Molecular cloning and localization of a novel cotton annexin gene expressed preferentially during fiber development.

Annexins constitute a family of multifunction and structurally related proteins. These proteins are ubiquitous in the plant kingdom, and are important calcium-dependent membrane-binding proteins that participate in the polar development of different plant regions such as rhizoids, root caps, and pollen tube tips. In this study, a novel cotton annexin gene (designated as GhFAnnx) was isolated from a fiber cDNA library of cotton (Gossypium hirsutum). The full-length cDNA of GhFAnnx comprises an open reading frame of 945 bp that encodes a 314-amino acid protein with a calculated molecular mass of 35.7 kDa and an isoelectric point of 6.49. Genomic GhFAnnx sequences from different cotton species, TM-1, Hai7124 and two diploid progenitor cottons, G. herbaceum (A-genome) and G. raimondii (D-genome) showed that at least two copies of the GhFAnnx gene, each with six exons and five introns in the coding region, were identified in the allotetraploid cotton genome. The GhFAnnx gene cloned from the cDNA library in this study was mapped to the chromosome 10 of the A-subgenome of the tetraploid cotton. Sequence alignment revealed that GhFAnnx contained four repeats of 70 amino acids. Semi-quantitative reverse transcriptase-polymerase chain reaction revealed that GhFAnnx is preferentially expressed in different developmental fibers but its expression is low in roots, stems, and leaves. Subcellular localization of GhFAnnx in onion epidermal cells and cotton fibers suggests that this protein is ubiquitous in the epidermal cells of onion, but assembles at the edge and the inner side of the apex of the cotton fiber tips with brilliant spots. In summary, GhFAnnx influences fiber development and is associated with the polar expansion of the cotton fiber during elongation stages.

Construction of a bacterial artificial chromosome library of TM-1, a standard line for genetics and genomics in Upland cotton.

A bacterial artificial chromosome (BAC) library was constructed for Gossypium hirsutum acc. TM-1, a genetic and genomic standard line for Upland cotton. The library consists of 147 456 clones with an average insert size of 122.8 kb ranging from 97 to 240 kb. About 96.0% of the clones have inserts over 100 kb. Therefore, this library represents theoretically 7.4 haploid genome equivalents based on an AD genome size of 2 425 Mb. Clones were stored in 384 384(-) well plates and arrayed into multiplex pools for rapid and reliable library screening. BAC screening was carried out by four-round polymerase chain reactions using 23 simple sequence repeats (SSR) markers, three sequence-related amplified polymorphism markers and one pair of primers for a gene associated with fiber development to test the quality of the library. Correspondingly, in total 92 positive BAC clones were identified with an average four positive clones per SSR marker, ranging from one to eight hits. Additionally, since these SSR markers have been localized to chromosome 12 (A12) and 26 (D12) according to the genetic map, these BAC clones are expected to serve as seeds for the physical mapping of these two homologous chromosomes, sequentially map-based cloning of quantitative trait loci or genes associated with important agronomic traits.

Establishment of a multi-color genomic in situ hybridization technique to simultaneously discriminate the three interspecific hybrid genomes in gossypium.

To identify alien chromosomes in recipient progenies and to analyze genome components in polyploidy, a genomic in situ hybridization (GISH) technique that is suitable for cotton was developed using increased stringency conditions. The increased stringency conditions were a combination of the four factors in the following optimized state: 100:1 ratio of blocking DNA to probe, 60% formamide wash solution, 43 degrees C temperature wash and a 13 min wash. Under these specific conditions using gDNA from Gossypium sturtianum (C(1)C(1)) as a probe, strong hybridization signals were only observed on chromosomes from the C(1) genome in somatic cells of the hybrid F(1) (G. hirsutum x G. sturtianum) (A(t)D(t)C(1)). Therefore, GISH was able to discriminate parental chromosomes in the hybrid. Further, we developed a multi-color GISH to simultaneously discriminate the three genomes of the above hybrid. The results repeatedly displayed the three genomes, A(t), D(t), and C(1), and each set of chromosomes with a unique color, making them easy to identify. The power of the multi-color GISH was proven by analysis of the hexaploid hybrid F(1) (G. hirsutum x G. australe) (A(t)A(t)D(t)D(t)G(2)G(2)). We believe that the powerful multi-color GISH technique could be applied extensively to analyze the genome component in polyploidy and to identify alien chromosomes in the recipient progenies.

