Development of EST‐SSRs in Cucumis sativus from sequence database

Simple sequence repeat markers derived from expressed sequence tags (EST‐SSR) are potentially valuable tools for plant breeding and germplasm collection conservation, and increasingly, efforts have been made for developing this type of marker. We have identified 20 polymorphic SSR markers from cucumber ESTs deposited in public sequence database. The average allele number was 3.3 per locus, ranging from two to six alleles during screening 20 cucumber genotypes with the mean expected heterozygosity of 0.477. Amplification products were also detected by 13 pairs of primer in Cucumis melo. These informative EST‐SSR markers can be used in cucumber genetic improvement projects.     

Development of polymorphic markers from expressed sequence tags of Manihot esculenta Crantz

In this study, 49 primers were designed from sequences containing di-, tri-, tetra-, penta- and hexanucleotide motifs with a minimum of four repeats and presence of motif size polymorphisms (insertion/deletion) from cassava (Manihot esculenta Crantz) expressed sequence tags deposited in public sequence database. Each locus was subsequently screened on 29 M. esculenta Crantz obtained from 15 different countries. Cross-amplification was tested with M. esculenta Crantz (ssp. flabellifolia) and four different Manihot species, M. chlorosticta, M. carthaginensis, M. filamentosa and M. tristis. Of these, nine loci showed polymorphic profiles within M. esculenta Crantz, which revealed two to four alleles per locus. The average unbiased and direct count heterozygosities were 0.4901 and 0.5674, respectively.     

Plastome data analysis of Cucumis melo subsp.agrestis

It is of interest to refine the taxonomic status of C. melo ssp. agrestis using its plastome data. The chloroplast size and GC% was found to be 1,56,016 bp and 36.92% respectively in Cucumis melo subsp. agrestis. The plastome of C. melo subsp. agrestis comprises of two inverted repeat (IR) regions of 25,797 bp each. It consisted of 133 genes with 88 protein-coding genes, 8 rRNA genes and 37 tRNA genes. Analysis of the C. melo ssp. agrestis plastome data will help breeders to improve the yield the crop.     

Microsatellite markers for Fragaria from ‘Strawberry Festival’ expressed sequence tags

We present 37 microsatellite primer pairs developed from a cDNA library of Fragaria ^xananassa Duch. cv. Strawberry Festival. Polymorphism was high and the number of presumptive alleles of 13 expressed sequence tag–simple sequence repeats (EST–SSRs) in 70 strawberry cultivars ranged from five to 32 per primer pairs, averaging 16.1. Cross‐species amplification was also high and ranged from 89% in Fragaria vesca L. to 100% in the progenitor species of octoploid strawberry, Fragaria chiloensis (L.) Duch. and Fragaria virginiana Duch.     

Development of simple sequence repeat markers from expressed sequence tags of the black tiger shrimp (Penaeus monodon)

In this study, we describe the development of expressed sequence tag-simple sequence repeat markers from expressed sequence tags of the black tiger shrimp (Penaeus monodon) deposited in public sequence databases. A total of 46 primer pairs were designed and screened on 26 individuals of P. monodon from a natural population. Of these, 16 primer pairs showed polymorphic profiles with between two and five alleles per locus. The average unbiased and direct count heterozygosities were 0.4662 and 0.3516, respectively. Cross-amplification was tested with five individuals of Penaeus vannamei and polymorphic products were detected at five loci.     

Fifteen polymorphic simple sequence repeat markers from expressed sequence tags of Liriodendron tulipifera

We developed and evaluated simple sequence repeat (SSR) markers derived from expressed sequence tags (ESTs) of Liriodendron tulipifera. Characteristics of 15 EST‐SSR loci were investigated using 33 L. tulipifera individuals. The number of alleles per locus ranged from two to five. The expected and observed heterozygosities ranged from 0.216 to 0.751 and from 0.182 to 0.97, respectively. These loci were further tested for their cross‐species transferability to Liriodendron Chinense. Because of their high level of polymorphism and transferability, our 15 single‐locus EST‐SSR markers will be valuable tools for research on mating system, population genetics and systemic evolution of Liriodendron.     

