Xingxing Wang

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.

Effect of ulinastatin on HMGB1 expression in rats with acute lung injury induced by sepsis

The aim of this study was to investigate the influence of ulinastatin (UTI) on high mobility group box 1 (HMGB1), tumor necrosis factor (TNF)-α, and interleukin (IL)-6 expression in acute lung injury (ALI) rats with sepsis caused by cecal ligation and puncture (CLP) surgery, as well as to examine the underlying biological mechanism. Thirty rats were randomly and evenly divided into sham (control), CLP, and CLP + UTI groups. Thirty minutes after the surgery, the rats in the CLP + UTI group received UTI via the caudal vein, while normal saline was administered to rats in the other groups. Blood, lung tissues, and bronchoalveolar lavage fluid (BALF) were collected at different time points (6, 12, 24, and 48 h) after surgery for determination of related indicators. Compared with the CLP group, rats in the CLP + UTI group exhibited higher seven day survival rates, less lung injury, and decreased HMGB1 expression in the lung tissue, serum, and BALF. In addition, the levels of TNF-α and IL-6 at 24 h in the CLP + UTI group were markedly lower than those in the CLP group. These results suggest that by deregulation, UTI might decrease the lung injury and increase the survival time of ALI rats by downregulating HMGB1 expression as well as by inhibiting TNF-α and IL-6 levels in serum and BALF.

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.

Hepatitis B virus X protein activates human hepatic stellate cells through upregulating TGFβ1

We investigated the effects of the hepatitis B virus X gene (HBV X) on the activation of human hepatic stellate cells (HSCs) and the possible mechanisms underlying the pathway. Recombinant plasmid pHBV-X-IRES2-EGFP was constructed and transfected into HL-7702 cells using a lipid-mediated method. Transfected cells were screened by G418, which detected stable expression of the X gene by reverse transcription (RT)-PCR and Western blot analysis, and named L02/x. Cells not subjected to G418-selection were analyzed to confirm the transient expression of the X gene and named L02/48x. Subsequently, L02/x and L02/48x, together with non-HBx-expressing cells, were co-cultured with HSCs in a non-contact transwell system. After 36 h of co-culture, the proliferation and migration of HSCs was detected using different cell counting methods. Finally, the mRNA and protein levels of α-SMA, Col I, and TGFβ1 in HSCs were detected by real-time PCR and western blot analysis. RT-PCR and Western blot analysis showed that L02/x and L02/48x cells can express HBV X gene mRNA and protein. Additionally, HSCs co-cultured with L02/x or L02/48x cells showed significantly higher proliferation and migration levels than control groups. Real-time PCR and Western blot analysis showed that the mRNA and protein expressions of α-SMA, Col I, and TGFβ1 in HSCs co-cultured with HBx-expressing liver cells were higher than those in control groups. HBx protein activated HSCs in vitro, leading to increased proliferation and migration of HSCs and upregulation of α-SMA and Col I. The TGFβ1 gene may be involved in this pathway.

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.

XRCC1 rs25487 polymorphism is associated with lung cancer risk in epidemiologically susceptible Chinese people.

Base excision repair (BER) plays an important role in maintaining genome integrity and anti-cancer drug resistance. Single nu-cleotide polymorphisms (SNPs) in BER genes were detected in 500 lung cancer patients and 500 cancer-free controls. A logistic regression model was applied to analyze the relationship between lung cancer susceptibility and BER SNPs coupled with a wide range of epidemiological factors in a Chinese population. SNPs including rs25487 in the X-ray repair cross-complementing group 1 gene, rs1052133 in the 8-oxoguanine DNA gly-cosylase gene, and rs1136410 in the poly (ADP-ribose) polymerase 1 gene were identified. Multivariate analysis showed that the rs25487-AG geno-type was associated with a higher incidence of lung cancer compared with the GG genotype. The rs25487 SNP was associated with the pathological distribution of lung cancer. Moreover, rs1052133-GG was associated with early age of lung cancer onset compared with the CC genotype. Our data demonstrated that the SNPs rs25487 and rs1052133 are risk factors for lung cancer in epidemiologically susceptible Chinese people.