Simple sequence repeat genetic linkage maps of A-genome diploid cotton (Gossypium arboreum).

This study introduces the construction of the first intraspecific genetic linkage map of the A-genome diploid cotton with newly developed simple sequence repeat (SSR) markers using 189 F(2) plants derived from the cross of two Asiatic cotton cultivars (Gossypium arboreum L.) Jianglingzhongmian x Zhejiangxiaoshanlüshu. Polymorphisms between the two parents were detected using 6 092 pairs of SSR primers. Two-hundred and sixty-eight pairs of SSR primers with better polymorphisms were picked out to analyze the F(2) population. In total, 320 polymorphic bands were generated and used to construct a linkage map with JoinMap3.0. Two-hundred and sixty-seven loci, including three phenotypic traits were mapped at a logarithms of odds ratio (LOD) > or = 3.0 on 13 linkage groups. The total length of the map was 2 508.71 cM, and the average distance between adjacent markers was 9.40 cM. Chromosome assignments were according to the association of linkages with our backbone tetraploid specific map using the 89 similar SSR loci. Comparisons among the 13 suites of orthologous linkage groups revealed that the A-genome chromosomes are largely collinear with the A(t) and D(t) sub-genome chromosomes. Chromosomes associated with inversions suggested that allopolyploidization was accompanied by homologous chromosomal rearrangement. The inter-chromosomal duplicated loci supply molecular evidence that the A-genome diploid Asiatic cotton is paleopolyploid.

Cloning and characterization of cotton GhBG gene encoding beta-glucosidase.

Beta-1,4-glucosidase (BG, EC3.2.1.21), one of three cellulases, is a widespread family of enzymes involved in the metabolism of cell wall polysaccharides in both prokaryocytes and eukaryotes. Here, we report the isolation of a full-length cDNA encoding beta-1,4-glucosidase protein (designated as GhBG) and its putative function in the process of fiber development and in yeast. Through random sequencing of the cotton fiber cDNA library from 7235 germplasm line, with elite fiber quality in Gossypium hirsutum L. and utilizing the 5' rapid amplification of cDNA ends (RACE) technique, a 2133 bp cDNA clone encoding a cotton fiber specifically expressed protein (accession number: DQ103699) was isolated. GhBG was composed of a 1884 bp open reading frame (ORF) encoding 627 amino acid residues. This putative protein had an isoelectric point of 8.17, a calculated molecular weight of 68.78 KD and a signal peptide with 23 amino acid residues at the N-terminal. RT-PCR analysis indicated GhBG was specifically expressed in fiber cells and was highly abundant in 5-17 day post anthesis (DPA). It was not, however, expressed in root, hypocotyls or leaves. Southern blotting analysis showed there were two copies of GhBG in the upland cotton genome; most likely contained in sub-genome A and sub-genome D. GhBG was then integrated into a yeast expression vector, pREP-5N and electro-transformed into fission yeast Schizosaccharomyces pombe Q-01. The results demonstrated that GhBG led to a significant increase in cell length and width and a remarkable decrease of the length/width ratio. Compared to vector control transformants, cells were significantly larger and rounder and their growth velocity was also reduced.

QTL mapping for plant architecture traits in upland cotton using RILs and SSR markers.

Xiangzamian 2 (XZM2) is the most widely cultivated cotton hybrid in China. By crossing two parents Zhongmiansuo12 and 8891 and upon subsequent selfings, we got F8 and F9 populations having 180 recombinant inbred lines. Ten plant architecture traits were investigated in two years with this population. A genetic map was constructed mainly with SSR markers. Quantitative trait loci (QTL) conditioning plant architecture traits were determined at the single-locus and double-locus levels. The results showed that epistastic effects as well as additive effects of QTL played an important role as the genetic basis of cotton plant architecture. The QTL detected in our research might provide new information on improving plant architecture traits. The polymorphism of molecular markers between ZMS12 and 8891 were quite limited, while significant differences between their phenotypes were found and the hybrid XZM2 expressed high heterosis in yield. All these could be partly explained by the effect of epistatic QTL.

