors within the segregation distortion regions (SDRs). The 384 out of 2763 markers were distorted in 29 SDRs on 18 chromosomes. Good collinearity was observed among genetic and physical maps of G. hirsutum and G. barbadense syntenic blocks. Total 568 genes were identified from SDRs of 18 chromosomes. Out of these genes, 128 belonged to three top-ranked salt-tolerant gene families. The DUF597 contained 8 uncharacterized genes linked to Pkinase (PF00069) gene family in the phylogenetic tree, while 15 uncharacterized genes clustered with the zinc finger gene family. Two hundred thirty four miRNAs targeted numerous genes, including ghr-miR156, ghr-miR399 and ghr-miR482, while others targeted top-ranked stress-responsive transcription factors. Moreover, these genes were involved in the regulation of numerous stress-responsive cis-regulatory elements. The RNA sequence data of fifteen upregulated genes were verified through the RT-qPCR. The expression profiles of two highly upregulated genes (Gh_D01G2015 and Gh_A01G1773) in salt-tolerant G. darwinii showed antagonistic expression in G. hirsutum. The results indicated that salt-tolerant genes have been possibly transferred from the wild G. darwinii species. A detailed functional analysis of these genes can be carried out which might be helpful in the future for gene cloning, transformation, gene editing and the development of salt-resistant cotton varieties.

Wild cotton species have significant agronomic traits that can be introgressed into elite cultivated varieties. The use of a genetic map is important in exploring, identification and mining genes which carry significant traits. In this study, 188 F2mapping individuals were developed from Gossypium thurberi (female) and Gossypium trilobum (male), and were genotyped by using simple sequence repeat (SSR) markers. A total of 12,560 simple sequence repeat (SSR) markers, developed by Southwest University, thus coded SWU were screened out of which only 994 were found to be polymorphic, and 849 markers were linked in all the 13 chromosomes. The map had a length of 1,012.458 cM with an average marker distance of 1.193 cM. Segregation distortion regions (SDRs) were observed on Chr01, Chr02, Chr06, Chr07 Chr09, Chr10 and Chr11 with a large proportion of the SDR regions segregating towards the heterozygous allele. There was good syntenic block formation that revealed good collinearity between the genetic and physical map of G. raimondii, compared to the Dt_sub genome of the G. hirsutum and G. barbadense. A total of 2,496 genes were mined within the SSR related regions. The proteins encoding the mined genes within the SDR had varied physiochemical properties; their molecular weights ranged from 6.586 to 252.737 kDa, charge range of -39.5 to 52, grand hydropathy value (GRAVY) of -1.177 to 0.936 and isoelectric (pI) value of 4.087 to 12.206. The low GRAVY values detected showed that the proteins encoding these genes were hydrophilic in nature, a property common among the stress responsive genes. The RNA sequence analysis revealed more of the genes were highly upregulated in various stages of fiber development for instance; Gorai.002G241300 was highly up regulated at 5, 10, 20 and 25 day post anthesis (DPA). Validation through RT-qPCR further revealed that these genes mined within the SDR regions might be playing a significant role under fiber development stages, therefore we infer that Gorai.007G347600 (TFCA), Gorai.012G141600 (FOLB1), Gorai.006G024500 (NMD3), Gorai.002G229900 (LST8) and Gorai.002G235200 (NSA2) are significantly important in fiber development and in turn the quality, and further researches needed to be done to elucidate their exact roles in the fiber development process. The construction of the genetic map between the two wild species paves away for the mapping of quantitative trait loci (QTLs) since the average distance between the markers is small, and mining of genes on the SSR regions will provide an insight in identifying key genes that can be introgressed into the cultivated cotton cultivars.

