Development of EST‐SSRs in the Mediterranean blue mussel,Mytilus galloproviancialis

Eight polymorphic microsatellite repeat markers were identified from Mytilus galloproviancialis, expressed sequence tags (EST) deposited in public sequence database. Number of alleles per locus ranged from two to 10, and the observed and expected heterozygosities ranged from 0.029 to 0.872 and from 0.031 to 0.811, respectively. Three additional Mytiloida species assessed for cross‐species amplification revealed four loci could give positive amplifications. EST‐derived simple sequence repeats provide robust, informative and potentially transferable polymorphic markers suitable for population genetic, parentage, and mapping studies of M. galloproviancialis.     

Isolation and characterization of simple sequence repeat loci in Rubus hochstetterorum and their use in other species from the Rosaceae family

The identification of microsatellite loci in Rubus hochstetterorum provides an important tool for the characterization and conservation of wild populations of this species. Cross‐species amplification of markers may be of particular interest for the study of other Rubus species. In this study, 41 simple sequence repeat markers were identified in a genomic library of R. hochstetterorum. Fifteen of the identified microsatellite loci were characterized in a set of 30 samples and revealed to be polymorphic with three to 19 alleles per locus. All the identified markers allowed cross‐species amplification in at least one of the other three tested species from the Rosaceae family.     

Isolation and characterization of microsatellite markers in Koompassia malaccensis (Leguminosae), an important tropical timber species

This paper reports the isolation and characterization of 24 polymorphic microsatellite markers in an important tropical timber species, Koompassia malaccensis (Leguminosae). The primers were designed from a genomic library enriched for dinucleotide (CT) repeats and screened on 24 samples from a natural population. The number of alleles detected per locus ranged from two to 13 while the observed heterozygosity ranged from 0.042 to 1.000. Significant departure from Hardy–Weinberg equilibrium (P < 0.05) was detected in two loci. These microsatellite markers were tested across 13 timber species of the same family. The amplification success appeared to be associated with taxonomy classification at the genus but not subfamily levels.     

Twelve polymorphic microsatellite loci from the Asian citrus psyllid,Diaphorina citri Kuwayama,the vector for citrus greening disease,huanglongbing

Twelve polymorphic microsatellite markers were developed from microsatellite‐enriched DNA libraries and mined from an expressed sequence tags library of Diaphorina citri, the vector of the citrus greening disease (huanglongbing). Analysis of 288 individuals from Florida, Texas, and Brazil showed that allelic diversity ranged from three to eight alleles per locus and observed and expected heterozygosities ranged from 0.014 to 0.569 and from 0.052 to 0.653, respectively. These variable microsatellite loci can provide means for assessing overall genetic variation and migration patterns for this agriculturally important pest species. This information can be used to aid in developing successful management strategies.     

EST‐SSR markers from the Pacific oyster,Crassostrea gigas

Ten polymorphic microsatellite repeat markers were identified from Crassostrea gigas, expressed sequence tags (EST) deposited in public sequence database. Number of alleles per locus ranged from three to 18, expected and observed heterozygosities ranged from 0.071 to 0.738 and from 0.306 to 0.913, respectively. Marker transferability was tested on other two Crassostrea species and polymorphic products were detected at nine loci. EST‐derived simple sequence repeats provide robust, informative and potentially transferable polymorphic markers suitable for population genetic, parentage, and mapping studies of C. gigas.     

dbEST‐derived microsatellite markers in celery (Apium graveolens L. var. dulce)

We report on the development of 11 (from database expressed sequence tags) dbEST‐derived microsatellite markers in celery (Apium graveolens L. var. dulce). The sequences were obtained from DNA accessions available from GenBank and contained di‐, tri‐ and pentanucleotidic motifs. All the microsatellites were found in expressed sequence tags and they are expected to become useful tools for ecological, genetic and evolutionary studies, as well as for celery breeding. Polymorphism was explored in 16 celery commercial varieties, and marker transferability was tested on three accessions of celeriac (A. graveolens var. rapaceum). Primers and PCR conditions for microsatellite amplification are reported.     

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.     

