The application of SSRs characterized for grape (Vitis vinifera) to conservation studies in Vitaceae

The use of microsatellite loci developed from a single plant species across a number of related taxa is becoming increasingly widespread. This approach can provide highly informative markers even for species for which microsatellites have not been characterized. As a number of expressed sequence tag (EST)-derived and enrichment-derived microsatellites are available for grape (Vitis vinifera), this study set out to assess transferability of nine such loci across 25 species from five different Vitaceae genera. Intergeneric transfer success in Vitaceae was high (51.1%) and EST-derived loci performed better than enrichment-derived loci. Six loci were further tested across two Australian native species, Cissus hypoglauca and C. sterculiifolia, in order to assess the conservation of microsatellite repeats and their flanking sequences. Polymorphism of these selected loci was successfully tested for each species across a small, isolated rain forest population from northern New South Wales (Australia). Results from this preliminary investigation suggest that it is possible to use grape-derived simple sequence repeats (SSR) loci for population studies on Vitaceae. As Vitaceae are an important component of rain forest flora, the availability of such highly informative loci will be beneficial to future studies aimed at determining the genetic consequences of rain forest fragmentation.     

Conservation of a dinucleotide simple sequence repeat locus in sharks

Recent studies indicate that the flanking region and repeat motif structure of conserved microsatellite loci are useful for phylogenetic inference. Most comparative studies of microsatellite loci involve relatively closely related species, however, primarily because primers developed for one species often amplify only related species. We describe an analysis of a microsatellite locus in lamniform sharks that we estimate has been conserved for a billion years. Combined analysis of the flanking sequence and repeat motif structure resulted in a gene tree comparable to those reported from similar analyses of other genes. The conservation of the simple sequence repeat (SSR), and of the sequence flanking the SSR, is explained by a low substitution rate in sharks coupled with the possibility that mutations which interrupt perfect repeats are lost by replication slippage.     

桉树EST序列中微卫星含量及相关特征

通过对桉树属(Eucalyptus)的10 000条EST序列进行分析, 在其中的1 499条序列上共发现1 775个微卫星重复序列。含有微卫星的EST序列约占序列总数的15%。此外, 还发现桉树EST序列所含微卫星长度的变异速率与重复单元长度呈负相关; 微卫星的丰度与重复单元长度也呈负相关(三碱基重复微卫星除外)。在桉树EST序列中, 重复单元长度为三碱基的微卫星最为丰富。三碱基重复单元微卫星的过度富集可能是由于遗传密码选择所致。在微卫星的丰度及长度变异方面, 桉树EST序列与杨树(Populus trichocarpa)基因组注释的转录序列随重复单元长度的变化呈现出相同的规律, 但桉树EST序列中微卫星频率及三碱基重复微卫星的含量显著偏低, 推测含微卫星的基因表达丰度极有可能低于不含微卫星的基因。通过对发现的所有微卫星位点进行引物设计, 并对设计的引物进行PCR检测, 结果表明所设计的引物具有极高的扩增成功率。     

桉树EST序列中微卫星含量及相关特征

通过对桉树属（Eucalyptus）的10000条EST序列进行分析,在其中的1499条序列上共发现1775个微卫星重复序列。含有微卫星的EST序列约占序列总数的15%。此外,还发现桉树EST序列所含微卫星长度的变异速率与重复单元长度呈负相关;微卫星的丰度与重复单元长度也呈负相关（三碱基重复微卫星除外）。在桉树EST序列中,重复单元长度为三碱基的微卫星最为丰富。三碱基重复单元微卫星的过度富集可能是由于遗传密码选择所致。在微卫星的丰度及长度变异方面,桉树EST序列与杨树（Populus trichocarpa）基因组注释的转录序列随重复单元长度的变化呈现出相同的规律,但桉树EST序列中微卫星频率及三碱基重复微卫星的含量显著偏低,推测含微卫星的基因表达丰度极有可能低于不含微卫星的基因。通过对发现的所有微卫星位点进行引物设计,并对设计的引物进行PCR检测,结果表明所设计的引物具有极高的扩增成功率。     

