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1.
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.  相似文献   

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A collection of 5,659 expressed sequence tags (ESTs) from pineapple [Ananas comosus (L.) Merr.] was screened for simple sequence repeats (EST-SSRs) with motif lengths between 1 and 6 bp. Lower thresholds of 15, 7 and 5 repeat units were used to define microsatellites of the mono-, di-, and tri- to hexanucleotide repeat type, respectively. Based on these criteria, 696 SSRs were identified among 3,389 EST unigenes, together representing 2,840 kb. This corresponds to an average density of one SSR every 4.1 kb of non-redundant EST sequences. Dinucleotide repeats were most abundant (38.4% of all SSRs) followed by trinucleotide repeats (38.1%). Flanking primer pairs were designed for 537 EST-SSR loci, and 49 of these were screened for their functionality in 12 accessions of A. comosus, 14 accessions of 5 additional Ananas species and 1 species of Pseudananas. Distinct PCR products of the expected size range were obtained with 36 primer pairs. Eighteen loci analyzed in more detail were all polymorphic in pineapple, and primer pairs flanking these loci also generated PCR products from a wide range of genera and species from six subfamilies of the Bromeliaceae. The potential to reveal polymorphism in a heterologous target species was demonstrated in Deuterocohnia brevifolia (subfamily Pitcairnioideae).  相似文献   

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Analysis of a sugarcane (Saccharum spp.) EST (expressed sequence tag) library of 8678 sequences revealed approximately 250 microsatellite or simple sequence repeats (SSRs) sequences. A diversity of dinucleotide and trinucleotide SSR repeat motifs were present although most were of the (CGG)n trinucleotide motif. Primer sets were designed for 35 sequences and tested on five sugarcane genotypes. Twenty-one primer pairs produced a PCR product and 17 pairs were polymorphic. Primer pairs that produced polymorphisms were mainly located in the coding sequence with only a single pair located within the 5′ untranslated region. No primer pairs producing a polymorphic product were found in the 3′ untranslated region. The level of polymorphism (PIC value) in cultivars detected by these SSRs was low in sugarcane (0.23). However, a subset of these markers showed a significantly higher level of polymorphism when applied to progenitor and related genera (Erianthus sp. and Sorghum sp.). By contrast, SSRs isolated from sugarcane genomic libraries amplify more readily, show high levels of polymorphism within sugarcane with a higher PIC value (0.72) but do not transfer to related species or genera well.  相似文献   

4.
Expressed sequence tag (EST) derived simple sequence repeats (SSRs, microsatellites) were screened and identified from 3863 almond and 10 185 peach EST sequences, and the spectra of SSRs in the non-redundant EST sequences were investigated after sequence assembly. One hundred seventy-eight (12.07%) almond SSRs and 497 (9.97%) peach SSRs were detected. The EST-SSR occurs every 4.97 kb in almond ESTs and 6.57 kb in peach, and SSRs with di- and trinucleotide repeat motifs are the most abundant in both almond and peach ESTs. Twenty one EST-SSRs were thereafter, developed and used together with 7 genomic SSRs, to study the genetic relationship among 36 almond (P. communis Fritsch.) cultivars from China and the Mediterranean area, as well as 8 accessions of other related species from the genus Prunus. Both EST-derived and genomic SSR markers showed high cross-species transferability in the genus. Out of the 112 polymorphic alleles detected in the 36 cultivated almonds, 28 are specific to Chinese cultivars and 25 to the others. The 44 accessions were clustered into 4 groups in the phylogenetic tree and the 36 almond cultivars formed two distinct subgroups, one containing only Chinese cultivars and one of unknown origin and the other only those originating from the Mediterranean area, indicating that Chinese almond cultivars have a distinct evolutionary history from the Mediterranean almond. Our preliminary results indicated that common almond was more closely related to peach (P. persica (L.) Batsch.) than to the four wild species of almond, (P. mongolica Maxim., P. ledebouriana Schleche, P. tangutica Batal., and P. triloba Lindl.). The implications of these SSR markers for evolutionary analysis and molecular mapping of Prunus species are discussed.  相似文献   

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A total of 145 microsatellite primer pairs from Prunus DNA sequences were studied for transferability in a set of eight cultivars from nine rosaceous species (almond, peach, apricot, Japanese plum, European plum, cherry, apple, pear, and strawberry), 25 each of almond genomic, peach genomic, peach expressed sequence tags (EST), and Japanese plum genomic, 22 of almond EST, and 23 of apricot (13 EST and 10 genomic), all known to produce single-locus and polymorphic simple-sequence repeats in the species where they were developed. Most primer pairs (83.6%) amplified bands of the expected size range in other Prunus. Transferability, i.e., the proportion of microsatellites that amplified and were polymorphic, was also high in Prunus (63.9%). Almond and Japanese plum were the most variable among the diploid species (all but the hexaploid European plum) and peach the least polymorphic. Thirty-one microsatellites amplified and were polymorphic in all Prunus species studied, 12 of which, covering its whole genome, are proposed as the “universal Prunus set”. In contrast, only 16.3% were transferable in species of other Rosaceae genera (apple, pear, and strawberry). Polymorphic Prunus microsatellites also detected lower levels of variability in the non-congeneric species. No significant differences were detected in transferability and the ability to detect variability between microsatellites of EST and genomic origin.  相似文献   

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Expressed sequence tag (EST) databases provide a primary source of nuclear DNA sequences for genetic marker development in non-model organisms. To date, the process has been relatively inefficient for several reasons: 1) priming site polymorphism in the template leads to inferior or erratic amplification; 2) introns in the target amplicon are too large and/or numerous to allow effective amplification under standard screening conditions; and 3) at least occasionally, a PCR primer straddles an exon–intron junction and is unable to bind to genomic DNA template. The first is only a minor issue for species or strains with low heterozygosity but becomes a significant problem for species with high genomic variation, such as marine organisms with extremely large effective population sizes. Problems arising from unanticipated introns are unavoidable but are most pronounced in intron-rich species, such as vertebrates and lophotrochozoans. We present an approach to marker development in the Pacific oyster Crassostrea gigas, a highly polymorphic and intron-rich species, which minimizes these problems, and should be applicable to other non-model species for which EST databases are available. Placement of PCR primers in the 3′ end of coding sequence and 3′ UTR improved PCR success rate from 51% to 97%. Almost all (37 of 39) markers developed for the Pacific oyster were polymorphic in a small test panel of wild and domesticated oysters.  相似文献   

10.
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).  相似文献   

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