A bacterial artificial chromosome (BAC) library for G. mustelinum Miers ex G. Watt (AD4) was constructed. Intact nuclei from G. mustelinum (AD4) were used to isolate high molecular weight DNA, which was partially cleaved with Hind III and cloned into pSMART BAC (Hind III) vectors. The BAC library consisted of 208,182 clones arrayed in 542 384-microtiter plates, with an average insert size of 121.72 kb ranging from 100 to 150 kb. About 2% of the clones did not contain inserts. Based on an estimated genome size of 2372 Mb for G. mustelinum, the BAC library was estimated to have a total coverage of 10.50 × genome equivalents. The high capacity library of G. mustelinum will serve as a giant gene resource for map-based cloning of quantitative trait loci or genes associated with important agronomic traits or resistance to Verticillium wilt, physical mapping and comparative genome analysis.

Cotton is a pioneer of saline land crop, while salt stress still causes its growth inhibition and fiber production decrease. Phenotype identification showed better salt tolerance of a wild diploid cotton species Gossypium klotzschianum. To elucidate the salt-tolerant mechanisms in G. klotzschianum, we firstly detected the changes in hormones, H2O2 and glutathione (GSSH and GSH), then investigated the gene expression pattern of roots and leaves treated with 300 mM NaCl for 0, 3, 12, 48 h, and each time control by RNA-seq on the Illumina-Solexa platform. Physiological determination proved that the significant increase in hormone ABA at 48 h, while that in H2O2 was at 12 h, likewise, the GSH content decrease at 48 h and the GSSH content increase at 48 h, under salt stress. In total, 37,278 unigenes were identified from the transcriptome data, 8,312 and 6,732 differentially expressed genes (DEGs) were discovered to be involved in salt stress tolerance in roots and leaves, respectively. Gene function annotation and expression analysis elucidated hormone biosynthesis and signal transduction, reactive oxygen species (ROS), and salt overly sensitive (SOS) signal transduction related genes revealed the important roles of them in signal transmission, oxidation balance and ion homeostasis in response to salinity stress. This is a report which focuses on primary response to highly salty stress (upto 300 mM NaCl) in cotton using a wild diploid Gossypium species, broadening our understanding of the salt tolerance mechanism in cotton and laying a solid foundation of salt resistant for the genetic improvement of upland cotton with the resistance to salt stress.

The cotton genus (Gossypium spp.) contains 8 monophyletic diploid genome groups (A, B, C, D, E, F, G, K) and a single allotetraploid clade (AD). To gain insight into the phylogeny of Gossypium and molecular evolution of the chloroplast genome in this group, we performed a comparative analysis of 19 Gossypium chloroplast genomes, six reported here for the first time. Nucleotide distance in non-coding regions was about three times that of coding regions. As expected, distances were smaller within than among genome groups. Phylogenetic topologies based on nucleotide and indel data support for the resolution of the 8 genome groups into 6 clades. Phylogenetic analysis of indel distribution among the 19 genomes demonstrates contrasting evolutionary dynamics in different clades, with a parallel genome downsizing in two genome groups and a biased accumulation of insertions in the clade containing the cultivated cottons leading to large (for Gossypium) chloroplast genomes. Divergence time estimates derived from the cpDNA sequence suggest that the major diploid clades had diverged approximately 10 to 11 million years ago. The complete nucleotide sequences of 6 cpDNA genomes are provided, offering a resource for cytonuclear studies in Gossypium.

We investigated the locations of 5S and 45S rDNA in Gossypium diploid A, B, D, E, F, G genomes and tetraploid genome (AD) using multi-probe fluorescent in situ hybridization (FISH) for evolution analysis in Gossypium genus. The rDNA numbers and sizes, and synteny relationships between 5S and 45S were revealed using 5S and 45S as double-probe for all species, and the rDNA-bearing chromosomes were identified for A, D and AD genomes with one more probe that is single-chromosome-specific BAC clone from G. hirsutum (A1D1). Two to four 45S and one 5S loci were found in diploid-species except two 5S loci in G. incanum (E4), the same as that in tetraploid species. The 45S on the 7th and 9th chromosomes and the 5S on the 9th chromosomes seemed to be conserved in A, D and AD genomes. In the species of B, E, F and G genomes, the rDNA numbers, sizes, and synteny relationships were first reported in this paper. The rDNA pattern agrees with previously reported phylogenetic history with some disagreements. Combined with the whole-genome sequencing data from G. raimondii (D5) and the conserved cotton karyotype, it is suggested that the expansion, decrease and transposition of rDNA other than chromosome rearrangements might occur during the Gossypium evolution.

