Identification of Molecular Markers Associated with Semigamy in Cotton

Semigamy, controlled by one incomplete dominant gene (Se) in Pima cotton (Gossypium barbadense), is an aberrant form of sexual reproduction or apomixis, in which the sperm and egg nuclei fail to fuse and proceed to develop independently resulting in various outcomes, such as haploids, diploids, and chimeras. The objective of this study was to develop molecular markers linked to the semigamy gene using a (Pima S-1?×?57-4)?×?Pima S-1 BC₁F₁ mapping population consisting of 34 semigametic and 30 non-semigametic progenies. Random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), ATG-anchored AFLP (ATG-AFLP), sequenced tagged site (STS), and single-strand conformational polymorphism (SSCP) markers were tested. None of the apparent polymorphic RAPD, AFLP, and ATG-AFLP markers were linked to the semigamy gene, while three polymorphic markers developed from genes based on comparative microarray and differential display analyses, SSCP Se18-1800 and Se18-3000, and STS Se19, were mapped to a linkage group carrying the semigamy gene. The results present evidence that targeted gene mapping is an efficient method using markers developed from comparative gene expression studies. The current work represents the first study in identifying DNA markers associated with semigamy in plants, providing an incentive toward high-resolution mapping and isolation of the semigamy gene.     

Molecular Cloning and Characterization of GhGolS1, a Novel Gene Encoding Galactinol Synthase from Cotton (Gossypium hirsutum)

Galactinol synthase (GolS) is a key initial regulatory enzyme in the synthesis of raffinose family oligosaccharides (RFOs), sugars that play essential roles in plant growth and development. We have cloned the GhGolS1 gene (GenBank accession number: JF813792), which encodes the cotton GolS1 protein by rapid amplification of cDNA ends?Cpolymerase chain reaction (RACE?CPCR). The full-length cDNA is 1,729-bp long and encoded an open reading frame (ORF) of 343 amino acids with a deduced molecular weight of 39.2?kDa. The GhGolS1 protein shared 74?C78% identity at the amino acid level with GolS isolated from Ricinus communis, Populus trichocarpa, Solanum lycopersicum, Arabidopsis thaliana and Capsicum annuum. The corresponding genomic DNA, which contained three exons and two introns, was isolated and analyzed. The 5?? flanking region was also analyzed to identify a group of putative cis-acting elements. DNA gel blot analysis showed that in the cotton genome, the GhGolS gene family contains at least three members. Real-time PCR (RT-PCR) analysis revealed that GhGolS1 is expressed in cotton leaves, anthers and 35 DPA fibers, and its expression level in anthers is much higher than that in leaves and 35 DPA fibers. It was minimally expressed in other tissues. Additionally, the GhGolS1 protein was localized to the cell membrane. On the basis of these results, we propose that GhGolS1 has critical roles in cotton male fertility, fiber quality and seed development.     

Influence of Exogenous Fatty Acids on Cottonseed Germination

Effects of exogenous free fatty acid application on percentage germination and radicle length were investigated with more chill sensitive (Pima S-4) and less chill sensitive (Pima S-5) cottonseed (Gossypium barbadense L.) at chilling (14°C) and optimal (34°C) temperatures. Of the organic solvents able to solubilize free fatty acids, aqueous solutions as high as 5% dimethyl sulfoxide did not adversely affect germination at 34°C. Palmitic, oleic and linoleic acids (120 μM) were solubilized in 5% DMSO. At 14°C, percent germination of fatty acid treated Pima S-5 significantly increased, while no difference was observed for Pima S-4. Conversely, radicle length of fatty acid treated Pima S-4 significantly increased especially with oleate and linoleate, while no difference was detected for Pima S-5. Fatty acid supplementation influenced neither germination nor radicle length for Pima S-4 and Pima S-5 at 34°C.     

开放的差异基因表达技术研究进展

自 90年代早期发展以来 ,差异基因表达 (DGE)技术在许多领域得到了应用 .“开放”结构系统的DGE技术不需原始的生物学或序列信息 ,而且可应用于任何种群 .主要介绍 6项开放的DGE技术 :cDNA代表性差示分析 (cDNA RDA)、基因表达系统分析 (SAGE)、表达序列标签串联排列连接(TALEST) ,和早期的DGE技术差异显示 (DD)、随机引物聚合酶链反应 (AP PCR) ,以及一项受专利保护的技术———GeneCalling .通过几项重要的参数对这些技术进行了比较 ,认为DD虽然有其致命的弱点 ,但在目前仍然应用得非常广泛 .cDNA RDA能有效富增特异片段 ,扣除共有序列 ,如能和SAGE结合 ,将能进一步促进其发展 .TALEST和GeneCalling操作较简便 ,一次试验能获得大量的数据 ,但是分析这些数据比较麻烦 ,须借助另外的分析软件 .最后介绍了应用DGE技术取得的最新成果 .     