Development, characterization, and cross-species/genera transferability of SSR markers for rubber tree (Hevea brasiliensis)

Genomic simple sequence repeat (SSR) markers are particularly valuable in studies of genetic diversity, evolution, genetic linkage map construction, quantitative trait loci tagging, and marker-assisted selection because of their multi-allelic nature, reproducibility, co-dominant inheritance, high abundance, and extensive genome coverage. The traditional methods of SSR marker development, such as genomic-SSR hybrid screening and microsatellite enrichment, have the disadvantages of high cost and complex operation. The selectively amplified microsatellite method is less costly and highly efficient as well as being simple and convenient. In this study, 252 sequences with SSRs were cloned from the rubber tree (Hevea brasiliensis) genome from which 258 SSR loci were obtained. The average repeat number was six. There were only 10 (3.9%) mononucleotide, trinucleotide, and pentanucleotide repeats, whereas the remaining 248 (96.1%) were dinucleotide repeats, including 128 (49.6%) GT/CA repeats, 118 (45.7%) GA/CT repeats, and 2 (0.8%) AT/TA repeats. A total of 126 primer pairs (see ESM) were successfully designed of which 36 primer pairs generated polymorphic products from 12 accessions of the cultivated species, 4 related species, and 3 species of the family Euphorbiaceae. In addition, investigations based on four genomic SSRs (GAR4, ACR22, CTR25, and GTR28) by cloning and sequencing provided evidence for cross-species/genera applicability, and homologous sequences were obtained from the rubber tree and Euphorbiaceae. Further analysis about the variation of the flanking regions of the four markers was carried out.     

Reverse random amplified microsatellite polymorphism reveals enhanced polymorphisms in the 3' end of simple sequence repeats in the pepper genome

Microsatellites or simple sequence repeats (SSR) are widely distributed in eukaryotic genomes and are informative genetic markers. Despite many advantages of SSR markers such as a high degree of allelic polymorphisms, co-dominant inheritance, multi-allelism, and genome-wide coverage in various plant species, they also have shortcomings such as low polymorphic rates between genetically close lines, especially in Capsicum annuum. We developed an alternative technique to SSR by normalizing and alternating anchored primers in random amplified microsatellite polymorphisms (RAMP). This technique, designated reverse random amplified microsatellite polymorphism (rRAMP), allows the detection of nucleotide variation in the 3' region flanking an SSR using normalized anchored and random primer combinations. The reproducibility and frequency of polymorphic loci in rRAMP was vigorously enhanced by translocation of the 5' anchor of repeat sequences to the 3' end position and selective use of moderate arbitrary primers. In our study, the PCR banding pattern of rRAMP was highly dependent on the frequency of repeat motifs and primer combinations with random primers. Linkage analysis showed that rRAMP markers were well scattered on an intra-specific pepper map. Based on these results, we suggest that this technique is useful for studying genetic diversity, molecular fingerprinting, and rapidly constructing molecular maps for diverse plant species.     

<Emphasis Type="Italic">Prunus</Emphasis> microsatellite marker transferability across rosaceous crops

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.     

Evaluating the potential of SSR flanking regions for examining taxonomic relationships in the Vitaceae

Three EST-derived microsatellite loci from Vitis vinifera were amplified and sequenced across eight species of Vitaceae from four different genera. Phylogenetic analysis of the microsatellite’s flanking regions produced informative results in congruence with previous studies. Generic relationships were respected and the data produced sufficient inter-specific variation to distinguish between Cayratia acris and Cayratia saponaria, two very closely related species. Overall, the sequence alignments showed that priming sites were conserved, whereas microsatellite repeats were present in most cases but structurally variable. The sequence data provided information on the evolutionary patterns of various microsatellite repeats and their correlation to evolutionary relationships among taxa. Received: 15 December 2000 / Accepted: 12 April 2001     

