Development,characterization and variability analysis of microsatellites in lychee (<Emphasis Type="Italic">Litchi chinensis</Emphasis> Sonn., Sapindaceae)

We report 12 microsatellites enriched in CT repeats obtained from a genomic library of the lychee (Litchi chinensis Sonn.) cultivar Mauritius. The polymorphisms revealed by these microsatellites were evaluated in a collection of 21 lychee cultivars. A total of 59 fragments were detected with these 12 SSRs, with an average of 4.9 bands/SSR. Three primer pairs seem to amplify more than a single locus. The mean expected and observed heterozygosities over the 9 single-locus SSRs averaged 0.571 (range: 0.137–0.864) and 0.558 (range: 0.169–0.779) respectively. The total value for the probability of identity was 7.53×10^-5. In addition, the selected SSRs were used to amplify DNA from four longan cultivars. Eleven of the 12 SSRs produced amplification fragments in longan, and eight of these fragments were polymorphic. All except two of the products amplified from longan were the same size as those amplified from lychee, suggesting a close genetic proximity between the two species. The SSRs studied produced 22 different patterns, allowing the unambiguous identification of 16 lychee and the 4 longan cultivars studied. Discrimination was possible with just four selected microsatellites. Two groups with two and three undistinguishable cultivars were obtained, reflecting probable synonymies. Unweighted pair-group method of artimetic averages (UPGMA) cluster analysis divided the lychee cultivars studied into two main groups, one consisting of ancient cultivars and the other with more diverse recent cultivars. This is the first report of microsatellite development in the Sapindaceae, and the results demonstrate the usefulness of microsatellites for identification, similarity studies and germplasm conservation in lychee and related species.Communicated by H.F. Linskens     

Development of simple sequence repeats (SSRs) in olive tree (Olea europaea L.)

We report the development of microsatellites or simple sequence repeats (SSRs) in the olive tree (Olea europaea L.). Forty three positive clones obtained by the screening of a GA-enriched genomic library were sequenced and primers were designed for 13 microsatellite loci. Five primer pairs amplified polymorphic products of the expected size range. SSR polymorphism was explored in a set of 46 olive cultivars. A total of 26 alleles were detected for the five loci. Heterozygosity ranged from 0.46 to 0.71. Ninety one per cent of the cultivars had unique multilocus genotypes. Microsatellite segregation was studied in a complex population from a cross between the commercial cultivars ’Leccino’ and ’Dolce Agogia’. Received: 3 February 2000 / Accepted: 21 March 2000     

Development of microsatellite markers for mango (Mangifera indica L.)

Microsatellite markers for mango (Mangifera indica L.) were developed using a genomic library enriched for (GA)_n and (GT)_n dinucleotide repeats. A subset of 41 positive clones was sequenced and primers were designed. Twenty‐eight primer pairs produced polymorphic amplification products for a diversity sample including 15 mango cultivars and two accessions from the related species Mangifera laurina and Mangifera applanata. Nineteen simple sequence repeat (SSR) loci with clear scorable patterns were chosen to study diversity in the mango germplasm bank of Guadalupe (FWI). The number of alleles ranged from three to 13 with observed levels of heterozygosity ranging from 0.059 to 0.857.     

Isolation and characterization of microsatellites in Brassica rapa L

We report here the isolation and characterization of microsatellites, or simple sequence repeats (SSRs), in Brassica rapa. The size-fractionated genomic library was screened with (GA)(15) and (GT)(15) oligonucleotide probes. A total of 58 clones were identified as having the microsatellite repeats, and specific primer pairs were designed for 38 microsatellite loci. All primer pairs, except two, amplified fragments having the sizes expected from the sequences. Of the 36 primer pairs, 35 amplified polymorphic loci in 19 cultivars of B. rapa, while monomorphism was observed in only one primer pair. A total of 232 alleles was identified by the 36 primer pairs in 19 cultivars of B. rapa, and these primer pairs were examined also in nine Brassicaceae species. Most of the 36 primer pairs amplified the loci in the Brassicaceae species. Segregation of the microsatellites was studied in an F(2) population from a cross of doubled-haploid lines DH27 x G309. The microsatellites segregated in a co-dominant manner. These results indicate that the microsatellites isolated in this study were highly informative and could be useful tools for genetic analysis in B. rapa and other related species.     

