中国重要农业害虫韭菜迟眼蕈蚊多态性EST-SSR标记的开发

【背景】韭菜迟眼蕈蚊是我国重要的农业害虫,然而它的遗传资源有限。本研究旨在开发韭菜迟眼蕈蚊EST-SSR标记,为研究不同地区的韭菜迟眼蕈蚊种群结构和遗传多样性奠定基础。【方法】从韭菜迟眼蕈蚊的表达序列标签(EST序列)中设计16对简单重复序列(SSR)引物,进一步筛选出9对具有多态性的SSR引物。【结果】从42095条unigene中确定了3383个SSR位点。利用查找到的SSR位点共设计出16对引物,进一步检测筛选发现9对引物具有多态性,引物的每个位点平均有3.33个等位基因。利用9对引物对30头韭菜迟眼蕈蚊进行检测,共获得30个等位基因,观测杂合度和期望杂合度的范围分别为0.0000~0.6875和0.0370~0.6877;其中,9个位点中有5个位点显著偏离Hardy-Weinberg平衡。【结论与意义】本研究成功从迟眼蕈蚊EST序列中筛选出9个具有多态性的微卫星位点,这为进一步分析该害虫种群的遗传结构和遗传多样性奠定了基础。     

Isolation and characterization of polymorphic nuclear microsatellite loci in <Emphasis Type="Italic">Taxus baccata</Emphasis> L.

Seven polymorphic nuclear microsatellite markers for Taxus baccata L. (English yew) were developed using an enriched-library method. An additional polymorphic SSR was obtained by testing eight primer pairs from the congeneric species Taxus sumatrana. Mendelian inheritance for the seven Taxus baccata SSRs was proved by genotyping 17 individuals and 124 megagametophytes (conifer seed haploid tissue). A total of 96 individuals from 5 different populations (10–26 samples per population) were used to estimate genetic diversity parameters. High levels of genetic diversity, with values ranging from 0.533 to 0.929 (6–28 alleles per SSR) were found. No linkage disequilibrium between pairs of loci was detected. All loci but one showed significant departures from Hardy–Weinberg equilibrium. Excess of homozygosity was probably due to high inbreeding in English yew populations, an outcome of low effective population size and/or fragmented distribution. Highly polymorphic SSRs will be used to conduct population genetic studies at different geographical scales and to monitor gene flow.     

Mining and characterizing microsatellites from citrus ESTs

Freely available computer programs were arranged in a pipeline to extract microsatellites from public citrus EST sequences, retrieved from the NCBI. In total, 3,278 bi- to hexa-type SSR-containing sequences were identified from 56,199 citrus ESTs. On an average, one SSR was found per 5.2 kb of EST sequence, with the tri-nucleotide motifs as the most abundant. Primer sequences flanking SSR motifs were successfully identified from 2,295 citrus ESTs. Among those, a subset (100 pairs) were synthesized and tested to determine polymorphism and heterozygosity between/within two genera, sweet orange (C. sinensis) and Poncirus (P. trifoliata), which are the parents of the citrus core mapping population selected for an international citrus genomics effort. Eighty-seven pairs of primers gave PCR amplification to the anticipated SSRs, of which 52 and 35 appear to be homozygous and heterozygous, respectively, in sweet orange, and 67 and 20, respectively, in Poncirus. By pairing the loci between the two intergeneric species, it was found that 40 are heterozygous in at least one species with two alleles (9), three alleles (28), or four alleles (3), and the remaining 47 are homozygous in both species with either one allele (31) or two alleles (16). These EST-derived SSRs can be a resource used for understanding of the citrus SSR distribution and frequency, and development of citrus EST-SSR genetic and physical maps. These SSR primer sequences are available upon request. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Exploiting EST databases for the development and characterization of gene-derived SSR-markers in barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.)