Conservation of (GA)n microsatellite loci between Quercus species

Microsatellite (simple sequence repeat, SSR) amplification was performed in eight different members of the Fagaceae family by using sets of primers developed from sessile oak, Quercus petraea . In total, 136 cases of heterologous amplification were carried out, and 66% resulted in interpretable amplification products. From these, 12 PCR amplification products were sequenced and all 12 contained a sequence homologous to the original locus from Q. petraea . Although SSR primers worked even across different genera, with increasing evolutionary distance there was a clear tendency for decreasing ability to successfully amplify loci and a decreasing proportion of polymorphism amongst those markers which could be amplified. Two of the loci, ssrQpZAG46 and ssrQpZAG110, were polymorphic in all Quercus species tested. Only at one locus, ssrQpZAG58, a specific PCR product could be amplified in all species analysed. For four loci found in two species, we observed significant interspecies differences in the size range of the amplified alleles. Sequence analysis of two alleles showed that the size differences are not only due to variations in the number of (GA) repeats but also to an insertion of approximately 80 nucleotides in the flanking region. Our findings prove the usefulness of SSR markers within and amongst closely related genera of plants.     

SSR allelic variation in almond (Prunus dulcis Mill.)

Sixteen SSR markers including eight EST-SSR and eight genomic SSRs were used for genetic diversity analysis of 23 Chinese and 15 international almond cultivars. EST- and genomic SSR markers previously reported in species of Prunus, mainly peach, proved to be useful for almond genetic analysis. DNA sequences of 117 alleles of six of the 16 SSR loci were analysed to reveal sequence variation among the 38 almond accessions. For the four SSR loci with AG/CT repeats, no insertions or deletions were observed in the flanking regions of the 98 alleles sequenced. Allelic size variation of these loci resulted exclusively from differences in the structures of repeat motifs, which involved interruptions or occurrences of new motif repeats in addition to varying number of AG/CT repeats. Some alleles had a high number of uninterrupted repeat motifs, indicating that SSR mutational patterns differ among alleles at a given SSR locus within the almond species. Allelic homoplasy was observed in the SSR loci because of base substitutions, interruptions or compound repeat motifs. Substitutions in the repeat regions were found at two SSR loci, suggesting that point mutations operate on SSRs and hinder the further SSR expansion by introducing repeat interruptions to stabilize SSR loci. Furthermore, it was shown that some potential point mutations in the flanking regions are linked with new SSR repeat motif variation in almond and peach. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

In silico mining for simple sequence repeat loci in a pineapple expressed sequence tag database and cross-species amplification of EST-SSR markers across Bromeliaceae

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).     

What phylogeny and gene genealogy analyses reveal about homoplasy in citrus microsatellite alleles

Sixty-five microsatellite alleles amplified from ancestral citrus accessions classified in three separate genera were evaluated for sequence polymorphism to establish the basis of inter- and intra-allelic genetic variation, evaluate the extent of size homoplasy, and determine an appropriate model (stepwise or infinite allele) for analysis of citrus microsatellite alleles. Sequences for each locus were aligned and subsequently used to determine relationships between alleles of different taxa via parsimony. Interallelic size variation at each SSR locus examined was due to changes in repeat copy number with one exception. Sequencing these alleles uncovered new distinct point mutations in the microsatellite region and the region flanking the microsatellite. Several of the point mutations were found to be genus, species, or allele specific, and some mutations were informative about the inferred evolutionary relationships among alleles. Overall, homoplasy was observed in alleles from all three loci, where the core microsatellite repeat was changed causing alleles of the same size class to be identical in state but not identical by descent. Because nearly all changes in allele size (with one exception) were due to expansion or contraction of the repeat motif, this suggests that a stepwise mutation model, which assumes homoplasy may occur, would be the most appropriate for analyzing Citrus SSR data. The collected data indicate that microsatellites can be a useful tool for evaluating Citrus species and two related genera since repeat motifs were reasonably well retained. However, this work also demonstrated that the number of microsatellite alleles is clearly an underestimate of the number of sequence variants present.     