Early flowering and increased expression of a FLOWERING LOCUS T-like gene in chrysanthemum transformed with a mutated ethylene receptor gene mDG-ERS1(etr1-4)

Ethylene has an inhibitory effect on flowering in a short-day (SD) plant chrysanthemum (Chrysanthemum morifolium Ramat.). In this study, we used a hexaploid chrysanthemum ??Sei-Marine?? and found that its transgenic lines transformed with a mutated ethylene receptor gene mDG-ERS1(etr1?C4), which conferred reduced ethylene sensitivity (J. Plant Biol. 51: 424?C427, 2008), opened flowers earlier than the non-transformed control. We examined whether the accelerated flower induction in the transformant occurred through the enhanced expression of chrysanthemum genes homologous to FLOWERING LOCUS T (FT), a floral inducer gene in Arabidopsis. We cloned three cDNAs for FT homologs (CmFTL1, CmFTL2, and CmFTL3) from ??Sei-Marine??. CmFTL2 putatively encodes a non-functional gene product due to a frame shift caused by a 2 bp-deletion in the coding region. RT-PCR analysis revealed differential expression patterns of CmFTL genes in the transgenic and control lines, suggesting that these genes might be under the control of ethylene. CmFTL1/2 mRNA level was lower in a SD condition than a long-day (LD) condition. CmFTL3 mRNA accumulated abundantly under SD condition as compared with LD condition in the transgenic line. These results suggest the association of increased expression of CmFTL3 gene with the accelerated flowering in the transgenic line with reduced ethylene sensitivity.     

一个血清抑制基因的克隆

比较血清培养细胞和血清饥饿细胞的基因表达差异,获得了一段血清饥饿细胞中特异表达的cDNA序列,以此序列出发,通过搜索表达序列标签(EST),拼接出完整的基因序列,通过PCR分段克隆获得全长cDNA序列.该基因全长5 429 bp,编码框预测有791个氨基酸残基.GenBank搜索,该基因与已有的细胞周期调控基因没有同源性.所以,该基因是一个新的与细胞周期有关的基因（GenBank接受号：AY050169）.由于该基因最初发现在无血清培养条件下表达,故叫血清抑制基因（serum inhibit gene,Si-1基因）.     

Inheritance of stomatal conductance in cotton (Gossypium barbadense)

Stomatal conductance in improved Pima cotton cultivars (Gossypium barbadense) has been previously shown to be positively associated with heat resistance and yield potential. In the present study we determined the mode of inheritance of stomatal conductance in crosses of six G. barbadense parents varying in origin, degree of agronomic development and stomatal conductance. Parents included a primitive tropical cotton (B368), two obsolete cultivars (St Vincent V135, Pima 32), one modern commercial line (Pima S-6) and two elite genotypes of the Pima germplasm (P70, P73). These lines showed distinct differences in stomatal conductance, under greenhouse and field conditions. The primitive B368 had the lowest conductance, and the elite lines the highest. Generation means analysis was used to quantify genetic effects in the crosses P70 × St Vincent V135, Pima S-6 × B368, Pima S-6 × Pima 32, P73 × Pima 32 and P73 × Pima S-6. Best-fit models of the inheritance of stomatal conductance varied in complexity from a simple additive-dominance model in the cross P70 × St. Vincent V135 to models displaying digenic epistatic interactions in the remaining crosses. Significant additive mean effects for stomatal conductance were detected in all crosses. Dominance mean effects were significant in the crosses P73 × Pima 32 and P73 × Pima S-6. Broadsense heritability estimates of stomatal conductance were relatively low (0.16–0.44) in all crosses except Pima S-6 × B368 (0.74). Results also show that the mode of inheritance of stomatal conductance is multigenic, and may have maternal as well as nuclear components. Recouping higher stomatal conductance levels from genetically wider crosses appears feasible and could proceed at a moderate rate. Fixing higher levels of stomatal conductance in populations from crosses of elite germplasm may be more difficult because of the presence of dominant mean effects and digenic epistatic interactions.     

The marine red alga Chondrus crispus has a highly divergent β-tubulin gene with a characteristic 5′ intron: functional and evolutionary implications

We characterized a nuclear gene and its corresponding cDNA encoding β-tubulin (gene TubB1) of the marine red alga Chondrus crispus. The deduced TubB1 protein is the most divergent β-tubulin so far reported with only 64 to 69% amino acid identity relative to other β-tubulins from higher and lower eukaryotes. Our analysis reveals that TubB1 has an accelerated evolutionary rate probably due to a release of functional constraints in connexion with a specialization of microtubular structures in rhodophytes. It further indicates that isoform diversity and functional differentiation of tubulins in eukaryotic cells may be controlled by independent selective constraints. TubB1 has a short spliceosomal intron at its 5′ end which seems to be a characteristic feature of nuclear protein-coding genes from rhodophytes. The splice junctions of the four known rhodophyte introns comply well with the corresponding consensus sequences of higher plants in agreement with previous suggestions from phylogenetic inference that red algae and green plants may be sister groups. The paucity and asymmetrical location of introns in rhodophyte genes can be explained by differential intron loss due to conversion of genes by homologous recombination with cDNAs corresponding to reverse transcribed mRNAs or partially spliced pre-mRNAs, respectively. The identification of an intron containing TubB1 cDNA in C. crispus confirms that pre-mRNAs can escape both splicing and degradation in the nucleus prior to transport into the cytoplasm. Differential Southern hybridizations under non-stringent conditions with homologous and heterologous probes suggest that C. crispus contains a second degenerate β-tubulin gene (or pseudogene?) which, however, is only distantly related to TubB1 as it is to the more conserved homologues of other organisms.