EST-derived Simple Sequence Repeat Markers Research Articles

Functional characteristics of EST-SSR markers available for Scots pine

EST-derived simple sequence repeat markers (EST-SSRs) are important tools for studies on genetic diversity, phylogeny, evolution, comparative genomics, QTL analysis, and gene-based associations. We have searched the literature for known EST-SSRs used for Scots pine (Pinus sylvestris L.) – one of the world’s major forest species. Then, 91 of 102 EST-SSRs suggested for Scots pine studies were manually aligned against the reference genome of Pinus taeda L. as well as available genes of P. sylvestris. For 83 EST-SSRs, genome location and consensus putative functions of the associated genes were identified through conservative domain analysis (CDD), functional analysis of known homologs in terms of Gene Ontology annotations, and KEGG pathway analysis. Many of the markers were located in untranslated regions (mostly in 3’-UTR), as well as in coding sequences of Scots pine and loblolly pine genes. For eight markers whose EST sequences were known no genes could be identified in any of the species. Seven of these markers were located in P. taeda scaffold regions carrying no genes in the current genome assembly (v.1.0). The results can be used in the future to improve the choice of markers for population genetic research, studies of adaptive traits, and QTL mapping of P. sylvestris, as well as other pine species.

Development of EST-Molecular Markers from RNA Sequencing for Genetic Management and Identification of Growth Traits in Potato Grouper (Epinephelus tukula)

Simple SummaryThe potato grouper is a novel aquaculture species in Taiwan. Due to the lack of genetic information concerning this species, we have developed molecular markers based on transcriptome sequencing and further characterized their association with gene diversity and growth traits of this species. Ultimately, these markers could be utilized as accurate and efficient tools for genetic management and marker-assisted selection of potato grouper with distinct growth traits.The accuracy and efficiency of marker-assisted selection (MAS) has been proven for economically critical aquaculture species. The potato grouper (Epinephelus tukula), a novel cultured grouper species in Taiwan, shows large potential in aquaculture because of its fast growth rate among other groupers. Because of the lack of genetic information for the potato grouper, the first transcriptome and expressed sequence tag (EST)-derived simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers were developed. Initially, the transcriptome was obtained from seven cDNA libraries by using the Illumina platform. De novo transcriptome of the potato grouper yielded 51.34 Gb and 111,490 unigenes. The EST-derived SSR and SNP markers were applied in genetic management, in parentage analysis, and to discover the functional markers of economic traits. The F1 juveniles were identified as siblings from one pair of parents (80 broodstocks). Fast- and slow-growth individuals were analyzed using functional molecular markers and through their association with growth performance. The results revealed that two SNPs were correlated with growth traits. The transcriptome database obtained in this study and its derived SSR and SNP markers may be applied not only for MAS but also to maintain functional gene diversity in the novel cultured grouper.

Characterization of new microsatellite markers from sugarcane (Saccharum officinarum) transcriptome

Microsatellites, or simple sequence repeats (SSR), developed by expressed sequence tag (EST) databases is an economical and efficient tools that can be used to perform genetic investigations at a functional level. Here, a new sugarcane database of transcriptome from our variety, GT35, was examined for the presence of SSRs. To test the utility of EST-derived SSR markers, a total of 51 new EST-SSRs were identified for possible use as potential genetic markers from no redundant SSR-positive ESTs, which were unmapped with the sequences available in the NCBI’EST database of sugarcane by BLASTN. Polymorphisms of the identified 51 EST-SSR markers were evaluated in 21 sugarcane genotypes, planted and collected in 2013 at Sugarcane Research Institute of Guangxi Academy of Agricultural Sciences (SRI-GXAAS) in China. High polymorphisms were detected in terms of number of alleles ranging from 5-36 with an average of 16.8 per locus and polymorphism information content values ranging from 0.74 to 0.95 with a mean of 0.92. Average transferability to Erianthus arundinaceus and Narenga porphyrocoma was 23.9% and 34.4%, respectively. The ability to establish genetic relationship was analyzed by cluster analysis, the result of which revealed that the major grouping was in accordance with taxonomical classification. The development of new EST-SSR markers presented in this work will have important implications for genetic analysis and breeding.

Characterization and development of EST-derived SSR markers in Sinowilsonia henryi (Hamamelidaceae).