Diallel analysis of superior fiber quality properties in selected upland cottons.

Twenty cross combinations were produced using a complete diallel-mating system with five varieties or lines that differed in fiber properties in Upland cotton to determine the inheritance and breeding merits of superior fiber properties. Evaluations of parents and F1 ' s were conducted in two years. The results showed that fiber length uniformity was greatly affected by environmental factors, whereas the other fiber properties were mainly controlled by genetic factors. There were no significant interaction effect of environment with genotype for fiber strength or length, but there were significant environment interactions with additive and maternal affects for Micronaire, and with the dominance effects for elongation. There were no maternal effect, and additive effects predominated for the all fiber properties. Additive heritability was high for fiber strength and length, 77.6% and 73.2% respectively; for Micronaire, it was 45.2%, while the dominance effect was 11.5%, which was the highest among fiber properties. Micronaire had significant heterosis over mid-parent based on population mean (3.2%), while the other fiber properties showed no heterosis. Therefore, the performance of fiber properties in F1 ' s can be predicted from the average value of both parents. Since the additive heritability of strength, length, and fineness of fiber were high, these traits can be selected in early generations in breeding for high quality fiber properties.

Molecular marker assisted selection and pyramiding of two QTLs for fiber strength in upland cotton.

Based on two major QTLs that control high fiber strength which originated from an elite fiber germ-plasm line 7235 (Gossypium hiusutum L.), the efficiency of molecular marker-assisted selection (MAS) was investigated using two populations from pedigree selection and modified backcrossing pyramiding developed for the breeding purpose. Simian 3 (SM3), a widely planted variety in the Yangtze River Valley, and 7235 were used as parents to develop the two populations. In the two major QTLs for fiber strength from 7235, QTLfs-1 could explain more than 30% of the phenotypic variation (PV) in the (7235 x TM-1) F2 population. QTLfs-2 was at first identified in another super quality fiber line HS427-10 from (HS427-10 x TM-1) F2 population with 12.5% of PV explanation,which was further also identified in 7235 line but was non-allelic with QTLfs-1. The result of molecular marker-assisted selection for fiber strength showed that the genetic effect of the QTLfs-1 was stable under different environmental conditions, and its molecular marker-assisted selection showed significant selective efficiency among breeding populations with different genetic backgrounds. QTLfs-2 also showed high selective efficiency in advanced generation populations though its effect was a little lower than the former. When QTLfs-1 was selected simultaneously with 2 molecular markers with known genetic distance, the selection efficiency for the fiber strength was greatly increased. The pyramiding for two QTLs that control high fiber strength by MAS greatly improved the selection efficiency for cotton fiber strength. This report provides a successful example of MAS pyramiding for QTL for favorable traits in breeding programs.

Gossypium barbadense genome sequence provides insight into the evolution of extra-long staple fiber and specialized metabolites.

Of the two cultivated species of allopolyploid cotton, Gossypium barbadense produces extra-long fibers for the production of superior textiles. We sequenced its genome (AD)2 and performed a comparative analysis. We identified three bursts of retrotransposons from 20 million years ago (Mya) and a genome-wide uneven pseudogenization peak at 11-20 Mya, which likely contributed to genomic divergences. Among the 2,483 genes preferentially expressed in fiber, a cell elongation regulator, PRE1, is strikingly At biased and fiber specific, echoing the A-genome origin of spinnable fiber. The expansion of the PRE members implies a genetic factor that underlies fiber elongation. Mature cotton fiber consists of nearly pure cellulose. G. barbadense and G. hirsutum contain 29 and 30 cellulose synthase (CesA) genes, respectively; whereas most of these genes (>25) are expressed in fiber, genes for secondary cell wall biosynthesis exhibited a delayed and higher degree of up-regulation in G. barbadense compared with G. hirsutum, conferring an extended elongation stage and highly active secondary wall deposition during extra-long fiber development. The rapid diversification of sesquiterpene synthase genes in the gossypol pathway exemplifies the chemical diversity of lineage-specific secondary metabolites. The G. barbadense genome advances our understanding of allopolyploidy, which will help improve cotton fiber quality.

[A and D genome evolution in Gossypium revealed using SSR molecular markers].