Development of 16 polymorphic simple sequence repeat markers for Lycoris longituba from expressed sequence tags

Lycoris longituba is a tulip-like ornamental plant in China. We report on the data mining of L. longituba expressed sequence tags (ESTs) to generate simple sequence repeat (SSRs) markers. Eighteen EST-SSRs were isolated and validated for 32 individuals. These markers will be valuable for studying the genetic structure and diversity of populations for L. longituba.     

Seed coat structure and oxygen availability control lowtemperature germination of melon (Cucumis melo) seeds

The involvement of the seed coat in low-temperature germination of melon seeds was examined in two accessions differing in their ability to germinate at 14°C: Noy Yizre'el (a cold-sensitive cultivar) and Persia 202 (a cold-tolerant breeding line). Decoating resulted in full germination of Noy Yizre'el at 14°C, but splitting the coat increased germination only partially. Thus, the inhibition of Noy Yizre'el germination at 14°C is not due to physical constraint on radicle protrusion. At 25°C, seeds of both accessions submerged in water or agar germinated fully as long as the hilum aperture remained uncovered. Submerging the whole seed, or covering the hilum with lanolin, strongly depressed germination of Noy Yizre'el but not of Persia 202. Accessions differed in germination response to decreasing O₂ concentration, with Noy Yizre'el showing higher sensitivity to hypoxia. These differences were correlated with differences in seed coat structure as well as in embryo sensitivity to hypoxia. Intercellular spaces in the outer layer of the seed coat were evident in the more tolerant Persia 202, while in the sensitive Noy Yizre'el this layer was completely sealed. Sensitivity to hypoxia increased at 15°C as compared with 25°C, the increase being greater in Noy Yizre'el. It is proposed that the seed coat-imposed dormancy at low temperature in Noy Yizre'el is the combined result of more restricted oxygen diffusion through the seed coat and a greater embryo sensitivity to hypoxia, rather than to physical constraints of radicle break-through or impairment of imbibition.     

Role of endogenous gibberellins in germination of melon (Cucumis melo) seeds

The effect of the plant growth retardants ancymidol. mefluidide and uniconazole on germination of two melon accessions differing in their ability to germinate at 14°C was examined. The accessions were the cold sensitive Noy Yizre'el and the cold tolerant Persia 202. The three growth retardants were able to delay the germination of intact Noy Yizre'el seeds, but did not affect that of intact Persia 202 seeds. On the other hand germination of decoated seeds of both accessions was unaffected by these inhibitors at normal oxygen concentration, but was inhibited at 5% oxygen. When gibberellin-like activity was measured by a dwarf rice biological assay following HPLC fractionation, it was found that seeds of Persia 202 contained much more gibberellin-like activity than Noy Yizre'el seeds. Among the extracted compounds several endogenous gibberellins were identified by combined gas chromatography-mass spectrometry (GC-MS). They included GA₄, GA₂₀, GA₁ and GA₃ in Noy Yizre'el and GA₃₄, GA₂₀, GA₁ and GA₈ in Persia 202. It is suggested that the better germination of intact Persia 202 seeds, compared to Noy Yizre'el seeds at low temperature and low oxygen concentration, is due to a higher endogenous level of GA and a better seed coat permeability to oxygen.     