Fluorescence in situ hybridization (FISH) has become one of the most important techniques applied in plant molecular cytogenetics. However, the application of this technique in cotton has lagged behind because of difficulties in chromosome preparation. The focus of this article was FISH performed not only on cotton pachytene chromosomes, but also on cotton extended DNA fibers. The cotton pollen mother cells (PMCs) instead of buds or anthers were directly digested in enzyme to completely breakdown the cell wall. Before the routine acetic acid treatment, PMCs were incubated in acetic acid and enzyme mixture to remove the cytoplasm and clear the background. The method of ice-cold Carnoy's solution spreading chromosome was adopted instead of nitrogen removed method to avoid chromosomes losing and fully stretch chromosome. With the above-improved steps, the high-quality well-differentiated pachytene chromosomes with clear background were obtained. FISH results demonstrated that a mature protocol of cotton pachytene chromosomes preparation was presented. Intact and no debris cotton nuclei were obtained by chopping from etiolation cotyledons instead of the conventional liquid nitrogen grinding method. After incubating the nuclei with nucleus lysis buffer on slide, the parallel and clear background DNA fibers were acquired along the slide. This method overcomes the twist, accumulation and fracture of DNA fibers compared with other methods. The entire process of DNA fibers preparation requires only 30 min, in contrast, it takes 3 h with routine nitrogen grinding method. The poisonous mercaptoethanol in nucleus lysis buffer is replaced by nonpoisonous dithiothreitol. PVP40 in nucleus isolation buffer is used to prevent oxidation. The probability of success in isolating nuclei for DNA fiber preparation is almost 100% tested with this method in cotton. So a rapid, safe, and efficient method for the preparation of cotton extended DNA fibers suitable for FISH was established.

BACKGROUND: Cotton (Gossypium spp.) is a model system for the analysis of polyploidization. Although ascertaining the donor species of allotetraploid cotton has been intensively studied, sequence comparison of Gossypium chloroplast genomes is still of interest to understand the mechanisms underlining the evolution of Gossypium allotetraploids, while it is generally accepted that the parents were A- and D-genome containing species. Here we performed a comparative analysis of 13 Gossypium chloroplast genomes, twelve of which are presented here for the first time. METHODOLOGY/PRINCIPAL FINDINGS: The size of 12 chloroplast genomes under study varied from 159,959 bp to 160,433 bp. The chromosomes were highly similar having >98% sequence identity. They encoded the same set of 112 unique genes which occurred in a uniform order with only slightly different boundary junctions. Divergence due to indels as well as substitutions was examined separately for genome, coding and noncoding sequences. The genome divergence was estimated as 0.374% to 0.583% between allotetraploid species and A-genome, and 0.159% to 0.454% within allotetraploids. Forty protein-coding genes were completely identical at the protein level, and 20 intergenic sequences were completely conserved. The 9 allotetraploids shared 5 insertions and 9 deletions in whole genome, and 7-bp substitutions in protein-coding genes. The phylogenetic tree confirmed a close relationship between allotetraploids and the ancestor of A-genome, and the allotetraploids were divided into four separate groups. Progenitor allotetraploid cotton originated 0.43-0.68 million years ago (MYA). CONCLUSION: Despite high degree of conservation between the Gossypium chloroplast genomes, sequence variations among species could still be detected. Gossypium chloroplast genomes preferred for 5-bp indels and 1-3-bp indels are mainly attributed to the SSR polymorphisms. This study supports that the common ancestor of diploid A-genome species in Gossypium is the maternal source of extant allotetraploid species and allotetraploids have a monophyletic origin. G. hirsutum AD1 lineages have experienced more sequence variations than other allotetraploids in intergenic regions. The available complete nucleotide sequences of 12 Gossypium chloroplast genomes should facilitate studies to uncover the molecular mechanisms of compartmental co-evolution and speciation of Gossypium allotetraploids.