QTL Mapping for Fiber Quality and Yield Traits Based on Introgression Lines Derived from Gossypium hirsutum × G. tomentosum.

The tetraploid species Gossypium hirsutum is cultivated widely throughout the world with high yield and moderate fiber quality, but its genetic basis is narrow. A set of 107 introgression lines (ILs) was developed with an interspecific cross using G. hirsutumacc. 4105 as the recurrent parent and G. tomentosum as the donor parent. A specific locus amplified fragment sequencing (SLAF-seq) strategy was used to obtain high-throughput single nucleotide polymorphism (SNP) markers. In total, 3157 high-quality SNP markers were obtained and further used for identification of quantitative trait loci (QTLs) for fiber quality and yield traits evaluated in multiple environments. In total, 74 QTLs were detected that were associated with five fiber quality traits (30 QTLs) and eight yield traits (44 QTLs), with 2.02-30.15% of the phenotypic variance explained (PVE), and 69 markers were found to be associated with these thirteen traits. Eleven chromosomes in the A sub-genome (At) harbored 47 QTLs, and nine chromosomes in the D sub-genome (Dt) harbored 27 QTLs. More than half (44 QTLs = 59.45%) showed positive additive effects for fiber and yield traits. Five QTL clusters were identified, with three in the At, comprised of thirteen QTLs, and two in the Dt comprised of seven QTLs. The ILs developed in this study and the identified QTLs will facilitate further molecular breeding for improvement of Upland cotton in terms of higher yield with enhanced fiber quality.

Overexpression of rice NAC gene SNAC1 improves drought and salt tolerance by enhancing root development and reducing transpiration rate in transgenic cotton.

The SNAC1 gene belongs to the stress-related NAC superfamily of transcription factors. It was identified from rice and overexpressed in cotton cultivar YZ1 by Agrobacterium tumefaciens-mediated transformation. SNAC1-overexpressing cotton plants showed more vigorous growth, especially in terms of root development, than the wild-type plants in the presence of 250 mM NaCl under hydroponic growth conditions. The content of proline was enhanced but the MDA content was decreased in the transgenic cotton seedlings under drought and salt treatments compared to the wild-type. Furthermore, SNAC1-overexpressing cotton plants also displayed significantly improved tolerance to both drought and salt stresses in the greenhouse. The performances of the SNAC1-overexpressing lines under drought and salt stress were significantly better than those of the wild-type in terms of the boll number. During the drought and salt treatments, the transpiration rate of transgenic plants significantly decreased in comparison to the wild-type, but the photosynthesis rate maintained the same at the flowering stage in the transgenic plants. These results suggested that overexpression of SNAC1 improve more tolerance to drought and salt in cotton through enhanced root development and reduced transpiration rates.

The flavonoid pathway regulates the petal colors of cotton flower.

Although biochemists and geneticists have studied the cotton flower for more than one century, little is known about the molecular mechanisms underlying the dramatic color change that occurs during its short developmental life following blooming. Through the analysis of world cotton germplasms, we found that all of the flowers underwent color changes post-anthesis, but there is a diverse array of petal colors among cotton species, with cream, yellow and red colors dominating the color scheme. Genetic and biochemical analyses indicated that both the original cream and red colors and the color changes post-anthesis were related to flavonoid content. The anthocyanin content and the expression of biosynthesis genes were both increased from blooming to one day post-anthesis (DPA) when the flower was withering and undergoing abscission. Our results indicated that the color changes and flavonoid biosynthesis of cotton flowers were precisely controlled and genetically regulated. In addition, flavonol synthase (FLS) genes involved in flavonol biosynthesis showed specific expression at 11 am when the flowers were fully opened. The anthocyanidin reductase (ANR) genes, which are responsible for proanthocyanidins biosynthesis, showed the highest expression at 6 pm on 0 DPA, when the flowers were withered. Light showed primary, moderate and little effects on flavonol, anthocyanin and proanthocyanidin biosynthesis, respectively. Flavonol biosynthesis was in response to light exposure, while anthocyanin biosynthesis was involved in flower color changes. Further expression analysis of flavonoid genes in flowers of wild type and a flavanone 3-hydroxylase (F3H) silenced line showed that the development of cotton flower color was controlled by a complex interaction between genes and light. These results present novel information regarding flavonoids metabolism and flower development.

A genetic and metabolic analysis revealed that cotton fiber cell development was retarded by flavonoid naringenin.