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.     

Multiple methods for the identification of polymorphic simple sequence repeats (SSRs) in sorghum [Sorghum bicolor (L.) Moench]

Simple sequence repeats (SSRs), also known as microsatellites, are highly variable DNA sequences that can be used as markers for the genetic analysis of plants. Three approaches were followed for the development of PCR primers for the amplification of DNA fragments containing SSRs from sorghum [Sorghum bicolor (L.) Moench]: a search for sorghum SSRs in public DNA databases; the use of SSR-specific primers developed in the Poaceae species maize (Zea mays L.) and seashore paspalum grass (Paspalum vaginatum Swartz); and the screening of sorghum genomic libraries by hybridization with SSR oligonucleotides. A total of 49 sorghum SSR-specific PCR primer pairs (two designed from GenBank SSR-containing sequences and 47 from the sequences of genomic clones) were screened on a panel of 17 sorghum and one maize accession. Ten primer pairs from paspalum and 90 from maize were also screened for polymorphism in sorghum. Length polymorphisms among amplification products were detected with 15 of these primer pairs, yielding diversity values ranging from 0.2 to 0.8 with an average diversity of 0.56. These primer pairs are now available for use as markers in crop improvement and conservation efforts.     

Development of Simple Sequence Repeat Markers from Functional Genes and Establishment of Molecular Identity for Tree Peony

Gene-derived simple sequence repeats (genic SSRs), also known as functional markers, are generally superior to random markers because they are located in genes and therefore may affect gene expression or function. However, extremely limited genic SSRs are available for tree peony. We used the functional gene sequences available from Paeonia to develop genic SSRs. A total of 132 SSR loci were identified from 35 cDNA sequences, of which trinucleotide (58, 43.9%) and hexanucleotide repeat (37, 28.0%) were dominant. Moreover, 121 primer pairs were successfully designed and synthesized, of which 49 primer pairs (40.5%) provided efficient and reliable amplification. By screening 16 tree peony varieties, we developed eight polymorphic genic SSRs with 37 alleles, ranging from 2 to 11 for each marker. Transferability analysis indicated that 100% of the genic SSRs could be amplified in eight other Paeonia samples. Based on eight polymorphic genic SSRs and 12 polymorphic EST-SSRs developed by predecessors, the molecular identity of 190 tree peony cultivars was constructed by capillary electrophoresis. The results showed that 146 alleles and 338 genotypes were detected, with 2–13 alleles and 3–36 genotypes for each marker. All cultivars were completely identified and exhibited unique DNA identity. In addition, the identification efficiency of different primers combinations was analyzed, and 190 germplasms were identified using 6 core primers. This study provides valuable genic SSR resources for marker-assisted selection breeding of the genus Paeonia. The DNA identity of cultivars is of great significance for the protection, utilization and management of tree peony resources.

     

Development of transcript-associated microsatellite markers for diversity and linkage mapping studies in hop (<Emphasis Type="Italic">Humulus lupulus</Emphasis> L.)

