Development and characterization of 20 new polymorphic microsatellite markers from Mangifera indica (Anacardiaceae)

? Premise of the study: Twenty microsatellite loci for mango (Mangifera indica), an important commercial fruit tree in East Asia, were developed to evaluate the genetic diversity and identification of cultivars. ? Methods and Results: The 20 new microsatellite markers were isolated from mango using a magnetic bead enrichment method, and polymorphisms were identified in 22 mango cultivars. The number of alleles ranged from one to nine, with expected heterozygosity ranging from 0 to 0.826. The polymorphism information content ranged from 0 to 0.756 with a mean of 0.525. ? Conclusions: These new microsatellite loci should be useful and convenient for further studies of the genetic diversity and identification of cultivars in mango.     

The development of oat microsatellite markers and their use in identifying relationships among Avena species and oat cultivars

Microsatellites have many desirable marker properties. There has been no report of the development and utilization of microsatellite markers in oat. The objectives of the present study were to construct oat microsatellite-enriched libraries, to isolate microsatellite sequences and evaluate their level of polymorphism in Avena species and oat cultivars. One hundred clones were isolated and sequenced from three oat microsatellite-libraries enriched for either (AC/TG) _n , (AG/TC) _n or (AAG/TTC) _n repeats. Seventy eight clones contained microsatellites. A database search showed that 42% of the microsatellite flanking sequences shared significant homology with various repetitive elements. Alu and retrotransposon sequences were the two largest groups associated with the microsatellites. Forty four primer sets were used to amplify the DNA from 12 Avena species and 20 Avena sativa cultivars. Sixty two percent of the primers revealed polymorphism among the Avena species, but only 36% among the cultivars. In the cultivars, the microsatellites associated with repetitive elements were less polymorphic than those not associated with repetitive elements. Only 25% of the microsatellites associated with repetitive elements were polymorphic, while 46% of the microsatellites not associated with repetitive elements showed polymorphism in the cultivars. An average of four alleles with a polymorphism information content (PIC) of 0.57 per primer set was detected among the Avena species, and 3.8 alleles with a PIC of 0.55 among the cultivars. In addition, 54 barley microsatellite primers were tested in Avena species and 26% of the primers amplified microsatellites from oat. Using microsatellite polymorphisms, dendrograms were constructed showing phylogenetic relationships among Avena species and genetic relationships among oat cultivars. Received: 1 November 1999 / Accepted: 14 April 2000     

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.     

Development and genetic mapping of 127 new microsatellite markers in barley

To enhance the marker density of existing genetic maps of barley (Hordeum vulgare L.), a new set of microsatellite markers containing dinucleotide motifs was developed from genomic clones. Out of 254 primer pairs tested, a total of 167 primer pairs were classifed as functional in a panel of six barley cultivars and three H. spontaneum accessions, and of those, 127 primer pairs resulting in 133 loci were either mapped or located onto chromosomes. The polymorphism information content (PIC) ranged from 0.05 to 0.94 with an average of 0.60. The number of alleles per locus varied from 1 to 9. On average, 3.9 alleles per primer pair were observed. The RFLP frameworks of two previously published linkage maps were used to locate a total of 115 new microsatellite loci on at least one mapping population. The chromosomal assignment of 48 mapped loci was corroborated on a set of wheat-barley chromosome addition lines; 18 additional loci which were not polymorphic in the mapping populations were assigned to chromosomes by this method. The microsatellites were located on all seven linkage groups with four significant clusters in the centromeric regions of 2H, 3H, 6H and 7H. These newly developed microsatellites improve the density of existing barley microsatellite maps and can be used in genetic studies and breeding research.Communicated by G. Wenzel     

Microsatellite retrieval in lettuce (Lactuca sativa L.).