The cotton (Gossypium spp.) fiber is a unique elongated cell that is useful for investigating cell differentiation. Previous studies have demonstrated the importance of factors such as sugar metabolism, the cytoskeleton, and hormones, which are commonly known to be involved in plant cell development, while the secondary metabolites have been less regarded. By mining public data and comparing analyses of fiber from two cotton species (Gossypium hirsutum and Gossypium barbadense), we found that the flavonoid metabolism is active in early fiber cell development. Different flavonoids exhibited distinct effects on fiber development during ovule culture; among them, naringenin (NAR) could significantly retard fiber development. NAR is a substrate of flavanone 3-hydroxylase (F3H), and silencing the F3H gene significantly increased the NAR content of fiber cells. Fiber development was suppressed following F3H silencing, but the overexpression of F3H caused no obvious effects. Significant retardation of fiber growth was observed after the introduction of the F3H-RNA interference segment into the high-flavonoid brown fiber G. hirsutum T586 line by cross. A greater accumulation of NAR as well as much shorter fibers were also observed in the BC1 generation plants. These results suggest that NAR is negatively associated with fiber development and that the metabolism mediated by F3H is important in fiber development, thus highlighting that flavonoid metabolism represents a novel pathway with the potential for cotton fiber improvement.

GbTCP, a cotton TCP transcription factor, confers fibre elongation and root hair development by a complex regulating system.

As the most important natural raw material for textile industry, cotton fibres are an excellent model for studying single-cell development. Although expression profiling and functional genomics have provided some data, the mechanism of fibre development is still not well known. A class I TCP transcription factor (designated GbTCP), encoding 344 amino acids, was isolated from the normalized cDNA library of sea-island cotton fibre (from -2 to 25 days post anthesis). GbTCP was preferentially expressed in the elongating cotton fibre from 5 to 15 days post anthesis. Some expression was also observed in stems, apical buds, and petals. RNAi silencing of GbTCP produced shorter fibre, a reduced lint percentage, and a lower fibre quality than the wild-type plants. Overexpression of GbTCP enhanced root hair initiation and elongation in Arabidopsis and regulated branching. Solexa sequencing and Affymetrix GeneChip analysis indicated that GbTCP positively regulates the level of jasmonic acid (JA) and, as a result, activates downstream genes (reactive oxygen species, calcium signalling, ethylene biosynthesis and response, and several NAC and WRKY transcription factors) necessary for elongation of fibres and root hairs. JA content analysis in cotton also confirmed that GbTCP has a profound effect on JA biosynthesis. In vitro ovule culture showed that an appropriate concentration of JA promoted fibre elongation. The results suggest that GbTCP is an important transcription factor for fibre and root hair development by regulating JA biosynthesis and response and other pathways, including reactive oxygen species, calcium channel and ethylene signalling.

Molecular diversity, genomic constitution, and QTL mapping of fiber quality by mapped SSRs in introgression lines derived from Gossypium hirsutum × G. darwinii Watt.

Because the genetic basis of current upland cotton cultivars is narrow, exploring new germplasm resources and discovering novel alleles relevant to important agronomic traits have become two of the most important themes in the field of cotton research. In this study, G. darwinii Watt, a wild cotton species, was crossed with four upland cotton cultivars with desirable traits. A total of 105 introgression lines (ILs) were successfully obtained. By using 310 mapped SSRs evenly distributed across the interspecific linkage map of G. hirsutum × G. barbadense, these 105 ILs and their corresponding parents were analyzed. A total of 278 polymorphic loci were detected among the 105 ILs, and the average length of introgression segments accumulated to 333.5 cM, accounting for 6.7 % of the whole genome. These lines included many variations. However, high similarity coefficients existed between lines, even between those derived from different parents. Finally, all the ILs and their upland cotton parents were used for association mapping of fiber quality in three environments. A total of 40 SSRs were found to be associated with five fiber quality indexes (P < 0.05) with some being detected in multiple environments and traits. The contribution rate for trait variation was 6.31 % on average, ranging from 2.00 to 14.79 %. This study develops novel ILs for cotton genetics and breeding and provides the basis for future research into fine mapping of genes related to fiber quality, analyses of candidate gene association, and molecular marker-assisted breeding.

GbPDF1 is involved in cotton fiber initiation via the core cis-element HDZIP2ATATHB2.

Cotton (Gossypium spp.) fiber cells are seed trichomes derived from the epidermal layer of the cotton seed coat. The molecular components responsible for regulating fiber cell differentiation have not been fully elucidated. A cotton PROTODERMAL FACTOR1 gene (GbPDF1) was found to be expressed preferentially during fiber initiation and early elongation, with highest accumulation in fiber cells 5 d post anthesis. PDF1 silencing caused retardation of fiber initiation and produced shorter fibers and lower lint percentage compared with the wild type, indicating that the gene is required for cotton fiber development. Further analysis showed that a higher accumulation of hydrogen peroxide occurred in the RNA interference transgenic cotton lines. Meanwhile, the expression of several genes related to ethylene and pectin synthesis or sugar transport during cotton fiber growth was found to be significantly reduced in the PDF1-suppressed cotton. Three proteins interacting with GbPDF1 in yeast and in planta might involve cellular signaling or metabolism. GbPDF1 promoter::GUS constructs in transgenic cotton were predominantly expressed in the epidermis of ovules and developing fibers. Progressive deletions of the GbPDF1 promoter showed that a 236-bp promoter fragment was sufficient for basal GbPDF1 transcription in cotton. Mutation of putative regulatory sequences showed that HDZIP2ATATHB2, an element within the fragment, was essential for PGbPDF1-1 expression. The binding activity between this cis-element and nuclear extracts from fiber-bearing cotton ovules at 5 d post anthesis was specific. We conclude that GbPDF1 plays a critical role together with interaction partners in hydrogen peroxide homeostasis and steady biosynthesis of ethylene and pectin during fiber development via the core cis-element HDZIP2ATATHB2.

