Detection of genetic diversity using RAPD-PCR and sugar analysis in watermelon [Citrullus lanantus (Thunb.) Mansf.] germplasm

RAPD (random amplified polymorphic DNA) markers generated by 15 arbitrary decamers were used to determine the frequency of DNA polymorphism in 39 watermelon [Citrullus lanantus (Thunb.) Mansf.] germplasms. Of the 15 primers tested, all except 1 (primer 275) directed the amplification of polymorphic products. A total of 162 amplification products were generated across all 39 genotypes. Among the 162 fragments, 35 (21%) appeared to be reliable polymorphic markers. The mean value by marker difference in this comparison was 0.24, and the highest, 0.69. Eight RAPD markers could be utilized in the unique variety discrimination 8 watermelon genotypes. From the phenograms constructed by UPGMA based on the comparison of RAPD markers, four clusters were resolved. Each group was also characterized and identified with morphological and genetic characteristics for each genotype. The free sugars of the edible parts of watermelons were analyzed by HPLC (high-performance liquid chromatography). Results from the phylogenetic analysis of band sharing data were consistent with sweetness as measured by HPLC. In conclusion, RAPD assays can be used for providing alternative markers for identifying genotypes and quantitative characteristics in watermelon.     

Identification of expressed sequence tags of watermelon (Citrullus lanatus) leaf at the vegetative stage

A cDNA library was constructed using mRNA prepared from leaves of watermelon [Citrullus lanatus (Thunb.) Matsum&Nakai] at the vegetative stage. Randomly selected cDNA clones were sequenced in order to identify potentially informative genes. Database comparisons indicated that out of the 704 watermelon cDNA clones, 399 clones (56.7 %) revealed a high degree of sequence similarity to genes from other organisms. These expressed sequence tag clones were divided into ten categories depending upon gene function. Since this kind of experiment has not previously been carried out in this genome, random nucleotide sequencing of these cDNAs could contribute considerable information concerning the novel genes in this organism. Received: 10 July 1999 / Revision received: 20 December 1999 / Accepted: 11 January 2000     

Partial sequence analysis of 130 randomly selected maize cDNA clones.

As part of a project to identify novel maize (Zea mays L. cv B73) genes functionally, we have partially sequenced 130 randomly selected clones from a maize leaf cDNA library. Data base comparisons revealed seven previously sequenced maize cDNAs and 18 cDNAs with sequence similarity to related maize genes or to genes from other organisms. One hundred five cDNAs show little or no similarity to previously sequenced genes. Our results also establish the suitability of this library for large-scale sequencing in terms of its large insert size, proper insert orientation, and low duplication rate.     

Genetics of seed-coat colour in Citrullus lanatus (Thunb.) Mansf.

Summary Intervarietal crosses in watermelon, Citrullus lanatus (Thunb.) Mansf., involving six parents with black (J18-1 and J 75), brown (J56-1 and N.H. Midget), red (Bykovski-199) or light cream (Red Nectar) seed-coat colour were made. Parents, F₁, F₂ and backcross populations were evaluated for their phenotypic expressions with regard to the seed-coat colours involved. Black colour was monogenically dominant over brown light cream and red colour of seed-coat separately or independently. Red colour was dominant over light cream colour of seed-coat by a single pair of genes. The light cream colour was recessive to the brown seed-coat colour of watermelon where a single pair of genes was involved.     