By using enriched genomic libraries, microsatellite-containing sequences were isolated from lettuce (Lactuca sativa) with high efficiency. With this approach, a sizeable fraction (up to 55%) of the clones contained a microsatellite. In about half of these clones, primers could be designed for PCR amplification of the microsatellite. This yielded 28 primer sets amplifying unambiguously scorable products, of which 26 showed polymorphisms in a test set of six lettuce varieties. Practically all microsatellite-amplifying primer sets yielded products in lettuce's nearest relative, L. serriola, but only half of the primer sets yielded products in the more distant species L. saligna and L. virosa. An average polymorphism information content (PIC) value of 0.55 and an average number of 3.5 alleles per locus were in the normal range for a self-fertilizing species like lettuce. In addition, the incidental cloning of a microsatellite-containing repeat family, apparently specific for Lactuca, is reported and the implications for the efficient retrieval of single-locus microsatellite sequences are discussed. The microsatellite loci isolated will be useful for distinguishing lettuce cultivars and for screening diversity of genetic resources.     

Isolation and characterization of polymorphic microsatellite markers from European pear (Pyrus communis L.)

This study reports the development and characterization of 19 microsatellite primer pairs developed from genomic DNA of European pear (Pyrus communis) and their transferability to other Pyrus and Malus material. The primers were designed from two different genomic libraries enriched for di‐ and trinucleotide repeats. When tested in six P. communis cultivars and 15 other Pyrus species, 13 primers revealed single‐locus polymorphism and six showed more complex patterns that suggest multiple loci. Two to 18 alleles were detected per locus and two primer pairs were sufficient to discriminate all accessions. Transferability of nine primer pairs to Malus was demonstrated through amplification of discrete products in two accessions.     

Use of short microsatellites from database sequences to generate polymorphisms among Lycopersicon esculentum cultivars and accessions of other Lycopersicon species

A search of nearly 2000 sequences from Solanaceae species in the EMBL and Genbank databases yielded 220 microsatellites. Among these were 80 microsatellites from 675 Lycopersicon entries. Dinucleotide repeats, as well as (CAA)_n and (TAA)_n repeats, were over-represented in non-coding DNA. The other trinucleotide repeats were predominantly found in exonic DNA. PCR analysis of 44 of the microsatellite-containing Lycopersicon loci identified 36 primer pairs that yielded well-scorable fragments, or groups of fragments, in L. esculentum cultivars and accessions of Lycopersicon species. Twenty-nine of these amplified bands that were polymorphic among the four Lycopersicon species. Ten primer pairs generated polymorphic bands among seven tomato cultivars. Upon examining the number of microsatellites and the degree of polymorphisms in relation to the repeat type and motif, the type of DNA the microsatellite resided in, the length of the microsatellite, and the presence of imperfections in the microsatellite, only two significant correlations were found. (i) Imperfect repeats were less polymorphic among species than perfect repeats. (ii) The percentage of loci polymorphic among cultivars increased from 6% for the shortest loci (with eight or less repeat units) to 60% for the group with the longest repeats (12 repeat units or longer). Among the species, however, all length classes contained about 83% polymorphic loci. In general, 2–4 alleles were found for each locus among the samples of the test set. In a few cases, up to eight alleles were found. A combination of these microsatellite loci can therefore be useful in distinguishing cultivars of tomato, which are genetically very closely related to each other. Received: 9 August 1996 / Accepted: 23 August 1996     

Development and characterization of new microsatellite markers in <Emphasis Type="Italic">Panax</Emphasis><Emphasis Type="Italic">ginseng</Emphasis> (C.A. Meyer) from BAC end sequences

Panax ginseng, commonly known as Korean ginseng, is a valued source of herbal medicine in Korea and China. We have developed and characterized 35 microsatellite markers in P. ginseng from available BAC end sequences. Characterization of these 35 SSR primer pairs in 14 cultivars of P. ginseng showed 12 primer pairs to be polymorphic and 19 primer pairs to be monomorphic, while the remaining four primer pairs did not produce any product. The number of alleles amplified ranged from 4 to 8 per primer pair, with an average of six alleles per locus. The expected and observed heterozygosities ranged from 0.7500 to 0.9678 and 0.5645 to 0.7109, respectively. None of these loci deviated from Hardy–Weinberg equilibrium. All of the functional primer pairs of P. ginseng showed cross-species transferability with Panax quinquefolium. The cross-species transferable markers could be used for ginseng cultivar identification, for genomic studies, including mapping of specific trait QTL/genes, and for measuring conservation of ginseng.     