Genetic diversity analysis of diploid and allotetraploid cotton species was carried out using SSR molecular markers by selecting representative species having A and (or) D genome in Gossypium. Ten diploid cotton species in A and D genomes had high polymorphism and the molecular cluster was consistent with Gossypium classification previously reported by Fryxell. From molecular level, G. gossypioides belonging to D genome had low similarity matrix compared with other diploid D genome species. Diploid cotton species separately having A or D genome had their high similarity matrix. The research supported that G. gossypioides was the most original cotton species among all D genome species, different genomes in Gossypium had common origin and made evolution separately. To better understand genetic events that accompany allopolyploid formation, we add 2 cultivated allotetraploid cotton species in our research materials. However, the results showed that it was not appropriate to study evolution of A and D genome in Gossypium using cultivated allotetraploid cotton species. In order to solve the question, original arboreum, herbaceum and wild types of allotetraploid cotton species should be adopted. Further evolution research on cotton species based on the transcription level of cotton genome is being carried on.

Cloning and expression analysis of a bZIP cDNA in Gossypium hirsutum L.

A new full-length cDNA clone was isolated from the fiber cDNA library of Gossypium hirsutum L. The cDNA, designated GhbZIP,encoded a polypeptide of 645 amino acids. GhbZIP protein had the structure characteristics of plant bZIP proteins, including two conserved domains, DUF630 and DUF632 with unknown functions, a proline-rich domain and a phenylalanine-rich domain. Meanwhile, the protein contained a leucine zipper-like motif in DUF632. The hydropathy analysis showed that the GhbZIP was a membrane protein. GhbZIP gene was preferentially expressed in ovule and fiber cells since three days post-anthesis, as indicated it might be involved in the transcription regulation of genes during cotton fiber elongation.

[Major-polygene effect analysis of super quality fiber properties in upland cotton (G. hirsutum L.)].

The modern textile industry depends on the improvement of fiber quality, especially strength to meet the needs of higher spinning speed. Inheritance of super quality fiber properties in Upland cotton was conducted in the present paper. P1, P2, F1, B1, B2 and F2 of eight crosses from five parents with different fiber strength, i.e. 7235 x TM1, TM1 x 7235, HS42 x TM1, PD69 x TM1, MD51 x TM1, 7235 x HS42, 7235 x PD69 and HS42 x PD69, and F2:3 for 7235 x TM1, were used in the study. The materials were planted in Nanjing or Hainan in 1998 and 1999, the individual plant fiber samples were tested with HVI system in Cotton Research Institute of CAAS at Anyang. The segregation analysis methods for major genes plus polygene mixed inheritance model developed by Gai were used to identify the genetic system of fiber qualities. The results from joint analyses of multiple segregating generations as well as single segregating generations, especially for F2:3, showed one major gene plus polygene mixed inheritance model in all fiber quality characters. The heritability values of major gene in F2 of 7235 x TM1 with great parent difference were estimated as 19.6% for fiber strength, 32.0% for micronaire and 13.9% for fiber length, but little in B1 and B2 for fiber qualities. The fiber length showed high and positive dominant effect, but negative value or zero of major or polygene dominant effects for other fiber qualities. Therefore, Mid-parent value or tendency to lower parent in F1 for most of fiber qualities lead to low selection efficiency, which suggests that molecular assisted selection should be considered at first in the improvement of fiber qualities.

[Cloning and characterization of two beta-mannosidase cDNAs in Gossypium hirsutum L].

By using the method of PCR-based cDNA library screening, two beta-mannosidase clones, GhManA1 and GhManA2, had been isolated. GhManA1 had a length of 2692 bp coding for a polypeptide of 834 amino acids, and GhManA2 was 3209 bp which encoded a polypeptide of 976 amino acids. GhManA1 and GhManA2 shared an identical sequence of 747 amino acids in their carboxyl-terminals, but were distinctly different in their amino-terminals. Both beta-mannosidases were members of glycosyl hydrolase family 2, which had two conserved glutamine residues in their sequences as the acid-base catalyst and nucleophilic group, respectively. Most surprisingly, the first 93 amino acids in the amino-terminal of GhManA1 was highly homologous to the beta-barrel domain of ATP synthase alpha-/beta-subunit, but an analogous domain has never been found in the sequence of other non-ATP synthase protein. GhManA1 was constitutively expressed in different cotton tissues, and GhManA2 was specifically expressed in fiber cells.