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.

Isolation and characterization of genes associated to cotton somatic embryogenesis by suppression subtractive hybridization and macroarray.

Somatic embryogenesis (SE) is the developmental reprogramming of somatic cells toward the embryogenesis pathway and is a notable illustration of cell totipotency. To identify genes involved in SE, subtractive polymerase chain reaction (PCR) was performed to generate transcripts highly enriched for SE-related genes, using cDNA prepared from a mixture of embryogenic callus and pre-globular somatic embryos, as the tester, and cDNA from non-embryogenic callus, as the driver. After differential screening and subsequent confirmation by reverse Northern blot analysis, a total of 671 differentially expressed cDNA fragments were identified, and 242 uni-genes significantly up-regulated during cotton SE were recovered, as confirmed by Northern blot and reverse-transcription PCR analysis of representative cases, including most previously published SE-related genes in plants. In total, more than half had not been identified previously as SE-related genes, including dominant crucial genes involved in transcription, post-transcription, and transportation, and about one-third had not been reported previously to GenBank or were expected to be unknown, or newly identified genes. We used cDNA arrays to further investigate the expression patterns of these genes in differentiating gradient culture, ranging from pro-embryogenic masses to somatic embryos at every stage. The cDNA collection is composed of a broad repertoire of SE genes which is an important resource for understanding the genetic interactions underlying SE signaling and regulation. Our results suggested that a complicated and concerted mechanism involving multiple cellular pathways is responsible for cotton SE. This report represents a systematic and comprehensive analysis of genes involved in the process of somatic embryogenesis.

Somatic embryogenesis and plant regeneration from different wild diploid cotton (Gossypium) species.

Calli were successfully induced from hypocotyls of eight wild diploid cotton species (Gossypium) on MSB (MS salts and B(5) vitamins) medium supplemented with 0.09 microM 2,4-D (2,4-dichlorophenoxyacetic acid) and 2.32 microM KT (kinetin). Plant growth regulator (PGR) combinations, adding GA(3) (Gibberellic acid), high inorganic salt stress, and PGR-free media were used to induce embryogenic calli from nonembryogenic calli. Embryogenic cultures were induced from G. aridum S. (D(4) genome), G. davidsonii K. (D(3)-d genome), G. klotzschianum A. (D(3)-k genome), G. raimondii U. (D(5) genome), and G. stocksii M. (E(1) genome). We then observed somatic embryogenesis in the five species while calli of G. africanum V. (A(1)-2 genome), G. anomalum W. (B(1) genome), and G. bickii P. (G genome) remained nonembryogenic. Somatic embryogenesis was adjusted by changing sugar sources, regulating combinations of PGRs, and using cell suspension culture. Embryos at various developmental stages produced mature and germinating embryos when cultured on filter paper placed on the media containing different sugar sources. The utility of different sugar sources promoted globular embryos developing into cotyledonary stage and increased the frequency of cotyledonary embryos developing into normal plants. Normal plantlets were regenerated from G. davidsonii, G. klotzschianum, G. raimondii, and G. stocksii. Only abnormal plantlets were obtained in G. aridum. This work will contribute to broadening the number of regenerable cotton species and provide foundations for somatic hybridization in cotton to create new germplasm.

[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.

Production and characterization of somatic hybrids between upland cotton (Gossypium hirsutum) and wild cotton (G. klotzschianum Anderss) via electrofusion.

Symmetric somatic hybrid plants between Gossypium hirsutum Coker 201 and G. klotzschianum were obtained through electrofusion. The fusion products were cultured in KM8P medium supplemented with 2.685 microM alpha-naphthaleneacetic acid and 0.465 microM kinetin, and the regenerated plants were morphologically, genetically, and cytologically characterized. Nuclear-DNA flow cytometric analysis revealed that the plants tested (31 of 40) had a relative DNA content close to the total DNA contents of the two parents. Subsequent genome DNA analysis using random amplified polymorphic DNA markers revealed 16 of 18 plants were true somatic hybrids. Cytological investigation of the metaphase root-tip cells of seven hybrids revealed there were 72-81 chromosomes in the hybrids, a value close to the expected 78 chromosomes. The morphology of the hybrids was distinct from that of the parents and from that of the regenerants from protoplasts of Coker 201. Somatic hybridization represents a potent and novel tool for transferring genomes of wild cottons containing economically important traits to cultivars in breeding programs. This is the first report on the regeneration of somatic hybrids via protoplast fusion in 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.