PERMANENT GENETIC RESOURCES: Development of microsatellite markers for the guava rust fungus, Puccinia psidii

We developed and characterized 15 polymorphic microsatellite markers present in the genome of the guava rust fungus, Puccinia psidii. The primers for these microsatellite markers were designed by sequencing clones from a genomic DNA library enriched for a simple sequence repeat (SSR) motif of (AG). All these 15 primer pairs successfully amplified DNA fragments from a sample of 22 P. psidii isolates, revealing a total of 71 alleles. The observed heterozygosity at the 15 loci ranged from 0.05 to 1.00. The SSR markers developed would be useful for population genetics study of the rust fungus.     

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.     

Genetic Relationships Among 22 Taxa of Bamboo Revealed by ISSR and EST-Based Random Primers

Genetic relationships among 22 taxa of bamboo were evaluated using 12 inter simple sequence repeats (ISSR) and four expressed sequence tag (EST)-based random primers, resulting in amplification of 220 loci. The grouping of species based on Jaccard's similarity matrix using UPGMA and principal coordinate analysis agreed with earlier published reports on molecular phylogenetic studies in bamboos with few deviations. In the dendrogram, however, species of one genus were placed in different clusters along with members of other genera. This calls for correct taxonomic delineation at the genus and species level using both vegetative and reproductive characters and correlation of molecular data with morphologically definable taxonomic groupings at the proper taxonomic level.     

In silico whole-genome EST analysis reveals 2322 novel microsatellites for the silver-lipped pearl oyster, Pinctada maxima

Molecular stock improvement techniques such as marker assisted selection have great potential in accelerating selective breeding programmes for animal production industries. However, the discovery and application of trait/marker associations usually requires a large number of genome-wide polymorphic loci. Here, we present 2322 unique microsatellites for the silver-lipped pearl oyster, Pinctada maxima, a species of aquaculture importance throughout the Indo-Australian Archipelago for production of the highly valued South Sea pearl. More than 1.2 million Roche 454 expressed sequence tag (EST) reads were screened for microsatellite repeat motifs. A total of 12,604 sequences contained either a di, tri, tetra, penta or hexa microsatellite repeat motif (n ≥ 6), with 6435 of these sequences having sufficient flanking regions for primer development. All identified microsatellites with designed primers were condensed into 2322 unique clusters (i.e., unique loci) of which 360 were shown to be polymorphic based on multiple sequence reads with different repeat motifs. Genotyping of five microsatellite loci demonstrated that in silico evaluation of polymorphism levels was a very useful method for identification of polymorphic loci, with the variation uncovered being a lower bound. Gene Ontology annotations of sequences containing microsatellites suggest that most are derived from a diverse array of unique genes. This EST derived microsatellite database will be a valuable resource for future studies in genetic map construction, diversity analysis, quantitative trait loci analysis, association mapping and marker assisted selection, not only for P. maxima, but also closely related species within the genus Pinctada.     

Microsatellite DNA markers in Populus tremuloides.

Markers for eight new microsatellite DNA or simple sequence repeat (SSR) loci were developed and characterized in trembling aspen (Populus tremuloides) from a partial genomic library. Informativeness of these microsatellite DNA markers was examined by determining polymorphisms in 38 P. tremuloides individuals. Inheritance of selected markers was tested in progenies of controlled crosses. Six characterized SSR loci were of dinucleotide repeats (two perfect and four imperfect), and one each of trinucleotide and tetranucleotide repeats. The monomorphic SSR locus (PTR15) was of a compound imperfect dinucleotide repeat. The primers of one highly polymorphic SSR locus (PTR7) amplified two loci, and alleles could not be assigned to a specific locus. At the other six polymorphic loci, 25 alleles were detected in 38 P. tremuloides individuals; the number of alleles ranged from 2 to 7, with an average of 4.2 alleles per locus, and the observed heterozygosity ranged from 0.05 to 0.61, with an average of 0.36 per locus. The two perfect dinucleotide and one trinucleotide microsatellite DNA loci were the most informative. Microsatellite DNA variants of four SSR loci characterized previously followed a single-locus Mendelian inheritance pattern, whereas those of PTR7 from the present study showed a two-locus Mendelian inheritance pattern in controlled crosses. The microsatellite DNA markers developed and reported here could be used for assisting various genetic, breeding, biotechnology, genome mapping, conservation, and sustainable forest management programs in poplars.     