Fingerprinting,embryo type and geographic differentiation in mango (<Emphasis Type="Italic">Mangifera indica</Emphasis> L., Anacardiaceae) with microsatellites

We report the sequence and variability parameters of 16 microsatellite primer pairs obtained from two mango (Mangifera indica L.) genomic libraries after digestion of DNA of the cultivar Tommy Atkins with HaeIII and RsaI and enrichment in CT repeats. Although no significant differences were recorded between the two libraries in the informativeness of the markers obtained, the RsaI library was shown to be more useful than the HaeIII taking into account the efficiency of the library and the feasibility of clone sequencing. The polymorphism revealed by those microsatellites was evaluated in a collection of 28 mango cultivars of different origins. A total of 88 fragments were detected with the 16 simple sequence repeats (SSRs) with an average of 5.5 bands/SSR. Two primer pairs amplified more than a single locus. The mean expected and observed heterozygosities over the 14 single-locus SSRs averaged 0.65 and 0.69 respectively. The total value for the probability of identity was 2.74 × 10⁻⁹. The SSRs studied allowed the unambiguous identification of all the mango genotypes studied and this discrimination can be carried out with just three selected microsatellites. UPGMA cluster analysis and Principal coordinates analysis group the genotypes according to their origin and their classification as monoembryonic or polyembryonic types reflecting the pedigree of the cultivars and the movement of mango germplasm. The results demonstrate the usefulness of microsatellites for studies on identification, variability, germplasm conservation, domestication and movement of germplasm in mango.     

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.     

Identification of microsatellite loci in apricot

The sequences of 21 primer pairs of microsatellite loci screened from a genomic library of apricot (Prunus armeniaca L.) are reported in this study. All the identified microsatellite loci were characterized in a set of 25 apricot cultivars and revealed to be polymorphic with 3–12 alleles per locus. These markers showed to be more informative than isozymes and restriction fragment length polymorphisms (RFLPs) reported in the literature.     

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.     

Microsatellite analysis of relationships within cultivated potato (Solanum tuberosum)

The potential of microsatellite markers for use in genetical studies in potato (Solanum tuberosum) was evaluated. Database searches revealed that microsatellite sequences were present in the non-coding regions of 24 potato genes. Twenty-two sets of primers were designed and products successfully amplified using 19 primer pairs. These were tested against a panel of 18 tetraploid potato cultivars. Four pairs of primers designed to amplify microsatellites from tomato were also used. Seven (including 2 of the tomato sequences) failed to reveal any variation in the accessions tested. Sixteen primer pairs did reveal polymorphism, detecting between 2 and 19 alleles at each locus. Of these, 3 gave rise to complex band patterns, suggesting that multiple polymorphic loci were being amplified using a single primer pair. Heterozygosity values ranged from 0.408 to 0.921. Phenetic analysis of the derived information allowed a dendrogram to be constructed depicting the relationships between the 18 potato cultivars. The potential of microsatellite markers for genetic analysis and satutory applications in potato is discussed in the context of these results. Furthermore, the potential of crossspecies amplification is highlighted as an additional source of microsatellite markers for genetic research in potato.     