4龄中华鲟垂体的EST分析

表达序列标签 (Expressed sequence tag, EST) 是鉴定基因表达规律和发现新基因的一种有效的分子生物学手段。为了能在中华鲟(Acipenser sinensis Gray) 中发现与生长和生殖内分泌调控相关的基因,我们构建了中华鲟垂体的SMART cDNA质粒文库。垂体是调节生长和生殖内分泌的重要器官。在本研究中,通过测序筛选得到了944个EST克隆,将所得EST 与 GenBank 数据库中的序列进行比对, 结果表明,802 (84.96%) 个克隆可以找到同源序列,共代表461个基因, 其中含132个已知功能基因;而 142 (15.04%) 个克隆不能找到同源序列。研究发现,在所有基因中,阿黑皮素原基因 (Proopiomelanocortin, POMC) 是出现次数最高的基因,占总EST数的10.17%, 显示出其在垂体中的重要地位。我们还发现了7个未知功能的基因并重点研究了其在心脏、肝脏、脾脏、肾脏、肌肉、精巢、卵巢和垂体等组织中的表达特异性。结果发现,4个基因：EG009334、EG009337、EG009338 和 EG009340为垂体特异性表达或垂体和卵巢特异性表达。对这些基因进一步的功能研究将有利于我们更好地了解中华鲟生长和生殖内分泌调控的分子机制。     

大片吸虫成虫cDNA表达文库的构建及其表达序列标签初步分析

为发现潜在的抗大片吸虫病的候选疫苗分子,控制大片形吸虫病,本研究以大片吸虫成虫为材料,应用SMARTTcDNA文库构建技术,构建了以表达载体λTriplEx2为基础的大片吸虫成虫cDNA表达文库。经测定,库容量为1·08×106PFU/ml ,重组率为96·6 %,扩增后的文库滴度为2·41×109PFU/ml ,插入片段平均大小约为1 000 bp;经大肠杆菌BM25·8质粒化后,从文库中随机挑选40个重组克隆测序,获得32条有效ESTs ;经BLASTX和BLASTn程序检索和分析,发现有9条ESTs代表已知基因, 16条ESTs相似性较低或无匹配,列为新基因。9条已知基因代表了半胱氨酸蛋白酶、卵壳蛋白、钙连接蛋白等三类功能蛋白,其余新基因也暗示与信号传导、蛋白合成、免疫刺激等基因相关,具有潜在的研究价值[动物学报51 (5) : 879 -883 , 2005]。     

西瓜种质资源遗传亲缘关系的RAPD分析

利用 RAPD 技术对国内外32份西瓜主栽品种与其骨干亲本及野生类型的遗传亲缘关系进行了研究。从720个随机引物(10bp)中筛选出15个能产生稳定多态性的引物用于 RAPD 反应,共扩增出104条 DNA 带,其中多态性 DNA 条带43条,占41.35%,平均每个引物扩增的 DNA 条带的数目为7.0条。聚类分析将供试材料分为6个类群:1个东亚生态型类群、1个美国生态型类群、2个中间生态型类群和2个非洲野生型类群,与传统的西瓜生态型分类基本吻合。每个生态型类群都有其特有的扩增(缺失)条带,同时分析了同一生态型中各个品种之间的亲缘关系及其品种的特异条带。本实验结果不仅从分子水平验证了西瓜是遗传基础狭窄的作物,而且在分子水平对西瓜传统分类与地理生态型分类进行了分析。     

Expressed sequence markers for genetic analysis of bulb onion (Allium cepa L.)