Premise of the study:Polymorphic microsatellite markers were developed to reveal the genetic diversity of extant populations and the mating system of Sinowilsonia henryi (Hamamelidaceae).Methods and Results:In this study, nuclear simple sequence repeat (SSR) markers were developed using the Illumina high-throughput sequencing technique (RNA-Seq). The de novo–assembled transcriptome generated a total of 64,694 unique sequences with an average length of 601 bp. A total of 2941 microsatellite loci were detected. Of the 121 tested loci, 13 loci were polymorphic and eight were monomorphic among 72 individuals representing three natural populations of the species. The number of alleles per locus ranged from one to four, and the observed and expected heterozygosity at population level were 0.00–1.00 and 0.10–0.66, respectively.Conclusions:The developed expressed sequence tag (EST)–SSRs will be useful for studying genetic diversity of S. henryi as well as assessing the mating system among Sinowilsonia species.

Development and characterization of EST-derived SSR markers in the cereal cyst nematode Heterodera avenae

The cereal cyst nematode Heterodera avenae is a pathogen of cereal crops and causes high yield losses worldwide. In this study, a collection of 37,348 expressed sequence tags (ESTs) of H. avenae was mined for EST-based simple sequence repeat (SSR) markers, which resulted in the identification of 5604 SSRs. A total of 210 pairs of SSR primers were further developed and used for validation of the amplification rate and assessment of the polymorphism. Eight SSR markers were finally identified and analyzed using 96 individual cysts of H. avenae sampled from three provinces in China. These loci were found to be moderately polymorphic with 3–8 alleles per locus. The observed and expected heterozygosity across the three populations ranged from 0.000 to 0.594 and from 0.000 to 0.731, respectively. The polymorphism information content (PIC) was medium and ranged from 0.080 to 0.562, with a mean of 0.409. The F ST ranged from 0.1034 to 0.1550, indicating moderate genetic differentiation among the three H. avenae populations. These EST-SSR markers can be used to study population genetic diversity and the dispersal route of H. avenae in China.

English

Niger (Guzoita abyssinica) is traditionally very important in the production of oil and as an oilseed crop in Ethiopia. The level of Niger genetic diversity is not studied at molecular level particularly with microsatellite markers which are cost effective, informative and co-dominant. In this study, contrasting samples of 65 Ethiopian Niger germplasm accessions collected from diversified eco-geographic regions were studied with eleven polymorphic EST derived SSR markers. Based on dissimilarity matrix ranging from 0.056 to 0.75, the accessions were grouped into three major and six sub clusters, showing the wealth of genetic diversity for exploitation in future breeding programs. The EST derived SSR markers used in this study also revealed high polymorphic information content (PIC) ranging from 0.2624 to 0.3677, the average being 0.3308 which indicate the usefulness of the primers in Niger germplasm characterization in the future by providing basic breeding information for breeders. The mean number of major allele frequency, gene diversity and heterozygosity using power marker v3.25, showed a value of 0.69, 0.42 and 0.50, respectively. The number of private alleles using GenAlEx 6.41 is 66 alleles with mean of 9 alleles per locus. Thus, utilizing EST SSR marker for diversity study lays basic foundation to understand the genetic distance of crop verities and greatly contributes for further improvement and preservation. Key words: Alleles, dendrogram, diversity, EST-SSR markers, heterozygosity, locus, Niger and polymorphic information content (PIC).

Development and validation of EST derived SSR markers with relevance to downy mildew (Sclerospora graminicola Sacc.) resistance in pearl millet [Pennisetum glaucum (L.) R. Br.

Molecular markers associated or linked with a particular trait are immensely essential for marker-assisted selection. Downy mildew is a severe disease in pearl millet causing huge production losses annually. There is a need to develop reliable functional markers associated with downy mildew resistance. In this study, expressed sequence tag-SSR (EST-SSRs) markers were developed from transcriptome data of downy mildew inoculated resistant and susceptible pearl millet genotypes. The high quality pearl millet reads were assembled by Trinity into 26,690 transcripts and 3586 (13.4%) transcripts harbored 4510 SSR motifs with the frequency of one SSR per 5.73 kb. The distribution of SSR motifs in different repeat type classes highlighted tri-nucleotide repeats as the highest (54.6% with CCG/CGG) followed by hexa-nucleotide (16.1% with CCGGCG/CCGGCG), penta-nucleotide (14.5% with CCGCG/CGCGG), tetra-nucleotides (8.0% with AGGG/CCCT) and di-nucleotide (6.8% with AG/CT) repeats. Total of 2419 EST-SSRs markers were developed and 230 markers based on their function in downy mildew-pearl millet interaction were validated in 12 pearl millet genotypes which are parents of mapping population. A total of 33 (20.62%) EST-SSR markers displayed polymorphism with a polymorphism information content (PIC) value ranging from 0.188 to 0.375 with an average of 0.354 and 2.2 alleles. The highest polymorphism was displayed by tri-nucleotide repeat EST-SSR markers followed by hexa-nucleotide, penta-nucleotide and di-nucleotide. The EST-SSR primers designed from eight transcripts coding for peroxidase 16-like, transcription factor myc7e, wound stress protein precursor, peroxidase 16-like, zinc finger ccch domain-containing, wrky transcription factor, beta purothionin were found to be more polymorphic than rest of the transcripts. The newly developed EST-SSR markers have added to the repository of molecular markers for pearl millet and can be employed for downy mildew resistance breeding.

