Mapping and quantitative trait loci analysis of verticillium wilt resistance genes in cotton.

Verticillium wilt is one of the most serious constraints to cotton production in almost all of the cotton-growing countries. In this study, "XinLuZao1" (XLZ1), a susceptible cultivar Gossypium hirsutum L. and "Hai7124" (H7124), a resistant line G. barbadense, and their F(2:3) families were used to map and study the disease index induced by verticillium wilt. A total of 430 SSR loci were mapped into 41 linkage groups; the map spanned 3,745.9 cM and the average distance between adjacent loci was 8.71 cM. Four and five quantitative trait loci (QTLs) were detected based on the disease index investigated on July 22 and August 24 in 2004, respectively. These nine QTLs explained 10.63-28.83% of the phenotypic variance, six of them were located on the D sub-genome. Two QTLs located in the same marker intervals may partly explain the significant correlation of the two traits. QTLs explaining large phenotypic variation were identified in this study, which may be quite useful in cotton anti-disease breeding.

Genes expression analyses of sea-island cotton (Gossypium barbadense L.) during fiber development.

Sea-island cotton (Gossypium barbadense L.) is one of the most valuable cotton species due to its silkiness, luster, long staples, and high strength, but its fiber development mechanism has not been surveyed comprehensively. We constructed a normalized fiber cDNA library (from -2 to 25 dpa) of G. barbadense cv. Pima 3-79 (the genetic standard line) by saturation hybridization with genomic DNA. We screened Pima 3-79 fiber RNA from five developmental stages using a cDNA array including 9,126 plasmids randomly selected from the library, and we selected and sequenced 929 clones that had different signal intensities between any two stages. The 887 high-quality expressed sequence tags obtained were assembled into 645 consensus sequences (582 singletons and 63 contigs), of which 455 were assigned to functional categories using gene ontology. Almost 50% of binned genes belonged to metabolism functional categories. Based on subarray analysis of the 887 high-quality expressed sequence tags with 0-, 5-, 10-, 15-, and 20-dpa RNA of Pima 3-79 fibers and a mixture of RNA of nonfiber tissues, seven types of expression profiles were elucidated. Furthermore our results showed that phytohormones may play an important role in the fiber development.

[Spontaneous and induced programmed cell death in suspension cell cultures of cotton (Gossypium hirsutum L.)].

Cotton suspension cells grew well in the MS medium supplemented with 0.1 mg/L 2,4 D and 0.1 mg/L KT. Senescence occurred when the cells were unsubcultured. The cells began to lose their viabilities on the 17th day, and on the 21th day oligonucleosomal sized DNA fragments ( DNA ladder) could be detected. Oligonucleosomal sized DNA fragments ( DNA ladder) was the hallmark of the programmed cell death. Programmed cell death of cotton suspension cells could be induced respectively by some stress factors which included heatshock (42+/-3 degrees C for 8 hours), 10 micromol/L camptothecin, 20 micromol/L fumonisin B1 and 50 mmol/L cycloheximide. The cotton suspension cells which grew in the MS medium supplemented with 0.1 mg/L 2,4 D and 0.1 mg/L KT differred physiologically from the cells in the MS medium supplemented with 0.1 mg/L IBA and 0.1 mg/L KT, and they responded differentially to the heatshock, 10 micromol/L camptothecin and 20 micromol/L fumonisin B1, while the same to 50 mmol/L cycloheximide.

[Construction and analysis of SSH library of Gossypium barbadense upon infection with Verticillium dahliae].

Roots were collected from the seedlings inoculated with pathogen Verticillium dahliae after 2, 4, 8, 12, 24, 48, 72 and 96 hours for total RNA extraction. The cDNAs from the inoculated seedlings were used as the tester and those from the control seedlings as the driver. SSH method was employed to find the differently expressed cDNAs responding to the pathogen. T/A clone library was constructed containing 534 clones. The cDNA inserts were amplified from the bacterial clones directly with M13 primers by PCR. The size of the products ranged 0.2 - 1.2 kb with an average size of 0.5 kb. The SSH products were dotted on nylon filters, and the positive clones were screened by virtual Northern blotting with probes of the two kinds of initiative cDNAs. Totally 78 clones which were up-regulated and putatively involved in the defense response of G. barbadense were identified and sequenced. Sequence similarity searches were performed with the Blastn and Blastx. Most of them showed high or partial homology to genes or ESTs induced by different stresses in Arabidopsis thaliana and other species,such as the pathogenesis-related 10 family of G. hirsumtum and disease resistance-responsive family protein in Arabidopsis thaliana. The results would be helpful to understand the molecular mechanisms of disease response in cotton.

[Withdrawal of exogenous auxin induces programmed cell death of cotton embryogenic suspension cultures].

In the establishment of cotton suspension culture, we had observed an interesting phenomenon that large-scale cell death occurred when the embryogenic cells were transferred from the medium MS supplemented with IBA 0.5 mg/L to fresh MS medium without IBA. Cytological study and genomic DNA electrophoresis showed that this kind of cell death was accompanied by such morphological characters as chromatin condensation, the maintenance of membrane continuity, a condensed cytoplasm and evident DNA fragmentation of multimers of 140-180 bp. Inhibitor studies suggested the proteolysis and the caspase-like proteases were involved in cell death. These results support that cell death caused by withdrawal of exogenous auxins is a kind of programmed cell death (PCD). So auxin is involved in the regulation of programmed cell death signal transduction pathways, and may be another plant-specific regulator beside ethylene, abscisic acid and gibberellin in PCD.