A software tool was developed for the identification of simple sequence repeats (SSRs) in a barley ( Hordeum vulgare L.) EST (expressed sequence tag) database comprising 24,595 sequences. In total, 1,856 SSR-containing sequences were identified. Trimeric SSR repeat motifs appeared to be the most abundant type. A subset of 311 primer pairs flanking SSR loci have been used for screening polymorphisms among six barley cultivars, being parents of three mapping populations. As a result, 76 EST-derived SSR-markers were integrated into a barley genetic consensus map. A correlation between polymorphism and the number of repeats was observed for SSRs built of dimeric up to tetrameric units. 3'-ESTs yielded a higher portion of polymorphic SSRs (64%) than 5'-ESTs did. The estimated PIC (polymorphic information content) value was 0.45 +/- 0.03. Approximately 80% of the SSR-markers amplified DNA fragments in Hordeum bulbosum, followed by rye, wheat (both about 60%) and rice (40%). A subset of 38 EST-derived SSR-markers comprising 114 alleles were used to investigate genetic diversity among 54 barley cultivars. In accordance with a previous, RFLP-based, study, spring and winter cultivars, as well as two- and six-rowed barleys, formed separate clades upon PCoA analysis. The results show that: (1) with the software tool developed, EST databases can be efficiently exploited for the development of cDNA-SSRs, (2) EST-derived SSRs are significantly less polymorphic than those derived from genomic regions, (3) a considerable portion of the developed SSRs can be transferred to related species, and (4) compared to RFLP-markers, cDNA-SSRs yield similar patterns of genetic diversity.     

Development,Characterization and Cross Species Amplification of Polymorphic Microsatellite Markers from Expressed Sequence Tags of Turmeric (<Emphasis Type="Italic">Curcuma longa</Emphasis> L.)

Expressed sequence tags (ESTs) from turmeric (Curcuma longa L.) were used for the screening of type and frequency of Class I (hypervariable) simple sequence repeats (SSRs). A total of 231 microsatellite repeats were detected from 12,593 EST sequences of turmeric after redundancy elimination. The average density of Class I SSRs accounts to one SSR per 17.96 kb of EST. Mononucleotides were the most abundant class of microsatellite repeat in turmeric ESTs followed by trinucleotides. A robust set of 17 polymorphic EST–SSRs were developed and used for evaluating 20 turmeric accessions. The number of alleles detected ranged from 3 to 8 per loci. The developed markers were also evaluated in 13 related species of C. longa confirming high rate (100%) of cross species transferability. The polymorphic microsatellite markers generated from this study could be used for genetic diversity analysis and resolving the taxonomic confusion prevailing in the genus.     

Exploiting EST databases for the development and characterization of EST-SSRs in the Pacific oyster (Crassostrea gigas)

A total of 147 microsatellite-containing expressed sequence tags (ESTs) (3.63%) were detected from 4053 ESTs of the Pacific oyster (Crassostrea gigas) in GenBank. The average density of simple sequence repeats (SSRs) was 1 per 8.25 kb of EST after redundancy elimination. Dinucleotide repeat motifs appeared to be the most abundant type. Sixteen new polymorphic EST-SSRs were developed. The number of alleles per locus varied from 3 to 12, with an average of 5.9 alleles per locus. Marker transferability was tested on 2 other Crassostrea species, and 14 loci gave successful amplifications in both species. Twenty EST-SSRs were tested on 3 families of C. gigas for examination of inheritance mode of EST-SSRs. Thirty-five tests of segregation ratios revealed 5 significant departures from expected Mendelian ratios, 4 of which confirmed Mendelian expectations when accounting for the presence of null alleles. Null alleles were detected at 3 loci (15.0%) of the 20 loci, and the frequency of null alleles at EST-SSRs was lower than that in genomic SSRs in C. gigas. The results obtained in this study suggest that C. gigas EST-SSRs will complement the currently available genomic SSR markers and may be useful for comparative mapping, marker-assisted selection, and evolutionary studies.     