Development and Characterization of Genic SSR Markers from Indian Mulberry Transcriptome and Their Transferability to Related Species of Moraceae.

Improving mulberry leaf production with enhanced leaf quality holds the key to sustain the ever increasing demand for silk. Adoption of modern genomic approaches for crop improvement is severely constrained by the lack of sufficient molecular markers in mulberry. Here, we report development and validation of 206 EST derived SSR markers using transcriptome data generated from leaf tissue of a drought tolerant mulberry genotype, Dudia white. Analysis of transcriptome data containing 10169 EST sequences, revealed 1469 sequences with microsatellite repeat motifs. We designed a total of 264 primers to the most appropriate repeat regions, of which 206 were locus specific. These markers were validated with 25 diverse mulberry accessions and their transferability to closely related species belonging to family Moraceae was examined. Of these markers, 189 revealed polymorphism with up to 8 allelic forms across mulberry species, genotypes and varieties with a mean of 3.5 alleles per locus. The markers also revealed higher polymorphic information content of 0.824 among the accessions. These markers effectively segregated the species and genotypes and hence, can be used for both diversity analysis and in breeding applications. Around 40% of these markers were transferable to other closely related species. Along with the other genic and genomic markers, we report a set of over 750 co-dominant markers. Using these markers we constructed the first genetic linkage map of mulberry exclusively with co-dominant markers.

Development and validation of EST-derived SSR markers and diversity analysis in cluster bean (Cyamopsis tetragonoloba)

Cluster bean/guar (Cyamopsis tetragonoloba), has an important place in industry because of its seeds, which contain galactomannan (guar gum) rich endosperm. Guar gum, an important ingredient of many products, is purely an export oriented commodity. Development of molecular markers for this crop is essential to accelerate breeding for guar gum content in seeds. A total of 100 novel primers pairs were developed from 16,476 expressed sequence tags (ESTs) sequence of cluster bean. A total of 50 primers pairs with function annotation of gum synthesis were selected and validated on a panel of 32 genotypes. Among the 50 primers 39 primers were amplified with a total of 45 loci. The polymorphic information content (PIC) ranged from 0.00 to 0.42 with an average of 0.13. With low polymorphic simple sequence repeats (SSRs) and narrow genetic base, most of the genotypes scattered into three clusters regardless of their geographical origin. Present study showed the existence of very low genetic diversity in cluster bean. The results indicated that there is need to explore SSR markers from whole genome or alternative marker systems like SNP (single nucleotide polymorphism) markers, for effective implication of markers in cluster bean breeding.

Analysis of inheritance mode in chrysanthemum using EST-derived SSR markers

To study the inheritance mode of hexaploid chrysanthemum (random or preferential chromosome pairing), a segregation analysis was carried out using SSR markers derived from chrysanthemum ESTs in the public domain. A total of 248 EST-SSR primer pairs were screened in chrysanthemum cultivars ‘Dancer’ and ‘Puma White’, of which 49 EST-SSRs were selected as polymorphic and informative markers. These polymorphic markers were used for genotyping a F1-pseudo test cross population derived from a cross between these two cultivars. The 49 EST-SSRs detected 210 marker alleles with an average number of 4.29 marker alleles per locus. For 180 of these polymorphic SSR marker alleles, segregation could be estimated using a χ2 goodness of fit test (α=0.05) with the expected segregation ratios for hexasomic or disomic inheritance. For 65 SSR marker alleles the segregation ratio was informative for the type of inheritance, 33 marker alleles gave a good fit to the expected segregation ratio for hexasomic inheritance and whereas 24 marker alleles gave a good fit for disomic inheritance showing a higher number of marker alleles supporting autopolyploid segregation in chrysanthemum. In addition, the observed ratio of non-simplex to simplex markers was 20:80 (25 vs. 99) supported hexasomic inheritance. Furthermore, random marker allele assortment was found within the six fully informative markers giving conclusive evidence for hexasomic inheritance in chrysanthemum at these chromosomal regions.