Function analysis of promoter trapping system after inserted into cotton (Gossypium hirsutum L. ) genome.

The technique of promoter trapping was developed to investigate its viability in cotton ( Gossypium hirsutum L.) functional genomics. 141 independent transformants of cotton were generated via Agrobacterium tumefaciens mediated transformation, of which 97% showed positive by PCR detection. The reporter, GUS gene, was expressed to different extent in different organs, with a frequency of 48% in roots, 9.2% in vascular bundles of stem, 5.2% in leaves, and 51% in flowers. Meanwhile, we discovered that there existed great differences in expression patterns among different transgenic lines. Their GUS expression patterns were organ- or tissue-specific or ubiquitous in all of the plants. The promoter trapping system developed here was characterized as an effective method for creating mutants with diverse reporter gene expression patterns, which laid a solid foundation for further research of functional genomics in cotton.

[Transgenic cotton plants of chitinase and glucanase genes and their performance of resistance to Verticillium dahliea].

The two disease-resistance genes chitinase and glucanase, which were respectively directed by commelina yellow mottle virus promoter (CoYMV, vascular specific) and CaMV35S promoter, were introduced into cotton genome via Agrobacterium tumefaciens. Transgenic plants were obtained from two popularly cultivated varieties Jihe321 and CRIC35. After screening by spraying kanamycin over unfolding leaves, the kanamycin resistance (KmR) plants were tested by PCR and Southern blot. The results showed that there were one or two inserts of transgenes in cotton genome. Performance test of resistance of T3 families in field and greenhouse showed that seven lines were resistant or tolerant to Verticillium dahliea. Meanwhile, the resistance at seedling stage in greenhouse was in accordance with that at the boll-setting stage in field. Among the seven lines, D9910, D9915 and D9919 had a disease resistance index of 6.5, 9.4 and 9.5, respectively in field, which showed a high resistance level. Genetics analysis of the three lines showed a classical Mendelian pattern of one pair of genes, which meant that each of the three lines contains one copy of transgene. Southern blotting also confirmed the copy number of inserts.

[Evaluation of application of a new molecular marker SRAP on analysis of F2 segregation population and genetic diversity in cotton].

SRAP (Sequence-related Amplified Polymorphism), a new marker system, was applied in cotton genome analysis. We developed an efficient PCR reaction system for detecting SRAP that showed reliable, effective and reproductive. SRAP marker was used to detect the polymorphisms between 'Pima90' (G. barbodense) and ' Handan208' (G. hirsutum) and showed comparatively high polymorphism. Among the 30 primer pairs, 29 pairs generated 149 polymorphic bands with an average of 5.14 polymorphic bands per primer pair. The primer pair showing most polymorphism was the combination of me3 and em2, which produced 13 polymorphic bands. The SRAP marker was also tested among 11 upland cotton cultivars, 15 of the tested 30 primer pairs showed polymorphism yielding 22 polymorphic genetic loci, which is relatively high among upland cotton cultivars. The results showed that SRAP marker could be widely used in molecular genetic map construction and germplasm evaluation of cotton.

Localization of QTLs for yield and fiber quality traits of tetraploid cotton cultivar.

Using SSR and RAPD as molecular markers, and the 69 F2 families from a cross between Handan208 (Gossypium hirsutum) and Pima90 (Gossypium barbadense) as a mapping population, a linkage map comprising 126 markers was constructed. With an average spacing 13.7 cM between markers, the linkage map spanned 1,717.0 cM, which covers approximately 34.34% of the total recombinational length of cotton genome. Based on the linkage map and the F2:3 phenotypic data, overall genome QTL screening was conducted by composite interval mapping method, then 29 QTLs related to cotton yield traits and fiber quality traits were found. There are 16 loci related to yield traits and 13 loci related to fiber quality. Particularly, two QTLs relevant to fiber strength were detected, which could explain phenotypic variance of 34.15% and 13.86%, respectively.

[Sequence analysis of Bt insertion flanking fragments in transgenic Bt cotton].

The copies of outside gene and DNA structure of integration locus are important in high expression and avoidance of gene silence. Many research results showed that outside genes were inserted into the plant genome with recombinant type, and the integration was related to border T-DNA sequence in the course of Agrobaterium mediated transformation. SAR structure was also found in the integration location of transformants with direct DNA transformation method. The pollen-tube pathway transformation, one of direct DNA transformation method, was very successfully used in Bt transgenic cotton in China. But until now there has been no report about Bt gene integration. The aim of this research was to investigate flanking DNA structure in order to explain the mechanism of direct transformation method in the future. The structure of flanking DNA fragments of Bt integration in four different transgenic Bt cotton varieties including Simian-3, 161 resistant breeding line, 161 sensitive breeding line and Guokangmian-1 was analyzed with TAIL-PCR and nested PCR. The flanking DNA sequences of different self-crossed progenies from one plant are the same. In contrary, their DNA sequences are diverse for different breeding lines. Upstream flanking fragments in some transgenic cotton contained short transformation plasmid sequences, downstream flanking fragments in all the transgenic cotton varieties were composed of high percentage AT. The percentage of AT in Simian-3 transgenic Bt variety was as high as 92%. All the flanking fragments were of multi-copies and similar to repetitive sequences. No recognition sites of TOP enzyme were found in these fragments.