Data mining for simple sequence repeats in expressed sequence tags from barley,maize, rice,sorghum and wheat

Plant genomics projects involving model species and many agriculturally important crops are resulting in a rapidly increasing database of genomic and expressed DNA sequences. The publicly available collection of expressed sequence tags (ESTs) from several grass species can be used in the analysis of both structural and functional relationships in these genomes. We analyzed over 260000 EST sequences from five different cereals for their potential use in developing simple sequence repeat (SSR) markers. The frequency of SSR-containing ESTs (SSR-ESTs) in this collection varied from 1.5% for maize to 4.7% for rice. In addition, we identified several ESTs that are related to the SSR-ESTs by BLAST analysis. The SSR-ESTs and the related sequences were clustered within each species in order to reduce the redundancy and to produce a longer consensus sequence. The consensus and singleton sequences from each species were pooled and clustered to identify cross-species matches. Overall a reduction in the redundancy by 85% was observed when the resulting consensus and singleton sequences (3569) were compared to the total number of SSR-EST and related sequences analyzed (24606). This information can be useful for the development of SSR markers that can amplify across the grass genera for comparative mapping and genetics. Functional analysis may reveal their role in plant metabolism and gene evolution.     

Characterization of expressed sequence tag‐derived microsatellites from the kuruma prawn,Marsupenaeus japonicus

The kuruma prawn, Marsupenaeus japonicus, is a commercially important benthic marine crustacean in East Asia. Understanding the species’ population structures will be very important for its proper stock assessment and management strategy. Herein, 13 polymorphic microsatellite loci originating from expressed sequence tag libraries of M. japonicus were isolated and characterized. Number of alleles per locus ranged from two to 15, observed and expected heterozygosities ranged from 0.250 to 0.906 and from 0.310 to 0.932, respectively. The adequate level of variability within the population renders these microsatellite loci ideal markers for population genetic analysis of M. japonicus.     

甜瓜EST序列中微卫星的分布特征

GenBank中35547条甜瓜EST经去冗余处理后,得到总长度为250.3Mb的无冗余EST34438条。这些序列中有2813个微卫星简单重复序列（Simple sequence repeat,SSR）,分布于2107条EST中,出现频率为8.16%,平均分布距离为8.90kb。三核苷酸重复是主导重复类型,占SSR总数的47.14%;其次是二核苷酸和单核苷酸重复,分别占SSR总数的20.72%和16.99%。AAG/TTC是优势重复基元,占微卫星总数的29.26%,AG/CT和A/T分别占14.61%和16.25%。在所有的SSR中,重复次数为4～10次的占70.32%,长度为12～20bp的占51.12%。并对这些SSR的多态性潜能进行了评价。     

甜瓜离体再生继代培养中玻璃化现象的研究

为提高甜瓜离体培养的再生率和转基因效率,以优质甜瓜品种‘伽师瓜’(‘卡拉库赛’)离体再生不定芽为外植体,通过连续多代继代培养,对引起玻璃化苗现象的几个主要因素进行了研究。结果表明,在甜瓜离体再生继代培养中,外植体继代次数是影响玻璃化发生的主要因素,同时培养基中的6-BA浓度偏高、琼脂浓度偏低以及蔗糖浓度偏低或偏高等可导致玻璃化苗的增加。培养基中较低的6-BA浓度(0~0.2 mg/L),琼脂浓度为6 g/L,蔗糖浓度为25 g/L以及添加活性炭等措施可有效地降低甜瓜玻璃化苗的发生。     

Identification and characterization of 18 novel polymorphic microsatellite makers derived from expressed sequence tags in the Pacific oyster Crassostrea gigas

We report the development of 18 new polymorphic microsatellite DNA markers derived from Crassostrea gigas expressed sequences tags. Genotyping of 48 wild adult oysters sampled from Marennes-Oléron bay (France) revealed 12 to 48 alleles per locus. Observed and expected heterozygosity levels ranged from 0.64 to 1 and from 0.77 to 0.97, respectively. The development of these new markers creates a useful complementary tool for population genetics studies, parentage analysis and mapping in Pacific oyster, a species of major aquacultural and ecological importance.     