Data mining of gene sequences available from various projects dealing with the development of expressed sequence tags (ESTs) can contribute to the discovery of new microsatellite markers. Our aim was to develop new microsatellite markers in hop isolated from an enriched cDNA library and from coding GenBank sequences and to test their suitability in hop diversity studies and for construction of a linkage map. In a set of 614 coding GenBank sequences, 72 containing microsatellites were found (11.7%); the most frequent were trinucleotide repeats (54.0%) followed by dinucleotide repeats (34.5%). Additionally, 11 sequences containing microsatellites were isolated from an enriched cDNA library. A total of 34 primer pairs were designed, 29 based on GenBank sequences and five on sequences from the cDNA enriched library. Twenty-seven (79.4%) coding microsatellites were successfully amplified and used in diversity and linkage mapping studies. Eleven primer pairs amplified 12 coding microsatellite loci suitable for mapping and were placed on female and male linkage maps. We were able to extend previous simple sequence repeat (SSR) female, male and integral maps by 38.8, 25.8 and 40.0 cM, respectively. In the diversity study, 36 diverse hop genotypes were analyzed. Twenty-four coding microsatellites were polymorphic, 17 showing co-dominant behavior and 7 primer pairs amplifying three or more bands in some hop genotypes. Altogether, 143 microsatellite DNA fragments were amplified and they revealed a clear separation of hop genotypes according to geographical region, use or breeding history. In addition, a discussion and comparison of results with other plant coding/EST SSR studies is presented. Our results showed that these microsatellite markers can enhance hop diversity and linkage mapping studies and are a comparable marker system to non-coding SSRs.     

Identification and characterization of simple sequence repeat (SSR) markers from Fragaria×ananassa expressed sequences

The availability of expressed sequence data derived from gene discovery programmes enables mining for simple sequence repeats (SSRs), providing useful genetic markers for crop improvement. These markers are inexpensive, require minimal labour to produce and can frequently be associated with functionally annotated genes. This study reports on the development and characterization of expressed sequence tag (EST)–SSR markers in the cultivated strawberry, Fragaria×ananassa. Fourteen primer pairs were assessed for polymorphism in 13 F.×ananassa genotypes. The markers show reliable amplification and considerable polymorphism, demonstrating the utility of EST–SSRs for genetic analysis of commercial strawberry germplasm.     

Eleven new microsatellites for hop (Humulus lupulus L.)

We present a new set of 11 polymorphic microsatellite primer sequences for use with Humulus lupulus. Microsatellite‐enriched libraries for GA_n and GT_n types of repeats were produced. Sequencing of 72 clones revealed 42 unique inserts containing microsatellites, out of which 19 primer pairs were designed and microsatellite amplification was tested on 39 wild hops and cultivars. Eleven primer pairs showed single locus amplification with 2–13 alleles, average 7.2, of which 17 unique alleles were discovered. One primer pair amplified too strong stutter bands, one locus was monomorphic and multilocus amplification was obtained with the remaining six primer pairs.     

Citrullus simple sequence repeats markers from sequence databases

Publically available cDNA sequence data of Citrullus lanatus were searched for simple sequence repeats (SSRs). Nineteen microsatellites were identified and primer pairs were designed to amplify those loci. Primers were evaluated for their ability to detect polymorphisms within a set of several watermelon varieties and local landraces, C. colocynthis, and interspecific hybrids. Eighteen polymorphic SSR loci were identified. These polymorphic loci can be used for varietal identification and other uses.     

<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.     

Microsatellite markers isolated in olive ( Olea europaea L.) are suitable for individual fingerprinting and reveal polymorphism within ancient cultivars