Sequencing of cDNA clones previously screened for ability to reveal RFLPs in bulb onion has been completed and a further 128 ESTs from 111 clones have been deposited in public databases. A putative function was assigned to 66% (84/128) of ESTs by BLASTX searches against public databases and FASTA comparisons were used to determine similarity among clones, including those which detected linked RFLP loci. Cleavage amplified polymorphisms (CAPs) and single-stranded conformation polymorphisms (SSCP) were evaluated as strategies for converting onion expressed sequence tags (ESTs) into PCR-based assays for gene mapping. We screened 14 ESTs with 8 to 12 restriction enzymes and detected two CAPs, which mapped in the ’Brigham Yellow Globe’ (BYG15–23)×’Ailsa Craig’ (AC43) mapping population. A wider survey of CAPs for ESTs among eight bulb onion populations with six frequently cutting restriction enzymes detected variation, but too little to be practical for routine gene mapping. By contrast, non-radioactive SSCP of amplicons from 3′ UTRs of ESTs was found to detect useful levels of variation within bulb onion germplasm. In addition to SSCPs, homo- and hetero-duplex polymorphisms (duplex polymorphisms) were also frequently observed on the same gels. Of a total of 31 ESTs surveyed, 26 exhibited SSCP/duplex variation among bulb onion populations. SSCP/duplex polymorphisms in 11 ESTs were mapped in the ’BYG15–23’×’AC43’ family and, of these, ten were linked to an RFLP locus revealed by the original cDNA. The SSCP/duplex assays of five additional ESTs showed Mendelian segregations in the ’Colossal Grano’×’Pukekohe Longkeeper’ (P12) F₂ population. Two of these markers were linked, as predicted from linkage of their corresponding RFLPs in the ’BYG15–23’×’AC43’ family. Ninety two percent (12/13) of EST PCR products that amplified in Allium roylei exhibited marked differences in SSCP patterns from bulb onion. ESTs for invertase and sucrose-sucrose fructosyltransferase were mapped by SSCP and an ATP sulfurylase gene cloned by RT-PCR revealed SSCP/ duplex polymorphism within bulb onion. These results demonstrate that SSCP/duplex is an efficient and economical technique for exploiting onion EST information for gene mapping in onion. Received: 18 September 2000 / Accepted: 15 February 2001     

Mycorrhizal colonisation improves fruit yield and water use efficiency in watermelon (Citrullus lanatus Thunb.) grown under well-watered and water-stressed conditions

The effect of arbuscular mycorrhizal (AM) colonisation by Glomus clarum on fruit yield and water use efficiency (WUE) was evaluated in watermelon (Citrullus lanatus) cv. Crimson Sweet F1 under field conditions. Treatments were: (1) well-watered plants without mycorrhizae (WW-M), (2) well-watered plants with mycorrhizae (WW+M), (3) water- stressed plants without mycorrhizae (WS-M) and (4) water-stressed plants with mycorrhizae (WS+M). When soil water tension readings reached –20 and –50 kPa for well-watered (WW) and water-stressed (WS) treatments, respectively, irrigation was initiated to restore the top soil to near field capacity. Water stress reduced watermelon shoot and root dry matter, fruit yield, water use efficiency but not total soluble solids (TSS) in the fruit, compared with the non-stressed treatments. Mycorrhizal plants had significantly higher biomass and fruit yield compared to nonmycorrhizal plants, whether plants were water stressed or not. AM colonisation increased WUE in both WW and WS plants. Macro- (N, P, K, Ca and Mg) and micro- (Zn, Fe and Mn) nutrient concentrations in the leaves were significantly reduced by water stress. Mycorrhizal colonisation of WS plants restored leaf nutrient concentrations to levels in WW plants in most cases. This is the first report of the mitigation of the adverse effect of water stress on yield and quality of a fruit crop.     

Two cDNA clones coding for the legumin protein of Pisum sativum L. contain sequence repeats

The sequence of two cDNA clones coding for the whole of the -subunit and most of the -subunit of legumin are presented together with a considerable amount of protein sequence data to confirm the predicted amino acid sequence. A unique feature shown by these cDNAs is the presence of three 56 base pair tandem repeats in the region encoding the C terminal of the polypeptide. The tandem repeats are also exhibited in the predicted polypeptide sequence as three 18 amino acid repeats which contain extremely high proportions of polar, mainly acidic, residues. The new sequences are compared to the previously published sequence of some shorter legumin cDNAs (Nature 295: 76–79). In the region where the sequences overlap, the previous cDNAs differ from the new ones by only a few base substitutions but most of the repeated region is not present though the sequences on either side are. The possibility that the absence of the repeats may reflect the difference between two types of legumin gene, rather than an artefact of the cloning of the cDNAs, is discussed.