Development of ten microsatellite markers for <Emphasis Type="Italic">Quercus mongolica</Emphasis> var. <Emphasis Type="Italic">crispula</Emphasis> by database mining

Simple sequence repeats (SSRs) in the NCBI dbEST database were surveyed to identify potential SSR markers for Quercus mongolica. In total, 2,691 gene sequences, mainly from expressed sequence tags (ESTs) for Q. robur and Q. petraea had been registered. Twenty-two PCR primers were designed for SSRs in these sequences and screened for polymorphisms in 16 Q. mongolica trees. Ten loci were easily genotyped and showed polymorphism, with numbers of alleles and expected heterozygosity ranging from 3 to 15 and 0.28 to 0.94, respectively. These EST-SSR markers should be useful for studying the genetic diversity of Quercus species.     

Mining and charactering microsatellites in Cucumis melo expressed sequence tags from sequence database

Simple sequence repeats (SSRs) derived from expressed sequence tags (ESTs) are valuable markers because they represent transcribed regions and often have putative functions. We mined and characterized microsatellites in melon ESTs. Three hundred and eighty‐three SSR loci were identified in 309 of 3188 unigenes assembled by 5747 EST and mRNA sequences in GenBank with occurring frequency of 1/4.7 kb. Twenty‐two polymorphic EST‐SSR markers were developed with the mean allele number of 2.9 per locus and mean expected heterozygosity of 0.442. Amplification products were also detected by 15 pairs of primer in Cucumis sativus. Those informative EST‐SSR markers can be used in melon genetic improvement projects.     

Identification and development of polymorphic EST-SSR markers by sequence alignment in pepper, Capsicum annuum (Solanaceae)

? Premise of the study: The redundancies in expressed sequence tags (ESTs) in the National Center for Biotechnology Information sequence database were used to identify and develop polymorphic simple sequence repeat (SSR) markers for pepper (Capsicum annuum). ? Methods and Results: Sixty-eight polymorphic SSR loci were identified in the contigs (containing redundant ESTs) generated by assembling 118060 pepper ESTs from the public sequence database. Thirty-three SSR markers exhibited polymorphism among 31 pepper varieties, with alleles per SSR marker ranging from two to six. The mean observed and expected heterozygosity were 0.28 and 0.39, respectively. There were 18 SSR markers with a motif repeat number of less than five, accounting for 55% of the total. ? Conclusions: We demonstrated the value of mining the redundant sequences in public sequence databases for the development of polymorphic SSR markers, which can be used for marker-assisted breeding in pepper.     

Genetic mapping of EST-derived microsatellites from the diploid<Emphasis Type="Italic"> Gossypium arboreum</Emphasis> in allotetraploid cotton

To increase the numbers of microsatellites available for use in constructing a genetic map, and facilitate the use of functional genomics to elucidate fiber development and breeding in cotton, we sampled microsatellite sequences from expressed sequence tags (ESTs) transcribed during fiber elongation in the A-genome species Gossypium arboreum to evaluate their frequency of occurrence, level of polymorphism and distribution in the At and Dt subgenomes of tetraploid cotton. From among ESTs derived from G. arboreum fibers at 7–10 days post anthesis (dpa), 931 ESTs were found to contain simple sequence repeats (SSRs); 544 (58.4%) EST-SSR primer pairs were developed, and 468 (86%) amplified PCR products from allotetraploid cotton ( G. hirsutum cv. TM-1 and G. barbadense cv. Hai7124). However, only 99 (18.2%) of these were found to be polymorphic and segregating in our interspecific BC₁ mapping population [(TM-1×Hai7124)×TM-1]. In these amplified and informative EST-SSRs, hexa- and tri-nucleotide repeat motifs were the most frequent, representing 40.1 and 30%, respectively, of the total. A total of 111 loci detected with these 99 EST-SSRs were integrated into our backbone map including 511 SSR loci. The distribution of the EST-SSRs appeared to be non-random, since 72 loci were anchored to the At and 37 to the Dt subgenome of allotetraploid cotton based on linkage tests. Interestingly, out of the 10 pairs of duplicate loci amplified, seven were mapped to the corresponding homeologous linkage groups and/or chromosomes. BLASTX analysis revealed that 69 of the 99 ESTs showed significant similarities to known genes. Some genes important for fiber development, such as sucrose synthase, were mapped to corresponding chromosomes. These EST-SSRs provide structural and functional genomic information that will be useful for understanding cotton fiber development.Communicated by R. Hagemann     