We have isolated and sequenced 52 microsatellites or simple sequence repeats (SSRs) from nearly 60 positive clones obtained from two ’Frantoio’ olive genomic libraries enriched in (AC/GT) and (AG/CT) repeats, respectively. The repeat-containing fragments obtained from genomic DNA restricted with Tsp509I were separated using a biotinylated probe bound to streptavidin-coated paramagnetic beads. Fragments were then cloned into lambda ZAPII vector and sequenced. Thirty of the 36 primer pairs which gave correct re-amplification in the source genome were used to assay the polymorphism of 12 olive cultivars, namely four well-known cultivars (’Coratina’, ’Frantoio’, ’Leccino’, ’Pendolino’) and eight ancient cultivars grown locally near Lake Garda (’Casaliva’, ’Favarol’, ’Fort’, ’Grignan’, ’Less’, ’Raza’, ’Rossanel’, ’Trep’). The local cultivars were each re- presented by two to four long-lived individuals. The analysis was carried out using ³³P-labelled primers and 6% polyacrylamide sequencing gels. All except two microsatellites showed polymorphism, the number of alleles varying from 1 to 5. The average genetic diversity (H) was 0.55. The power of discrimination (PD) was 0.60. All cultivars, including the local ones, were easily separated from each other. Variations in the SSR pattern were observed among individual plants of the same cultivar in four out of the eight local cultivars analysed. Several primer pairs (17%) amplified more than one locus. Received: 23 March 2001 / Accepted 17 May 2001     

Microsatellite markers from sugarcane (Saccharum spp.) ESTs cross transferable to erianthus and sorghum

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.     

Rapid microsatellite marker isolation for the Pink Stem Borer,Sesamia inferens (Lepidopetera: Noctuidae), through 454 GS-FLX Titanium pyrosequencing of enriched DNA libraries and cross amplification in related taxa

Next-generation Roche 454 pyrosequencing was used to rapidly identify polymorphic microsatellites from enriched DNA libraries for the pink stem borer, Sesamia inferens (Walker). A total of 1,459 simple sequence repeats (SSRs) were isolated from the microsatellite-enriched library using 454 sequencing. Thirty-nine microsatellite markers were selected to synthesize for further optimization, and 12 loci exhibited reliable amplification of a single product of expected size. The forward primer of 12 primer pairs was end labeled with a fluorescent dye. All of the 12 microsatellite loci were polymorphic, with 5–13 alleles per locus and observed heterozygosities ranging from 0.097 to 0.957. Here, we also tested these 12 SSRs for cross-species amplification in Chilo suppressalis (Walker), Tryporyza incertulas (Walker) and Cnaphalocrocis medinalis (Guenée). These polymorphic markers will be a valuable tool for analyses of population connectivity and genetic structure in this rice pest.     

Development and characterisation of simple sequence repeat (SSR) markers for white clover (Trifolium repens L.)

Highly informative molecular markers, such as simple sequence repeats (SSRs), can greatly accelerate breeding programs. The aim of this study was to develop and characterise a comprehensive set of SSR markers for white clover (Trifolium repens L.), which can be used to tag genes and quantitative trait loci controlling traits of agronomic interest. Sequence analysis of 1123 clones from genomic libraries enriched for (CA) _n repeats yielded 793 clones containing SSR loci. The majority of SSRs consisted of perfect dinucleotide repeats, only 7% being trinucleotide repeats. After exclusion of redundant sequences and SSR loci with less than 25 bp of flanking sequence, 397 potentially useful SSRs remained. Primer pairs were designed for 117 SSR loci and PCR products in the expected size range were amplified from 101 loci. These markers were highly polymorphic, 88% detecting polymorphism across seven white clover genotypes with an average allele number of 4.8. Four primer pairs were tested in an F₂ population revealing Mendelian segregation. Successful cross-species amplification was achieved in at least one out of eight legume species for 46 of 54 primer pairs. The rate of successful amplification was significantly higher for Trifolium species when compared to species of other genera. The markers developed in this study not only provide valuable tools for molecular breeding of white clover but may also have applications in related taxa. Received: 3 April 2000 / Accepted: 12 May 2000     

Identifying Novel Polymorphic Microsatellites from Cultivated Flax (Linum usitatissimum L.) Following Data Mining

