Kunbo Wang

Constructing a high-density linkage map for Gossypium hirsutum × Gossypium barbadense and identifying QTLs for lint percentage

To introgress the good fiber quality and yield from Gossypium barbadense into a commercial Upland cotton variety, a high-density simple sequence repeat (SSR) genetic linkage map was developed from a BC1 F1 population of Gossypium hirsutumu2009×u2009Gossypium barbadense. The map comprised 2,292 loci and covered 5115.16 centiMorgan (cM) of the cotton AD genome, with an average marker interval of 2.23 cM. Of the marker order for 1,577 common loci on this new map, 90.36% agrees well with the marker order on the D genome sequence genetic map. Compared with five published high-density SSR genetic maps, 53.14% of marker loci were newly discovered in this map. Twenty-six quantitative trait loci (QTLs) for lint percentage (LP) were identified on nine chromosomes. Nine stable or common QTLs could be used for marker-assisted selection. Fifty percent of the QTLs were from G. barbadense and increased LP by 1.07%-2.41%. These results indicated that the map could be used for screening chromosome substitution segments from G. barbadense in the Upland cotton background, identifying QTLs or genes from G. barbadense, and further developing the gene pyramiding effect for improving fiber yield and quality.

Role of interleukin-6 gene polymorphisms in the risk of coronary artery disease

We conducted a case-control study to investigate the association between IL-6 -174 G>C and -572 C>G polymorphisms and the risk of coronary artery disease (CAD). We genotyped IL-6 -174 G>C and -572 C>G in 402 patients with CAD and 402 control individuals. IL-6 -174 G>C (rs1800795) and -572 C>G (rs1800796) alleles were detected by polymerase chain reaction-restriction fragment length polymorphism. Patients with CAD were more likely to have a smoking habit, diabetes, and hypertension, a high level of triglycerides, and low levels of total cholesterol and high- and low-density lipoprotein cholesterol. Multivariate regression analyses showed that subjects carrying the IL-6 -174CC genotype had a small but significant increased risk of CAD (P = 0.004). Those carrying the IL-6 -174 G>C polymorphic variant had a slightly increased risk of CAD in both dominant and recessive models. However, we did not find significant association between the IL-6 -572 C>G polymorphism and risk of CAD. Moreover, a significant interaction was found between the IL-6 -174 G>C polymorphism, gender, and smoking habit. Our study, therefore, demonstrated that the IL-6 -174 G>C polymorphism is correlated with CAD risk, and that this polymorphism shows interactions with both gender and smoking.

Chromosomal Locations of 5S and 45S rDNA in Gossypium Genus and Its Phylogenetic Implications Revealed by FISH.

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.

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.

A high-density SSR genetic map constructed from a F2 population of Gossypium hirsutum and Gossypium darwinii.

The cultivated allotetraploid species Gossypium hirsutum, accounts for 90% of the world cotton production, has narrow genetic basis thats why its yield, quality or stress resistance breeding is stagnant. It is therefore, essential to explore desirable genes from Gossypium darwinii which has enviable traits such as high fiber fineness, drought tolerance, fusarium and verticillium resistance. We used G. darwinii as primary plant materials in this study not only to enrich the genetic diversity of exiting germplasm but also to better understand its genome structure. An interspecific high density linkage map of allotetraploid cotton was constructed using F2 population (G. hirsutum×G. darwinii). The map was based entirely on genome-wide simple sequence repeat (SSR) markers. A total of 2763 markers were mapped in 26 linkage groups (chromosomes) covering a genome length of 4176.7cM with an average inter-locus distance of 1.5cM. The length of the chromosomes ranged from 84.7 to 238.5cM with an average length of 160.6cM. At subgenome length was 2160.7cM with an average distance of 1.6cM, where as Dt genome length was 2016cM with an average distance of 1.4cM. There were 601 distorted SSR loci. Less number of segregation distortion loci were located in At subgenome than in Dt subgenome. Two post-polyploidization reciprocal translocations of A2/A3 and A4/A5 were suggested by 44 pairs of duplicate loci.

QTL mapping for salt tolerance at seedling stage in the interspecific cross of Gossypium tomentosum with Gossypium hirsutum

Soil salinity negatively affects growth and development as well as yield and fiber quality of cotton. The identification of quantitative trait loci (QTLs) for traits related to salt tolerance could facilitate the development of cotton cultivars with salt tolerance. The objective of this study was to map QTLs for salt tolerance in an F2:3 population derived from an interspecific cross between an upland cotton, CRI-12 (G09091801-2), of upland cotton (Gossypium hirsutum) and an accession, AD3-00 (P0601211), of wild cotton Gossypium tomentosum. 1295 simple sequence repeat markers, which amplified 1342 loci, distributed on 26 chromosomes and covered 3328.24xa0cM with an average inter-marker distance of 3.0xa0cM, were utilized for molecular genotyping. Salt tolerance was evaluated in a hydroponic at a young seedling stage for 2xa0weeks at 150xa0mM NaCl concentration in three environments. Mapping of QTLs related to salt tolerance was carried out on 7 traits by composite interval mapping using Windows QTL Cartographer 2.5. Eleven consistent QTLs were detected on 8 chromosomes (9, 11, 15, 16, 21, 23, 24 and 26) in at least two environments. qRL-Chr16-1 for RL was a major QTL explaining the Phenotypic variance of 11.97 and 18.44xa0% in two environments. Of the 11 QTLs, 10 were located on the D subgenome, indicating that genes responsible for salt tolerance in the allotetraploid cotton AD genome were mainly derived from the D subgenome. The information derived from these studies may be useful in facilitating breeding of salt tolerant cotton lines.

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.

Preparations of Meiotic Pachytene Chromosomes and Extended DNA Fibers from Cotton Suitable for Fluorescence In Situ Hybridization

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 Carnoys 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.

Primary investigation on GISH-NOR in cotton

Six loci of nucleolar organizer region (NOR) were detected in genomicin situ hybridization (GISH) of cotton (Gossypium). NOR was the characteristic of 45S rDNA but could be generated by genomic DNA (gDNA) extracted fromGossypium species as probe. With twice FISH to the same mitotic cell ofG. herbaceum orG. hirsutum, number, position and size for NORs generated from 45S rDNA and gDNA were identified largely similar or even the same. The NORs with gDNA as probe were therefore permanently defined as GISH-NORs. GISH-NORs fromG. hirsutum andG. raimondii mitotic images were all terminal types. Four and two GISH-NORs fromG. herbaceum (var.africanum) were terminal and centromere types, respectively. Six GISH-NORs inG. hirsutum were chromosome mapped with two in A- and four in D-subgenomes. There were also GISH-NORs in mitotic image ofG. raimondii with its own gDNA as probe. From mitotic image ofG. herbaceum with its own gDNA as probe, GISH-NOR could not be observed but non-whole-recovery of hybridized signals was distinguished. These non-whole-recovery of hybridized signals were detected on long arm terminals of most chromosomes and especially existed in nearly half long arm of a pair of chromosomes inG. herbaceum gDNA probed itself GISH image, which may be possibly induced by low copy genes within the regions rather than inter-subgenomic segment translocations. GISH-NORs in G.hirsutum mitotic images were dominantly observed when gDNAs from D and A genome species were used as probes and block, respectively, but not when the reverse probe and block gDNA from the two diploid progenitor genomes were designed. There may be two speculations to this special phenomenon: rDNA concerted evolution; content of rDNA in genome D more than genome A.

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.