Development of Juglans Regia SSR Markers by Data Mining of the EST Database

Walnut (Juglans regia), an economically important woody plant, is widely cultivated in temperate regions for its timber and nutritional fruits. Despite abundant studies in germplasm, systemic molecular evaluations of walnut are sparsely reported mainly due to the limited molecular markers available. Expressed sequence tags (EST) provide a valuable resource for developing simple sequence repeat (SSR) markers. In this study, a total of 5,025 walnut ESTs (covering 16.41 Mb) were retrieved from the National Center for Biotechnology Information database. The SSR motifs were then analyzed by the SSRHunter software. In total, 398 SSRs were obtained with an average frequency of 1/4.08 kb. Dinucleotide (di-) repeat motifs accounted for 69.85% of all SSRs, followed by trinucleotide (tri-) with a frequency of 27.64%, while low frequency (2.51%) of tetranucleotide (tetra-) to hexanucleotide (hexa-) was observed. Meanwhile, GCA and TC motifs were prevalent among di- and tri- loci, respectively. Subsequently, a total of 123 primer pairs were designed from the non-redundant SSR-containing unigenes with the selection threshold of SSR length set to 10 bp or more. To examine the efficiency of candidate markers, seven DNA pools were collected from geographically different accessions. Results demonstrated that 41 SSR primer sets could generate high polymorphic amplification products (33.3%), and these polymorphic loci were mainly located in the 3′-untranslated region. Annotation analysis revealed that only two of these 41 loci were located inside open reading frames of characterized proteins (E ≤ 1E−30).     

Development of EST-SSR Markers by Data Mining in Three Species of Shrimp: Litopenaeus vannamei, Litopenaeus stylirostris, and Trachypenaeus birdy

We report on the data mining of publicly available Litopenaeus vannamei expressed sequence tags (ESTs) to generate simple sequence repeat (SSRs) markers and on their transferability between related Penaeid shrimp species. Repeat motifs were found in 3.8% of the evaluated ESTs at a frequency of one repeat every 7.8 kb of sequence data. A total of 206 primer pairs were designed, and 112 loci were amplified with the highest success in L. vannamei. A high percentage (69%) of EST-SSRs were transferable within the genus Litopenaeus. More than half of the amplified products were polymorphic in a small testing panel of L. vannamei. Evaluation of those primers in a larger testing panel showed that 72% of the markers fit Hardy-Weinberg equilibrium, which shows their utility for population genetic analysis. Additionally, a set of 26 of the EST-SSRs were evaluated for Mendelian segregation. A high percentage of monomorphic markers (46%) proved to be polymorphic by singles-stranded conformational polymorphism analysis. Because of the high number of ESTs available in public databases, a data mining approach similar to the one outlined here might yield high numbers of SSR markers in many animal taxa.     

The development of simple sequence repeat markers for Magnaporthe grisea and their integration into an established genetic linkage map

Although microsatellite or simple sequence repeat (SSR) markers have several advantages, few have been developed in fungi. The goal of this study was to identify and characterize SSR-containing loci in the filamentous ascomycete Magnaporthe grisea, the causal agent of rice blast disease, and to add these markers to an integrated genetic map of this species [Theor. Appl. Genet. 95 (1997) 20]. We have constructed and screened a microsatellite-enriched small-insert genomic library as well as exploited both publicly available and one proprietary databases for identification of M. grisea SSR containing sequences. Twenty-four out of 49 primer pairs designed to amplify SSR, produced unambiguous polymorphic products in our test population of six isolates. The number of alleles at each locus ranged from two to six when assayed on 3% agarose gels. Twenty-three of the primer pairs amplified polymorphic products between Guy11 and 2539, the parents of a cross from which a genetic map for M. grisea has been established. Genetic analysis showed that all the markers segregated in the expected 1:1 ratio and map positions were determined for all 23 loci.     