‘弥河银瓜’高效植株再生体系的建立

建立了以薄皮甜瓜品种‘弥河银瓜’子叶为外植体的高效再生体系。取生长5d的甜瓜子叶为外植体,置于MS+2.0mg·L-16-BA+0.5mg·L-1IBA+3.5%蔗糖+0.7%琼脂(pH5.8)的诱导培养基上培养,可获得100%芽分化频率。在不定芽伸长过程中,生长调节剂最佳浓度为0.5mg·L-16-BA+0.1mg·L-1IAA。潮霉素是较合适的筛选抗生素。     

嫁接对薄皮甜瓜养分吸收、伤流液中激素含量和产量的影响

以薄皮甜瓜品种‘玉美人’作接穗,以白籽南瓜‘圣砧一号’为砧木进行嫁接,以自根苗为对照的结果表明,嫁接植株的南瓜根系主动吸收能力增强,其伤流量比自根植株的大,植株吸收氮钾的能力高于自根植株,而吸收磷的能力则有所降低。伤流液中玉米素（ZT）、赤霉素（GAs）和脱落酸（ABA）的浓度均低于自根植株,但ZT和GA的含量高于自根苗,而ABA的含量低于自根苗。嫁接植株的增产效果显著,其平均单瓜重和667m^2产量均高于自根植株。     

厚皮甜瓜(Cucumis melo var. reliculatus)的快速繁殖

以厚皮甜瓜 (Cucumismelovar.reliculatus)西薄洛托带腋芽茎段为外植体进行离体快速繁殖研究。结果表明 :在MS BA 0 .5～ 1 .0mg/L IAA 0 .1mg/L的培养基上利于诱导形成丛生芽 ,芽的月增殖系数达到 1 1以上 ;在 1 /2MS IAA 0 .5mg/L培养基上并经暗处理 3d最易生根 ,生根率 90 % ;在蛭石 :草炭土 =1 :1 (体积比 )基质中移栽驯化效果好。试管植株定植大田后种性不变 ,生长和结果习性优于种子苗。     

甜瓜组织培养过程中的染色体数目变异

以甜瓜组织培养过程中产生的不定芽为材料,观察了不同阶段不定芽中染色体数目发生变异的频率,同时比较了在无外加诱变剂的情况下,甜瓜根尖与组培不定芽之间最大分裂相时间的变化。     

Single-nucleotide polymorphisms detected in expressed sequence tags of melon (Cucumis melo L.).

A search was performed for single-nucleotide polymorphisms (SNP) and short insertions-deletions (indels) in 34 melon (Cucumis melo L.) expressed sequence tag (EST) fragments between two distantly related melon genotypes, a group Inodorus 'Piel de sapo' market class breeding line T111 and the Korean accession PI 161375. In total, we studied 15 kb of melon sequence. The average frequency of SNPs between the two genotypes was one every 441 bp. One indel was also found every 1666 bp. Seventy-five percent of the polymorphisms were located in introns and the 3'untranslated regions. On average, there were 1.26 SNPs plus indels per amplicon. We explored three different SNP detection systems to position five of the SNPs in a melon genetic map. Three of the SNPs were mapped using cleaved amplified polymorphic sequence (CAPS) markers, one SNP was mapped using the single primer extension reaction with fluorescent-labelled dideoxynucleotides, and one indel was mapped using polyacrilamide gel electrophoresis separation. The discovery of SNPs based on ESTs and a suitable system for SNP detection has broad potential utility in melon genome mapping.     

Microsatellite markers for mungbean developed from sequence database

A novel set of microsatellite markers for mungbean [Vigna radiata (L.) Wilczek] was developed from the public sequence database. Seventy-eight primers were designed and evaluated for polymorphism among 22 cultivated accessions. Eight polymorphic loci detected two to three alleles per locus with an average of 2.25. The observed heterozygosity varied from 0.00 to 0.18, while the expected heterozygosity ranged from 0.09 to 0.46. Among them, all eight loci showed significant departuring from Hardy-Weinberg equilibrium, while four pairs of loci displayed significant pairwise linkage disequilibrium values. All eight loci except DMB-SSR1 showed heterozygote deficiency.