Microsatellite loci in Bactris gasipaes (Arecaceae): their isolation and characterization

We constructed a microsatellite‐enriched genomic library for Bactris gasipaes, an economically important, domesticated palm. We developed 18 polymorphic microsatellite markers, and found an average of seven alleles per locus in a sample of 14 individuals selected from a germplasm bank. Cross‐species amplification was evaluated in six other Bactris species. The loci detected will permit population studies and germplasm characterization, and can be used for genetic analyses in related species.     

Analysis of expressed sequence tags (ESTs) from a normalized cDNA library and isolation of EST simple sequence repeats from the invasive cotton mealybug Phenacoccus solenopsis

The cotton mealybug, Phenacoccus solenopsis Tinsley, is a serious and invasive pest. At present, genetic resources for studying P. solenopsis are limited, and this negatively affects genetic research on the organism and, consequently, translational work to improve management of this pest. In the present study, expressed sequence tags (ESTs) were analyzed from a normalized complementary DNA library of P. solenopsis. In addition, EST‐derived microsatellite loci (also known as simple sequence repeats or SSRs) were isolated and characterized. A total of 1107 high‐quality ESTs were acquired from the library. Clustering and assembly analysis resulted in 785 unigenes, which were classified functionally into 23 categories according to the Gene Ontology database. Seven EST‐based SSR markers were developed in this study and are expected to be useful in characterizing how this invasive species was introduced, as well as providing insights into its genetic microevolution.     

Development of highly conserved primers for 12 new polymorphic microsatellite loci for the genus Rorippa Scop. (Brassicaceae), yellow‐cress

We isolated 12 highly conserved polymorphic microsatellite loci for the yellow‐cress species Rorippa amphibia and Rorippa sylvestris. We used a partial genomic library enriched for several repeat motifs. Obtained sequences containing repetitive elements were blasted and aligned with the Arabidopsis thaliana sequence. We evaluated the cross‐species compatibility of primers designed from sequences either aligning strongly or weakly with A. thaliana. The former proved much more efficient in obtaining primers that worked in both species. The developed conserved primers for microsatellite loci provide excellent markers for studying segregation, gene flow and hybridization in the genus Rorippa.     

Isolation and characterization of sequence‐tagged microsatellite sites markers in chickpea (Cicer arietinum L.)

In this study we report the isolation of microsatellite sequences and their conversion to sequence‐tagged microsatellite sites (STMS) markers in chickpea (Cicer arietinum L.). Thirteen putative recombinants isolated from a chickpea genomic library were sequenced, and used to design 10 STMS primer pairs. These were utilized to analyse the genetic polymorphism in 15 C. arietinum varieties and two wild varieties, C. echinospermum and C. reticulatum. All the primer pairs amplified polymorphic loci ranging from four to seven alleles per locus. The observed heterozygosity ranged from 0 to 0.6667. Most of the STMS markers also amplified corresponding loci in the wild relatives suggesting conservation of these markers in the genus. Hence, these polymorphic markers will be useful for the evaluation of genetic diversity and molecular mapping in chickpea.     

Isolation and characterization of microsatellite loci from a commercial cultivar of Musa acuminata

A genomic library from the commercial diploid cultivar ‘Ouro’ (Musa acuminata), enriched for CT‐ and GT‐repeats, was used to isolate and characterize 23 microsatellite loci. These loci were tested in 10 Musa genotypes, representing various Musa genomic groups with distinct ploidy level. The number of alleles per locus ranged from one to seven, and 20 loci were highly informative. Four loci appeared to amplify B genome‐specific alleles, while three loci seemed to be absent in the B genome. The polymorphism revealed by these loci will be extremely useful for genetic mapping, marker‐assisted selection, germplasm characterization and evolutionary studies in Musa.     

Identification and characterization of expressed sequence tags‐derived simple sequence repeats markers from robusta coffee variety ‘CxR’ (an interspecific hybrid of Coffea canephora × Coffea congensis)

SSR (simple sequence repeats) markers derived from ESTs (expressed sequence tags), commonly called EST‐SSRs or genic SSRs provide useful genetic markers for crop improvement. These are easy and economical to develop as by‐products of large‐scale EST resources that have become available as part of the functional genomic studies in many plant species. Here, we describe for the first time, nine genic‐SSRs of coffee that are developed from the microsatellite containing ESTs from a cDNA library of moisture‐stressed leaves of coffee variety, ‘CxR’ (a commercial interspecific hybrid between Coffea congensis and Coffea canephora). The markers show considerable allelic diversity with PIC values up to 0.70 and 0.75 for Coffea arabica and Coffea canephora, respectively, and robust cross‐species amplification in 16 other related taxa of coffee. The validation studies thus demonstrate the potential utility of the EST‐SSRs for genetic analysis of coffee germplasm.     