基于RAD-seq技术的异型花SSR信息分析

用RAD-seq（restriction-site associated DNA sequencing）对异型花（Sinoswertia tetraptera（Maxiowicz） T.N.Ho,S.W.Liu & J.Q.Liu）进行简化基因组测序,并借此分析异型花的SSR（simple sequence repeats）信息。利用SR search软件甄别所得序列中的SSR,得到了双端各有至少100 bp的SSR位点5 844个,其中5 339个（91.38%）成功设计引物,而三核苷酸SSR位点最多（3 323个）;在能成功设计引物的SSR位点中,重复序列长度包括17种（12~36 bp）;重复序列的基序共277种,其中五核苷酸基序种类最多（106种）;随机挑选10对SSR引物,用4个异型花居群的32个个体检测检测其可用性和多态性,经PCR和聚丙烯酰氨凝胶电泳检测,有4对（ST2、ST3、ST6和ST10）成功扩增并表现出多态性;经GENEPOP 4.4对4个位点分析,显示其等位基因数量均值为6,多态性较高且不连锁（P<0.01）;4个位点在多数居群中偏离哈迪温伯格平衡（P<0.01）且存在较高的纯合子数量（观测杂合度均值0.023）,该结果归因于异型花主要进行自花授粉,在自然界中很难形成进行自由交配的居群;此外,ST2和ST6可在椭圆叶花锚（Halenia elliptica D.Don）中成功扩增,具有潜在通用性。本研究将为日后基于异型花SSR标记的相关研究提供数据库支持。     

In silico mining of microsatellites and analysis of genetic diversity among inter‐ and intra‐generic aphids of the subfamily Aphidinae

Nearly 5 000 aphid species damage crops, either by sucking plant sap or as disease‐transmitting vectors. Microsatellites are used for understanding molecular diversity and eco‐geographical relationships among aphid species. Expressed sequence tag (EST)‐microsatellite motifs were identified through an in silico approach using inbuilt simple sequence repeat mining tools in aphid EST dataset. Microsatellite mining revealed one in every five aphid genes as containing a repeat motif, and out of 9 290 EST microsatellites mined from Aphis gossypii Glover and Acyrthosiphon pisum (Harris) (both Hemiptera: Aphididae), 80% were of A and/or T (AT, ATA, AAT, AATA, and ATTT) motifs, and the rest contained G and/or C motifs. All microsatellite sequences were annotated using BLAST. Primers for EST microsatellites were designed using the Primer 3.0 tool. 106 primer pairs of both dinucleotide repeats (DNRs) and trinucleotide repeats (TNRs), representing open reading frames (ORFs) and untranslated regions (UTRs), were synthesized to amplify 15 aphid species belonging to the subfamily Aphidinae, collected from diverse hosts. Four hundred forty‐five polymorphic alleles were amplified. Fifty TNR and 23 DNR microsatellites amplified across the species studied. Polymorphism information content values of microsatellites ranged from 0.23 to 0.91, amplifying 2–16 alleles. Genetic similarity indices were estimated using the ‘NTSYS‐pc’ software package. Unweighted pair group with arithmetic mean and principal component analysis resolved taxonomic relationships of the aphid species studied. The new aphid microsatellites developed will provide valuable information to researchers to study Indian aphid species diversity and genetic relationships.