Xiaoyan Cai

Genome Wide SSR High Density Genetic Map Construction from an Interspecific Cross of Gossypium hirsutum × Gossypium tomentosum

A high density genetic map was constructed using F2 population derived from an interspecific cross of G. hirsutum × G. tomentosum. The map consisted of 3093 marker loci distributed across all the 26 chromosomes and covered 4365.3 cM of cotton genome with an average inter-marker distance of 1.48 cM. The maximum length of chromosome was 218.38 cM and the minimum was 122.09 cM with an average length of 167.90 cM. A sub-genome covers more genetic distance (2189.01 cM) with an average inter loci distance of 1.53 cM than D sub-genome which covers a length of 2176.29 cM with an average distance of 1.43 cM. There were 716 distorted loci in the map accounting for 23.14% and most distorted loci were distributed on D sub-genome (25.06%), which were more than on A sub-genome (21.23%). In our map 49 segregation hotspots (SDR) were distributed across the genome with more on D sub-genome as compared to A genome. Two post-polyploidization reciprocal translocations of “A2/A3 and A4/A5” were suggested by seven pairs of duplicate loci. The map constructed through these studies is one of the three densest genetic maps in cotton however; this is the first dense genome wide SSR interspecific genetic map between G. hirsutum and G. tomentosum.

Chloroplast DNA Structural Variation, Phylogeny, and Age of Divergence among Diploid Cotton Species

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.

Characterization of the late embryogenesis abundant (LEA) proteins family and their role in drought stress tolerance in upland cotton

BackgroundLate embryogenesis abundant (LEA) proteins are large groups of hydrophilic proteins with major role in drought and other abiotic stresses tolerance in plants. In-depth study and characterization of LEA protein families have been carried out in other plants, but not in upland cotton. The main aim of this research work was to characterize the late embryogenesis abundant (LEA) protein families and to carry out gene expression analysis to determine their potential role in drought stress tolerance in upland cotton. Increased cotton production in the face of declining precipitation and availability of fresh water for agriculture use is the focus for breeders, cotton being the backbone of textile industries and a cash crop for many countries globally.ResultsIn this work, a total of 242, 136 and 142 LEA genes were identified in G. hirsutum, G. arboreum and G. raimondii respectively. The identified genes were classified into eight groups based on their conserved domain and phylogenetic tree analysis. LEA 2 were the most abundant, this could be attributed to their hydrophobic character. Upland cotton LEA genes have fewer introns and are distributed in all chromosomes. Majority of the duplicated LEA genes were segmental. Syntenic analysis showed that greater percentages of LEA genes are conserved. Segmental gene duplication played a key role in the expansion of LEA genes. Sixty three miRNAs were found to target 89 genes, such as miR164, ghr-miR394 among others. Gene ontology analysis revealed that LEA genes are involved in desiccation and defense responses. Almost all the LEA genes in their promoters contained ABRE, MBS, W-Box and TAC-elements, functionally known to be involved in drought stress and other stress responses. Majority of the LEA genes were involved in secretory pathways. Expression profile analysis indicated that most of the LEA genes were highly expressed in drought tolerant cultivars Gossypium tomentosum as opposed to drought susceptible, G. hirsutum. The tolerant genotypes have a greater ability to modulate genes under drought stress than the more susceptible upland cotton cultivars.ConclusionThe finding provides comprehensive information on LEA genes in upland cotton, G. hirsutum and possible function in plants under drought stress.

Molecular evolution of the plastid genome during diversification of the cotton genus

Cotton (Gossypium spp.) is commonly grouped into eight diploid genomic groups, designated A-G and K, and one tetraploid genomic group, namely AD. To gain insight into the phylogeny of Gossypium and molecular evolution of the chloroplast genome duringdiversification, chloroplast genomes (cpDNA) from 6 D-genome and 2 G-genome species of Gossypium (G. armourianum D2-1, G. harknessii D2-2, G. davidsonii D3-d, G. klotzschianum D3-k, G. aridum D4, G. trilobum D8, and G. australe G2, G. nelsonii G3) were newly reported here. In combination with the 26 previously released cpDNA sequences, we performed comparative phylogenetic analyses of 34 Gossypium chloroplast genomes that collectively represent most of the diversity in the genus. Gossypium chloroplasts span a small range in size that is mostly attributable to indels that occur in the large single copy (LSC) region of the genome. Phylogenetic analysis using a concatenation of all genes provides robust support for six major Gossypium clades, largely supporting earlier inferences but also revealing new information on intrageneric relationships. Using Theobroma cacao as an outgroup, diversification of the genus was dated, yielding results that are in accord with previous estimates of divergence times, but also offering new perspectives on the basal, early radiation of all major clades within the genus as well as gaps in the record indicative of extinctions. Like most higher-plant chloroplast genomes, all cotton species exhibit a conserved quadripartite structure, i.e., two large inverted repeats (IR) containing most of the ribosomal RNA genes, and two unique regions, LSC (large single sequence) and SSC (small single sequence). Within Gossypium, the IR-single copy region junctions are both variable and homoplasious among species. Two genes, accD and psaJ, exhibited greater rates of synonymous and non-synonymous substitutions than did other genes. Most genes exhibited Ka/Ks ratios suggestive of neutral evolution, with 8 exceptions distributed among one to several species. This research provides an overview of the molecular evolution of a single, large non-recombining molecular during the diversification of this important genus.