Control of cotton fibre elongation by a homeodomain transcription factor GhHOX3.

Cotton fibres are unusually long, single-celled epidermal seed trichomes and a model for plant cell growth, but little is known about the regulation of fibre cell elongation. Here we report that a homeodomain-leucine zipper (HD-ZIP) transcription factor, GhHOX3, controls cotton fibre elongation. GhHOX3 genes are localized to the 12th homoeologous chromosome set of allotetraploid cotton cultivars, associated with quantitative trait loci (QTLs) for fibre length. Silencing of GhHOX3 greatly reduces (>80%) fibre length, whereas its overexpression leads to longer fibre. Combined transcriptomic and biochemical analyses identify target genes of GhHOX3 that also contain the L1-box cis-element, including two cell wall loosening protein genes GhRDL1 and GhEXPA1. GhHOX3 interacts with GhHD1, another homeodomain protein, resulting in enhanced transcriptional activity, and with cotton DELLA, GhSLR1, repressor of the growth hormone gibberellin (GA). GhSLR1 interferes with the GhHOX3-GhHD1 interaction and represses target gene transcription. Our results uncover a novel mechanism whereby a homeodomain protein transduces GA signal to promote fibre cell elongation.

The Fusarium Graminearum virulence factor FGL targets an FKBP12 immunophilin of wheat.

Wheat scab, caused by the fungal pathogen Fusarium graminearum is a devastating disease worldwide. Despite an extensive and coordinated effort to investigate this pathosystem, little progress has been made to understand the molecular basis of host-pathogen interactions, for example how the pathogen causes disease in plant. Recently, a secreted lipase (FGL1) has been identified from the fungus and shown to be an important virulence factor; however, the intrinsic function of FGL1 in plant is unknown. Here, we report the identification of the molecular components that may possibly be involved in the FGL virulence pathway using yeast two hybrid system. FGL gene was amplified from a local virulent strain (F15) and shown to be 99.5% identical to the original published FGL at the amino acid level. We showed that transient expression of this FGL gene by Agroinfiltration in tobacco leaves causes cell death further implicating the role of FGL in virulence. To identify FGL initial physical target in plant, we screened two wheat cDNA libraries using the FGL protein as the bait. From both libraries, a small FKBP-type immunophilin protein, designated wFKBP12, was found to physically interact with FGL. The direct interaction of FGL with wFKBP12 was confirmed in living onion epidermal cells by biomolecular fluorescence complementation (BiFC) assay. To investigate further, we then used wFKBP12 protein as bait and identified an elicitor-responsive protein that contains a potential Ca(2+) binding domain. Semi-quantitative PCR showed that this elicitor-responsive gene is down-regulated during the F. graminearum infection suggesting that this protein may be an important component in FGL virulence pathway. This work serves as an initial step to reveal how fungal lipases act as a general virulence factor.

[Tagging and mapping of QTLs controlling lint yield and yield components in upland cotton (Gossypium hirsutum L.) using SSR and RAPD markers].

Using interval mapping and marker simple regression methods, the QTLs of yield and its components in (Simian 3 x TM-1) F2 and F2:3, were tagged and Mapped with 39 SSR and 10 RAPD markers having polymorphism between parents screened from 301 pair SSR primers and 1040 RAPD primers. Simian 3 is being grown extensively in Yangtze River cotton-growing valley characterized as high productivity with more bolls and higher lint percent, whereas TM-1, Genetic standard in Upland cotton with more heavy boll weight. In the present report, two QTLs controlling boll size with 18.2% and 21.0% phenotype variance explained in F2:3 generation, one QTL controlling lint percent with 24.9% phenotype variance explained in F2 generation and 5.9% in F2:3 generation and one QTL controlling 100-seed weight with 15.6% phenotype variance explained in F2:3 generation were mapped in Chromosome 9. Additionally, another QTL responsible for 100-seed weight was identified and mapped at the same position in Chromosome 9 in F2:3 generation. It is worth for further to be studied whether it is one QTL for pleiotrophism or two closely linked QTLs. The molecular markers mapped and tagged closely with main QTLs of yield traits in this paper can be used for MAS in cotton high-yield breeding program.