Isolation and characterization of microsatellite loci from Larix kaempferi

Microsatellites were isolated and characterized for Japanese larch, Larix kaempferi, a conifer species distributed in Japan. A larch genomic DNA library enriched for (AG)_n repeats was screened using the colony polymerase chain reaction method and 145 unique microsatellite containing sequences were obtained. Seventy‐two primer pairs were designed and 30 produced single‐locus products, and 19 of them were polymorphic. The expected heterozygosity ranged from 0.566 to 0.951. These 19 polymorphic microsatellite loci should be valuable markers for genetic studies on Japanese larch.     

Novel polymorphic microsatellites for the red‐legged partridge (Alectoris rufa) and cross‐species amplification in Alectoris graeca

The red‐legged partridge, Alectoris rufa, is an endemic species of the southwestern Mediterranean, and the most popular game bird in the Iberian Peninsula. A total of 27 microsatellite loci was isolated from an enriched genomic library of A. rufa. Six perfect GT microsatellites were characterized and optimized in 45 individuals of A. rufa. All loci revealed high levels of polymorphism with a number of alleles that ranged from three to 13. Observed heterozygosity ranged from 0.2 to 0.6. Cross‐species amplification showed that all loci were also polymorphic in rock partridge, Alectoris graeca. The new markers will be useful in determining hybridization between both species of Alectoris.     

EST and random genomic nuclear microsatellite markers for Streptocarpus

Microsatellite markers have been developed from standard enriched genomic libraries and a cDNA library for the genus Streptocarpus. Out of 15 loci derived from ESTs (expressed sequence tags), four gave working primer pairs, with expected heterozygosities (H_E) ranging from 0.42 to 0.86. Out of 89 genomic library derived loci, 6 gave working primer pairs, with H_E ranging from 0.63 to 0.93.     

Isolation,characterization and cross‐species amplification of eight microsatellite DNA loci in the migratory locust (Locusta migratoria)

Eight polymorphic di‐ and trinucleotide microsatellite loci suitable for population genetic analysis were developed in Locusta migratoria from a partial phagemid genomic library enriched for microsatellite inserts. The expected heterozygosity at these loci ranges from 0.45 to 0.97, with the observed allele numbers varying between nine and 45. The overall microsatellite cloning efficiency in L. migratoria is 14%, suggesting that in migratory locusts, microsatellite sequences are abundant and should provide a valuable and easily accessible source of nuclear markers for genetic studies. These microsatellite loci were highly Locusta‐specific, with only very limited cross‐species applicability.     

Fifteen polymorphic simple sequence repeat markers from expressed sequence tags of Liriodendron tulipifera

We developed and evaluated simple sequence repeat (SSR) markers derived from expressed sequence tags (ESTs) of Liriodendron tulipifera. Characteristics of 15 EST‐SSR loci were investigated using 33 L. tulipifera individuals. The number of alleles per locus ranged from two to five. The expected and observed heterozygosities ranged from 0.216 to 0.751 and from 0.182 to 0.97, respectively. These loci were further tested for their cross‐species transferability to Liriodendron Chinense. Because of their high level of polymorphism and transferability, our 15 single‐locus EST‐SSR markers will be valuable tools for research on mating system, population genetics and systemic evolution of Liriodendron.     

Isolation and characterization of microsatellite loci in the Bang's leaf‐nosed bat Hipposideros turpis

The Bang's leaf‐nose bat, Hipposideros turpis, is an endangered cave‐dwelling species inhabiting the southwesternmost islands of Japan. We isolated six dinucleotide microsatellite markers from the partial genomic library of the bat, and examined their allelic variation using a sample (N = 33) from the largest colony in Japan. All the loci showed a moderate allelic variation ranging from two to eight alleles, with the observed heterozygosities from 0.33 to 0.73, and conformed to Hardy–Weinberg expectations. The present microsatellite markers will be useful in assessing population genetic structure and gene flow among populations of this species.