Salt stress responsiveness of a wild cotton species (Gossypium klotzschianum) based on transcriptomic 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.

Construction of cytogenetic map of Gossypium herbaceum chromosome 1 and its integration with genetic maps

BackgroundCytogenetic map can provide not only information of the genome structure, but also can build a solid foundation for genetic research. With the developments of molecular and cytogenetic studies in cotton (Gossypium), the construction of cytogenetic map is becoming more and more imperative.ResultsA cytogenetic map of chromosome 1 (A101) of Gossypium herbaceum (A1) which includes 10 bacterial artificial chromosome (BAC) clones was constructed by using fluorescent in situ hybridization (FISH). Meanwhile, comparison and analysis were made for the cytogenetic map of chromosome 1 (A101) of G. herbaceum with four genetic linkage maps of chromosome 1 (Ah01) of G. hirsutum ((AD)1) and one genetic linkage map of chromosome 1 of (A101) G. arboreum (A2). The 10 BAC clones were also used to be localized on G. raimondii (D5) chromosome 1 (D501), and 2 of them showed clear unique hybridized signals. Furthermore, these 2 BAC clones were also shown localized on chromosome 1 of both A sub-genome and D sub-genome of G. hirsutum.ConclusionThe comparison of the cytogenetic map with genetic linkage maps showed that most of the identified marker-tagged BAC clones appearing same orders in different maps except three markers showing different positions, which might indicate chromosomal segmental rearrangements. The positions of the 2 BAC clones which were localized on Ah01 and Dh01 chromosomes were almost the same as that on A101 and D501 chromosomes. The corresponding anchored SSR markers of these 2 BAC clones were firstly found to be localized on chromosome D501 (Dh01) as they were not seen mapped like this in any genetic map reported.

Simple Sequence Repeat (SSR) Genetic Linkage Map of D Genome Diploid Cotton Derived from an Interspecific Cross between Gossypium davidsonii and Gossypium klotzschianum

The challenge in tetraploid cotton cultivars is the narrow genetic base and therefore, the bottleneck is how to obtain interspecific hybrids and introduce the germplasm directly from wild cotton to elite cultivars. Construction of genetic maps has provided insight into understanding the genome structure, interrelationships between organisms in relation to evolution, and discovery of genes that carry important agronomic traits in plants. In this study, we generated an interspecific hybrid between two wild diploid cottons, Gossypium davidsonii and Gossypium klotzschianum, and genotyped 188 F2:3 populations in order to develop a genetic map. We screened 12,560 SWU Simple Sequence Repeat (SSR) primers and obtained 1000 polymorphic markers which accounted for only 8%. A total of 928 polymorphic primers were successfully scored and only 728 were effectively linked across the 13 chromosomes, but with an asymmetrical distribution. The map length was 1480.23 cM, with an average length of 2.182 cM between adjacent markers. A high percentage of the markers on the map developed, and for the physical map of G. raimondii, exhibited highly significant collinearity, with two types of duplication. High level of segregation distortion was observed. A total of 27 key genes were identified with diverse roles in plant hormone signaling, development, and defense reactions. The achievement of developing the F2:3 population and its genetic map constructions may be a landmark in establishing a new tool for the genetic improvement of cultivars from wild plants in cotton. Our map had an increased recombination length compared to other maps developed from other D genome cotton species.