One of the major concerns in genetic characterization and breeding of cultivated flax is the lack of informative microsatellite markers (SSRs). In this regard, the development of SSRs using molecular methods might be time-consuming, laborious, and expensive. On the other hand, using bioinformatics to mine sequences in public databases enables a cost-effective discovery of SSRs. A total of 3,242 Linum usitatissimum genomic sequences were surveyed for the identification of SSRs. Among them, 118 non-redundant sequences containing repeats were selected for designing primers. The most abundant motifs were tri- (72.4%) and dinudeotide (16.6%), within which AGG/CCT and AG/CT were predominant. Primers were tested for polymorphism in 60 L. usitatissimum cultivars/accessions including 57 linseed and three fiber flax. Eighty-eight pairs gave amplifications within the expected size range while 60 pairs were found to be polymorphic. The mean number of alleles amplified per primer was 3.0 (range, 2–8; 180 total alleles). The mean polymorphism information content (PIC) value was 0.39 (range, 0.06–0.87), and the highest average PIC was observed in dinucleotide SSRs (0.41). The SSR data mining presented here demonstrates the usefulness of in silico development of microsatellites. These novel genomic SSR markers could be used in genetic diversity studies, the development of genetic linkage maps, quantitative trait loci mapping, association mapping, and marker-assisted selection.     

Transferability and Level of Heterozygosity of Microsatellite Markers in <Emphasis Type="Italic">Citrus</Emphasis> Species

Microsatellite markers are a powerful tool for genetic studies, including germplasm conservation, cultivar identification, and integration of linkage maps. Several works have shown that primer pairs designed for one species can be used in related species to facilitate wider application because it reduces the costs for primer development. The objective of this study was to evaluate the transferability of microsatellite primers which was previously developed from the genomic library of Pêra sweet orange (Citrus sinensis L. Osbeck) and to determine the level of heterozygosity between citrus accessions and related genera. Twenty-four microsatellite loci were evaluated on 12 genotypes of Citrus, Poncirus, and an intergeneric hybrid. All analyzed markers were transferable across all genotypes. Seventeen loci were polymorphic, and the number of alleles per loci ranged from one to six. The lowest level of heterozygosity was observed for Poncirus trifoliata (L.) Raf. cultivars while the highest level was for Swingle citrumelo. In general, microsatellite markers showed wide genetic variation and demonstrated that they can be useful in citrus breeding programs.     

Identification of polymorphic,conserved simple sequence repeats (SSRs) in cultivated Brassica species

The application of simple sequence repeat (SSR) genotyping for the characterization of genetic variation in crop plants has been hindered by ready access to useful primer pairs and potentially limited conservation of the repeat sequences among related species. In this phase of work, we report on the identification and characterization of SSRs that are conserved in Brassica napus L. (rapeseed) and its putative progenitors, B. oleracea L. (cabbage, and related vegetable types) and B. rapa (vegetable and oil types). Approximately 140 clones from a size-fractionated genomic library of B. napus were sequenced, and primer pairs were designed for 21 dinucleotide SSRs. Seventeen primer pairs amplified products in the three species and, among these, 13 detected variation between and within species. Unlike findings on SSR information content in human, no relationship could be established between the number of tandem repeats within the target sequence and heterozygosity. All primer pairs have been designed to work under identical amplification conditions; therefore, single-reaction, multiplex polymerase chain reaction (PCR) with these SSRs is possible. Once moderate numbers of primer pairs are accessible to the user community, SSR genotyping may provide a useful method for the characterization, conservation, and utilization of agricultural crop diversity.     

EST‐SSR markers from Fragaria vesca L. cv. Yellow Wonder

Fourteen microsatellite primer pairs were developed from a cDNA library of heat‐treated seedlings of Fragaria vesca cv. yellow wonder. Transferability to 13 species of Fragaria ranged from 71% in diploid species F. gracilis Losinsk., F. iinumae Makino, F. nilgerrensis Schltdl. ex J. Gay and F. nipponica Makino, to 100% in octoploid domestic strawberry and its progenitors. Polymorphism was high in polyploid Fragaria species. However, polymorphism and heterozygosity of eight EST‐SSRs (expressed sequence tag–simple sequence repeats) was low in 14 F. vesca genotypes.