Development of EST-derived SSR markers using next-generation sequencing to reveal the genetic diversity of 50 chrysanthemum cultivars

Chrysanthemum plants are popular worldwide as cut flowers, potted, and in gardens. Several hundred cultivars have been commercialized, indicating that there is substantial genetic variations that can be manipulated under cultivations to produce a wide array of phenotypic variation. To study the genetic diversity of chrysanthemum cultivars in Korea, we first identified simple sequence repeats from chrysanthemum expressed sequence tags generated by FLX 454 sequencing. A total of 1109 ESTs out of 18,226 chrysanthemum ESTs were identified to carry SSRs. A total of 16 out of 46 primer pairs exhibited several polymorphisms among 50 chrysanthemum cultivars. The number of alleles per locus varied from 1 to 15, with an average of 6.25 alleles. The expected heterozygosity ranged from 0 to 0.8958, whereas polymorphism information content ranged from 0 to 0.8872. Based on polymorphisms using 16 SSR markers, a phylogenetic tree was generated revealing four groups within the 50 cultivars showing various levels of genetic diversity. The 16 polymorphic chrysanthemum SSR markers generated in this study would be useful for studies of the genetic conservation, diversity, and population structure of commercial chrysanthemum cultivars as well as closely related species.

Comparison of gSSR and EST-SSR markers for analyzing genetic variability among tomato cultivars (Solanum lycopersicum L.).

In order to study genetic variability and develop better strategies for the utilization of 48 tomato cultivars from America, China, the Netherlands, and Portugal, genomic simple sequence repeat (gSSR) and EST-derived SSR (EST-SSR) markers were applied. In all, 15 of 82 gSSR and 18 of 115 EST-SSR markers showed polymorphic loci. There were 995 and 2072 clear fragments amplified by polymorphic gSSR and EST-SSR markers, respectively. The total and average number of alleles detected by EST-SSRs (75, 4.2) was more than gSSRs (54, 3.6) as a result of some multi-locus EST-SSRs. A lower polymorphism information content value was found in gSSRs (0.529) compared to EST-SSRs (0.620). Similarity coefficient matrixes of the 48 tomato cultivars were established based on the gSSRs and EST-SSRs, and UPGMA dendrograms were constructed from the gSSRs and EST-SSRs similarity coefficient matrixes. A high similarity was observed between the gSSRs and EST-SSRs dendrograms. Genetic variability of four tomato populations from different countries showed that the observed number of alleles and Nei's genetic diversity were highest in the American population, and the effective number of alleles was highest in the Dutch population. The estimated genetic structure showed some tomato cultivars from different countries shared a common genetic background, which might be related to gene flow. It was inferred that both gSSR and EST-SSR markers were effective to assess genetic variability of tomato cultivars, and the combination of both markers could be more effective for genetic diversity analysis in tomato.

Breeding malting barley for consistently low grain protein to sustain production against predicted changes from global warming

Climate change, as summarised by global warming, poses a substantial challenge to malting barley (Hordeum vulgare L.) production and the malting industries. This is because, in sensitive varieties, heat stress causes grain protein content (GPC) to exceed the maximum acceptable value for malting purposes. One way to avoid the problem is to use cultivars with inherently low-GPC and hence reduce the risk of approaching a GPC above 12 %. The present study aimed to characterise the genetic locus on 5HS linked to GPC in two-rowed barley and to combine it with the locus on 6H identified in six-rowed barley. Saturation mapping with EST-derived SSR markers identified four new markers (UMB704, K03390, UMB707, and HvSMEh297) within the target QTL region, which provides functional markers for selection. Over a 3-year period, doubled haploid lines differing in the presence/absence of the marker alleles at the 5HS locus were characterised for physiological differences related to GPC accumulation. Both genotype groups flowered at about the same time, but during grain filling, the low-GPC group accumulated less nitrogen in the spikes, and more dry matter, than the high-GPC group. The 5HS and 6H QTLs were combined into two-row backgrounds, and preliminary field evaluation showed that almost all of the recombinants had GPC levels that were lower than the commercial check (Schooner) by up to 4 % units. Of the 155 F2-derived F5 lines, 20 had grain protein levels that were lower than that of the low-GPC check (VB0229) by 1–2 %. This represents a new germplasm pool that will be useful for variety development, not just to lower GPC, but also to stabilise GPC variability across different environments.