Genome-Wide Analysis of Multidrug and Toxic Compound Extrusion (MATE) Family in Diploid Cotton, Gossypium raimondii and Gossypium arboreum and Its Expression Analysis Under Salt, Cadmium and Drought Stress

The extrusion of toxins and substances at a cellular level is a vital life process in plants under abiotic stress. The multidrug and toxic compound extrusion (MATE) gene family plays a large role in the exportation of toxins and other substrates. We carried out a genome-wide analysis of MATE gene families in Gossypium raimondii and Gossypium arboreum and assessed their expression levels under salt, cadmium and drought stresses. We identified 70 and 68 MATE genes in G. raimondii and G. arboreum, respectively. The majority of the genes were predicted to be localized within the plasma membrane, with some distributed in other cell parts. Based on phylogenetic analysis, the genes were subdivided into three subfamilies, designated as M1, M2 and M3. Closely related members shared similar gene structures, and thus were highly conserved in nature and have mainly evolved through purifying selection. The genes were distributed in all chromosomes. Twenty-nine gene duplication events were detected, with segmental being the dominant type. GO annotation revealed a link to salt, drought and cadmium stresses. The genes exhibited differential expression, with GrMATE18, GrMATE34, GaMATE41 and GaMATE51 significantly upregulated under drought, salt and cadmium stress, and these could possibly be the candidate genes. Our results provide the first data on the genome-wide and functional characterization of MATE genes in diploid cotton, and are important for breeders of more stress-tolerant cotton genotypes.

Cotton Late Embryogenesis Abundant (LEA2) Genes Promote Root Growth and Confers Drought Stress Tolerance in Transgenic Arabidopsis thaliana

Late embryogenesis abundant (LEA) proteins play key roles in plant drought tolerance. In this study, 157, 85 and 89 candidate LEA2 proteins were identified in G. hirsutum, G. arboreum and G. raimondii respectively. LEA2 genes were classified into 6 groups, designated as group 1 to 6. Phylogenetic tree analysis revealed orthologous gene pairs within the cotton genome. The cotton specific LEA2 motifs identified were E, R and D in addition to Y, K and S motifs. The genes were distributed on all chromosomes. LEA2s were found to be highly enriched in non-polar, aliphatic amino acid residues, with leucine being the highest, 9.1% in proportion. The miRNA, ghr-miR827a/b/c/d and ghr-miR164 targeted many genes are known to be drought stress responsive. Various stress-responsive regulatory elements, ABA-responsive element (ABRE), Drought-responsive Element (DRE/CRT), MYBS and low-temperature-responsive element (LTRE) were detected. Most genes were highly expressed in leaves and roots, being the primary organs greatly affected by water deficit. The expression levels were much higher in G. tomentosum as opposed to G. hirsutum. The tolerant genotype had higher capacity to induce more of LEA2 genes. Over expression of the transformed gene Cot_AD24498 showed that the LEA2 genes are involved in promoting root growth and in turn confers drought stress tolerance. We therefore infer that Cot_AD24498, CotAD_20020, CotAD_21924 and CotAD_59405 could be the candidate genes with profound functions under drought stress in upland cotton among the LEA2 genes. The transformed Arabidopsis plants showed higher tolerance levels to drought stress compared to the wild types. There was significant increase in antioxidants, catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD) accumulation, increased root length and significant reduction in oxidants, Hydrogen peroxide (H2O2) and malondialdehyde (MDA) concentrations in the leaves of transformed lines under drought stress condition. This study provides comprehensive analysis of LEA2 proteins in cotton thus forms primary foundation for breeders to utilize these genes in developing drought tolerant genotypes.

Genome-wide assessment of genetic diversity and fiber quality traits characterization in Gossypium hirsutum races

Abstract Gossypium hirsutum races are believed to be potential reservoirs of desirable traits, which can play crucial roles to overcome the existing narrow genetic base of modern Upland cotton cultivars. However, prior to utilizing the races in cotton improvement programs, understanding their genetic constitutions is needed. Thus, this study used molecular and morphological techniques to characterize 110 G. hirsutum germplasm including 109 semi-wild accessions and one Upland cotton cultivar, CRI12. In the study, 104 SSR markers detected 795 alleles, with an average of 7.64 alleles per marker, ranging from 3 to 14, and average polymorphism information content (PIC) value of 0.71. And 96 of the markers were found to be highly informative, with PIC value≥0.50. Pairwise genetic similarity coefficient across the accessions ranged from 0.19 to 1.00, with an average value of 0.46. Morphological characterization was done using fiber length, fiber strength, micronaire, fiber uniformity index, and fiber elongation. Pairwise taxonomic distance within the accessions ranged from 0.17 to 3.41, with a mean of 1.33. The SSR and fiber quality traits data set based unweighted pair group method of arithmetic mean (UPGMA) analysis grouped the accessions into 7 and 12 distinct clusters, respectively, that corresponds well with the results of principal component analysis (PCA). Our study revealed the existence of vast molecular and morphological diversities within the accessions and provided valuable information on each semi-wild accession for quick and better informed germplasm utilization in cotton breeding programs.