Development of 14 EST-SSRs for Betula maximowicziana and their applicability to related species

Simple sequence repeats (SSRs) markers were developed for Betula maximowicziana using 2698 expressed sequence tags (ESTs) from the NCBI database. Out of 112 designed primer pairs, 54 showed clear PCR amplification and 14 of these revealed polymorphism in eight individuals sampled across the species’ range. The number of alleles detected and the expected heterozygosity ranged from 1 to 3 and 0.000 to 0.570, respectively, when these 14 loci were examined in 49 individuals from a single population. In the cross species transferability test, eight of the 14 loci were also polymorphic in all four of the diploid, tetraploid and hexaploid Betula species examined. These results showed high transferability of the developed EST-SSRs and that these markers are likely to be useful in studies of the population genetics of species in the genus Betula.     

Identification and characterisation of nine new gene-associated microsatellite markers for Atlantic cod (<Emphasis Type="Italic">Gadus morhua</Emphasis> L.)

Nine polymorphic microsatellite markers were identified by screening of 2464 ESTs derived from a cDNA library of Atlantic cod (Gadus morhua L.). About 35 novel microsatellite loci were selected and characterised in 96 individual cod. Nine markers were successfully amplified with number of alleles from 3 to 18 per locus and the average heterozygosity was 0.57 in the panel examined (range 0.29–0.86). All loci followed the Hardy–Weinberg expectation and no significant linkage disequilibrium was found in a test including all pairwise combinations. The gene identity was determined at four of the loci, confirming the associated microsatellites as Type I markers.     

Development of SSR markers from an ectomycorrhizal fungus, <Emphasis Type="Italic">Suillus bovinus</Emphasis>

Nine simple sequence repeat (SSR) loci were isolated from an ectomycorrhizal fungus Suillus bovinus by dual-suppression PCR. Three of the SSR loci isolated were polymorphic. The number of alleles per locus was between two and seven, and expected heterozygosities ranged from 0.087 to 0.740. One of these was confirmed to be species specific and codominant, suggesting applicability for the analysis of belowground population structure and gene flow of S. bovinus.     

Development and characterization of simple sequence repeat (SSR) markers and their use in determining relationships among Lycopersicon esculentum cultivars

The simple sequence repeat (SSR) or microsatellite marker is currently the preferred molecular marker due to its highly desirable properties. The aim of this study was to develop and characterize more SSR markers because the number of SSR markers currently available in tomato is very limited. Five hundred DNA sequences of tomato were searched for SSRs and analyzed for the design of PCR primers. Of the 158 pairs of SSR primers screened against a set of 19 diverse tomato cultivars, 129 pairs produced the expected DNA fragments in their PCR products, and 65 of them were polymorphic with the polymorphism information content (PIC) ranging from 0.09 to 0.67. Among the polymorphic loci, 2-6 SSR alleles were detected for each locus with an average of 2.7 alleles per locus; 49.2% of these loci had two alleles and 33.8% had three alleles. The vast majority (93.8%) of the microsatellite loci contained di- or tri-nucleotide repeats and only 6.2% had tetra- and penta-nucleotide repeats. It was also found that TA/AT was the most frequent type of repeat, and the polymorphism information content (PIC) was positively correlated with the number of repeats. The set of 19 tomato cultivars were clustered based on the banding patterns generated by the 65 polymorphic SSR loci. Since the markers developed in this study are primarily from expressed sequences, they can be used not only for molecular mapping, cultivar identification and marker-assisted selection, but for identifying gene-trait relations in tomato.