Construction of full-length cDNA library and development of EST-derived simple sequence repeat (EST-SSR) markers in Senecio scandens.

Senecio scandens Buch.-Ham. ex D. Don (Compositae) is a crucial source of Chinese traditional medicine with antibacterial properties. We constructed a cDNA library and obtained expressed sequence tags (ESTs) to show the distribution of gene ontology annotations for mRNAs, using an individual plant with superior antibacterial characteristics. Analysis of comparative genomics indicates that the putative uncharacterized proteins (21.07%) might be derived from "molecular function unknown" clones or rare transcripts. Furthermore, the Compositae had high cross-species transferability of EST-derived simple sequence repeats (EST-SSR), based on valid amplifications of 206 primer pairs developed from the newly assembled expressed sequence tag sequences in Artemisia annua L. Among those EST-SSR markers, 52 primers showed polymorphic amplifications between individuals with contrasting diverse antibacterial traits. Our sequence data and molecular markers will be cost-effective tools for further studies such as genome annotation, molecular breeding, and novel transcript profiles within Compositae species.

Characterization of EST-derived and non-EST simple sequence repeats in an F₁ hybrid population of Vitis vinifera L.

Among different classes of molecular markers, expressed sequence tags (ESTs) are a new resource for developing simple sequence repeat (SSR) functional markers for genotyping and genetic mapping in F1 hybrid populations of Vitis vinifera L. Recently, because of the availability of an enormous amount of data for ESTs in the public domain, the emphasis has shifted from genomic SSRs to EST-SSRs, which belong to transcribed regions of the genome and may have a role in gene expression or function. The objective of this study was to assess the polymorphisms among 94 F1 hybrids from "Early Rose" and "Red Globe" using 25 EST-derived and 25 non-EST SSR markers. A total collection of 362,375 grape ESTs that were retrieved from the National Center for Biotechnology Information (NCBI) and 2522 EST-SSR sequences were identified. From them, 205 primer pairs were randomly selected, including 176 pairs that were EST-derived and 29 non-EST SSR primer pairs, for polymerase chain reaction amplification. A total of 131 alleles were amplified using 50 pairs of primers; 78 alleles were amplified using EST-derived SSR primers and 53 were from non-EST SSR primers. At most, 6 and 5 alleles were amplified by EST-derived and non-EST SSR primers, respectively. The EST-derived SSR markers showed a maximum polymorphic information content (PIC) value of 1 and a minimum of 0.33 while non-EST SSR markers had maximum and minimum PIC values of 1 and 0.25, respectively. The average PIC value was 0.56 for EST-derived SSR markers and 0.45 for non-EST SSR markers.

<i>In</i> <i>Silico</i> Mining of EST-SSRs in Jatropha curcas L. towards Assessing Genetic Polymorphism and Marker Development for Selection of High Oil Yielding Clones

In recent years, Jatropha curcas L. has gained popularity as a potential biodiesel plant. The varying oil content, reported between accessions belonging to different agroclimatic zones, has necessitated the assessment of the existing genetic variability to generate reliable molecular markers for selection of high oil yielding variety. EST derived SSR markers are more useful than genomic markers as they represent the transcriptome, thus, directly linked to functional genes. The present report describes the in silico mining of the microsatellites (SSRs) using J. curcas ESTs from various tissues viz. embryo, root, leaf and seed available in the public domain of NCBI. A total of 13,513 ESTs were downloaded. From these ESTs, 7552 unigenes were obtained and 395 SSRs were generated from 377 SSR-ESTs. These EST-SSRs can be used as potential microsatellite markers for diversity analysis, MAS etc. Since the Jatropha genes carrying SSRs have been identified in this study, thus, EST-SSRs directly linked to genes will be useful for developing trait linked markers.