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 polymorphic microsatellite DNA markers in the brown sole,Pleuronectes herzensteini

New microsatellite DNA markers from brown sole were developed and characterized. Fourteen primer sets were designed from 40 microsatellite regions. Eight of 14 loci exhibited variations comprising 8–31 alleles. Observed and expected heterozygosities ranged from 0.611 to 0.833 and from 0.647 to 0.968 among 36 individuals, respectively. Phz3, Phz8 and Phz12 significantly deviated from Hardy–Weinberg equilibrium, and there was a significant linkage disequilibrium between Phz2 and Phz12. Seven of eight loci conformed to the Mendelian manner of inheritance in a full‐sib family. Seven to four loci of three related species were cross‐amplified by primers for brown sole.     

Development and characterization of polymorphic microsatellites from Prunus avium‘Napoleon’

Primers were developed for 21 microsatellite loci isolated by enrichment from Prunus avium‘Napoleon’. Twelve loci contained uninterrupted dinucleotide repeats and nine were more complex. Nineteen primer pairs (EMPA001–019) showed single locus polymorphisms in a cultivar survey of 14 sweet cherries, with two to seven alleles per locus. Three primer pairs in combination (EMPA014, 015 and 018) discriminated all cultivars. Two primer pairs for loci monomorphic in P. avium were included: EMPA020 revealed segregation in an interspecific progeny and EMPA021 revealed polymorphism in P. dulcis. Twelve primer pairs reliably amplified products in three peach cultivars of which seven revealed polymorphisms.     

Characterization of dinucleotide microsatellite loci in big brown bats (Eptesicus fuscus), and their use in other North American vespertilionid bats

We describe the development of six microsatellite loci for big brown bats, Eptesicus fuscus. Microsatellite markers were isolated from a small insert genomic library, and tested on a population of 44 animals from the Pend d’Oreille Valley, in southeastern British Columbia, Canada. These six loci were highly variable, with 12–23 alleles per locus, and observed and expected heterozygosities of greater than 79.5%. The six primer sets, and three others that were not variable in E. fuscus, were tested on 11 other species in the families Vespertilionidae and Antrozoidae. All the tested loci amplified highly variable products in at least several other species.     

Isolation,characterization and cross‐species amplification of microsatellite DNA loci in the tropical American yam Dioscorea trifida

We developed microsatellite markers in American yam (Dioscorea trifida). A microsatellite sequence‐enriched genomic library was screened, and after sequencing, primers were designed for 20 microsatellites. Among these, eight primer pairs yielded amplification products that were both interpretable and polymorphic in 24 yam cultivars. The number of alleles per locus ranged from two to 13 and the overall expected heterozygosity was around 0.5. Six of the eight Dioscorea trifida microsatellite loci gave amplification products in other Dioscorea species.     

Eleven polymorphic microsatellite loci in Lindera benzoin,Lauraceae

Microsatellite markers were developed for studies of the genetic diversity and population substructure of Lindera benzoin, Lauraceae (spicebush). Nuclear microsatellite sequences were obtained from DNA libraries that were enriched for (CA), (GA), (AAG) and (ATG) repeat motifs. From 69 microsatellite sequences, 20 primer sets were developed. Of these, 11 primer pairs resulted in amplified polymorphic loci. In 29 samples of eastern Pennsylvania spicebush plants, the number of microsatellite alleles ranged from two to 16 per locus with observed heterozygosity values ranging from 0.10 to 0.82.     

Characterization of polymorphic microsatellites for the invasive grass Microstegium vimineum (Poaceae)

? Premise of the study: Microsatellite markers were developed for the invasive plant Microstegium vimineum (Poaceae) to assess its population structure and to facilitate tracking of invasion expansion. ? Methods and Results: Using 454 sequencing, 11 polymorphic and six monomorphic microsatellite primer sets were developed for M. vimineum. The primer sets were tested on individuals sampled from six populations in the United States and China. The polymorphic primers amplified di-, tri-, and tetranucleotide repeats with three to 10 alleles per locus. ? Conclusions: These markers will be useful for a variety of applications including tracking of invasion dynamics and population genetics studies.