EST-SSR and SSR analyses of genetic diversity in diploid cotton genotypes from Iran

There are 21 diploid species/genotypes of the cotton genus Gossypium L. that are cultivated in many regions of Iran. Due to continuous artificial selection and cultivation practice there is a decrease in its genetic diversity causing genetic erosion. Therefore, cotton breeders continue to make various cross combinations among available cultivars. Genetic diversity analysis was considered in 21 cotton genotypes by using SSR and EST-derived SSR markers. Amplification of the EST-SSR and SSR markers produced 19 reproducible alleles in the cotton plants studied. The mean number of effective alleles was 1.12, and the mean value of Shannon’s information index was 0.10. The mean value for genetic polymorphism was 20 % in cotton plants studied. AMOVA test showed that only 2 % of total genetic variation is accounted in 3 populations groups, of which 98 % is due to genetic variations among accessions in each population group. Neighbor Joining tree and PCoA plot showed genetic affinity between plants from different populations. STRUCTURE result confirmed AMOVA test and showed presence of genetic diversity within each presumed population groups. Reticulogram showed gene exchange among some of the cotton plants. Gene exchange/allelic similarity were observed between VTDL (G. arboreum) and Semnan (G. herbaceum) genotypes and between some interspecific hybrids.

A Novel Set of EST-Derived SSR Markers for Pear and Cross-Species Transferability in Rosaceae

Pear is a fruit species with worldwide distribution belonging to the Rosaceae family. In this study, transcriptomes of 12 different tissues of pear were newly assembled, with a total of 17,353 SSR loci identified. Within these, repetitive sequences with dinucleotide and trinucleotide motifs were more abundant than other types. We developed 194 EST-SSR makers from the assembled sequences to test on seven pear varieties from different genetic backgrounds, on which 132 EST-SSR primers had amplicons. There were 108 primers with dinucleotide and trinucleotide motifs detecting a total of 311 alleles, with an average per primer of 2.88 and 2.86, respectively. These had better quality than primers with tetranucleotides, pentanucleotides, and hexanucleotides. Fifty polymorphic markers divided 36 pear genotypes into two groups with a similarity coefficient of 0.62. Results showed that Japanese pears had a comparatively close relationship with Chinese pears, indicating the great potential of the novel EST-SSR makers in future evaluations of pear germplasm. Furthermore, 54 EST-SSR markers were selected to test transferability to 96 other Rosaceae cultivars from eight species, including Japanese apricot, plum, apricot, peach, cherry, apple, strawberry, and loquat. Seventeen EST-SSR primers had PCR amplification products in at least one of the 96 accessions, with the highest transferability was from pear to apple at 29.63 %. Transferability from pear to Japanese apricot, plum, apricot, peach, cherry, strawberry, and loquat was 9.26, 14.81, 11.11, 12.96, 5.56, 3.7, and 12.96 %, respectively. Therefore, the newly developed EST-SSR markers can contribute to exploring the genetic relationship, evolution and comparative genomics, and gene introgressions in the Rosaceae family.

Development of EST-derived SSR Markers with Long-core Repeat in Olive and Their Use for Paternity Testing

In the present work, a set of eight new hexa-nucleotide simple sequence repeats (SSRs) is reported in olive (Olea europaea L). These SSRs loci were generated on the basis of expressed sequence tag (EST) sequences in the frame of an olive genomic project. The markers showed a high level of polymorphism when tested on a set of cultivars used as genitors in the olive breeding program of Córdoba, Spain. The long-core repeat motif of these markers allows a wider separation among alleles, thus permitting an accurate genotyping. Besides, these markers showed comparable levels of polymorphism to di-nucleotide SSRs, the only ones so far reported in olive. Selected on the basis of their discrimination capacity, four of the eight SSRs were used to test their ability for paternity testing in a total of 81 seedlings coming from 12 crosses. The paternity testing showed that seven crosses matched the alleged paternity and the remaining five were products of illicit pollinations. These results exactly matched with previous paternity testing performed with di-nucleotide SSR markers. These results demonstrate the usefulness of the developed hexa-nucleotide repeated motifs for checking the paternity of breeding progenies and suggest their use on variability studies.

Identification of 12 EST-derived SSR markers in Lumbricus rubellus

Abstract While many species of earthworms are globally distributed, very little is known about the genetic population dynamics of this diverse group. We present the characterization of novel simple sequence repeat (SSR) markers, including primer information, number of alleles, repeat motif, and approximate size ranges, to be used in population genetic analyses of the earthworm Lumbricus rubellus Hoffmeister 1843. Specifically, we designed and characterized 12 novel, polymorphic markers derived from published expressed sequence tags (EST) for amplification in L. rubellus. The mean number of alleles per locus was 6.25 ± 1.91, indicating these markers will be sufficiently polymorphic for population genetic studies of this species.