Cross‐species amplification of 105 Lolium perenne SSR loci in 23 species within the Poaceae

Amplification of 105 Lolium perenne SSR markers was studied in 23 grass species representing seven tribes from three subfamilies of Poaceae. Twelve of the SSR markers are published for the first time. Between 2% and 96% of the SSR markers could be amplified within a given species. A subset of eight SSR markers was evaluated for polymorphism across nine of the 23 grass species. Four to seven of the markers were polymorphic within each species, with an average detection of 2.4 alleles per species.     

Development of novel microsatellite markers for the grassland species Lolium multiflorum,Lolium perenne and Festuca pratensis

Twelve microsatellite markers were isolated from Lolium multiflorum. Allelic variability and cross‐species amplification were assessed on 16 individuals of each of the three grassland species L. multiflorum, Lolium perenne and Festuca pratensis. Cross‐species amplification success was 100% for L. perenne and 83% for F. pratensis. The number of alleles detected ranged from one to 14 with an average of 3.4. While three microsatellite loci were polymorphic in all three species, one marker produced species‐specific alleles in all three species. These microsatellite markers provide a valuable tool for population genetic studies within and among species of the Festuca–Lolium complex.     

Cross-species amplification of human microsatellite markers using noninvasive samples from white-handed gibbons (Hylobates lar)

Analysis of the population genetic structure and reproductive strategies of various primate species has been facilitated by cross-species amplification (i.e., the use of microsatellite markers developed in one species for analysis of another). In this study we screened 47 human-derived markers to assess their utility in the white-handed gibbon (Hylobates lar). Only eight produced accurate, reliable results, and exhibited levels of polymorphism that were adequate for individual identification. This low success rate was surprising given that human microsatellite markers typically work well in species (such as macaques) that are evolutionarily more distant from humans than are gibbons. In addition, we experienced limited success in using a set of microsatellite markers that have been reported to be useful in the closely-related H. muelleri, and applying our set of microsatellite markers to samples obtained from one H. pileatus individual. Our results emphasize the importance of extensively screening potential markers in representatives of the population of interest.     

Development and testing of novel chloroplast microsatellite markers for Lolium perenne and other grasses (Poaceae) from de novo sequencing and in silico sequences

Twelve primers to amplify microsatellite markers from the chloroplast genome of Lolium perenne were designed and optimized using de novo sequencing and in silico sequences. With one exception, each locus was polymorphic with a range from two to nine alleles in L. perenne. The newly developed primer pairs cross‐amplified in different species of Lolium and in 50 other grass species representing nine grass subfamilies.     

Development and characterisation of simple sequence repeat (SSR) markers for perennial ryegrass (Lolium perenne L.)

Enrichment methods were optimised in order to isolate large numbers of simple sequence repeat (SSR) markers for perennial ryegrass (Lolium perenne L.), with the aim of developing a comprehensive set of loci for trait mapping and cultivar identification. Two libraries were constructed showing greater than 50% enrichment for a variety of SSR-motif types. Sequence characterisation of 1853 clones identified 859 SSR-containing clones, of which 718 were unique. Truncation of flanking sequences limited potential primer design to 366 clones. One-hundred selected SSR primer pairs were evaluated for amplification and genetic polymorphism across a panel of diverse genotypes. The efficiency of amplification was 81%. A relatively high level of SSR polymorphism was detected (67%), with a range of 2–7 alleles per locus. Mendelian segregation of alleles detected by selected SSR-locus primer pairs was demonstrated in the F₁ progeny of a pair cross. Cross-species amplification was detected in a number of related pasture and turfgrass species, with high levels of transfer to other Lolium species and members of the related genus Festuca. The identity of putative SSR ortholoci in these related species was confirmed by DNA sequence analysis. These loci constitute a valuable resource of ideal markers for the molecular breeding of ryegrasses and fescues. Received: 8 May 2000 / Accepted: 13 June 2000     

Sixty simple sequence repeat markers for use in the Festuca–Lolium complex of grasses

So far only very few simple sequence repeat (SSR) markers developed from grass species have had their primer sequences published. To make more markers available to the scientific community, we isolated and sequenced 256 microsatellite‐containing clones from four genome libraries of a Lolium multiflorum×Festuca glaucescens F₁ hybrid following enrichment in (TC)_n, (TG)_n, or both repeats. In this work, we report the primer sequences of 60 SSRs including preliminary results of polymorphism for mapping.     

New chloroplast microsatellite markers suitable for assessing genetic diversity of Lolium perenne and other related grass species

Background and Aims

Lolium perenne (perennial ryegrass) is the most important forage grass species of temperate regions. We have previously released the chloroplast genome sequence of L. perenne ‘Cashel’. Here nine chloroplast microsatellite markers are published, which were designed based on knowledge about genetically variable regions within the L. perenne chloroplast genome. These markers were successfully used for characterizing the genetic diversity in Lolium and different grass species.

Methods

Chloroplast genomes of 14 Poaceae taxa were screened for mononucleotide microsatellite repeat regions and primers designed for their amplification from nine loci. The potential of these markers to assess genetic diversity was evaluated on a set of 16 Irish and 15 European L. perenne ecotypes, nine L. perenne cultivars, other Lolium taxa and other grass species.

Key Results

All analysed Poaceae chloroplast genomes contained more than 200 mononucleotide repeats (chloroplast simple sequence repeats, cpSSRs) of at least 7 bp in length, concentrated mainly in the large single copy region of the genome. Nucleotide composition varied considerably among subfamilies (with Pooideae biased towards poly A repeats). The nine new markers distinguish L. perenne from all non-Lolium taxa. TeaCpSSR28 was able to distinguish between all Lolium species and Lolium multiflorum due to an elongation of an A₈ mononucleotide repeat in L. multiflorum. TeaCpSSR31 detected a considerable degree of microsatellite length variation and single nucleotide polymorphism. TeaCpSSR27 revealed variation within some L. perenne accessions due to a 44-bp indel and was hence readily detected by simple agarose gel electrophoresis. Smaller insertion/deletion events or single nucleotide polymorphisms detected by these new markers could be visualized by polyacrylamide gel electrophoresis or DNA sequencing, respectively.

Conclusions

The new markers are a valuable tool for plant breeding companies, seed testing agencies and the wider scientific community due to their ability to monitor genetic diversity within breeding pools, to trace maternal inheritance and to distinguish closely related species.     

Isolation and characterization of microsatellite markers for red elm (Ulmus rubra Muhl.) and cross-species amplification with Siberian elm (Ulmus pumila L.)

Ulmus pumila is an elm species, non-native to the USA that hybridizes with Ulmus rubra. In order to study the genetic structure and hybridization patterns between these two elm species, we developed 15 primer pairs for microsatellite loci in U. rubra and tested their cross-amplification in U. pumila. All 15 primers amplified in both species, 11 of which possessed species-specific alleles. Eight loci were polymorphic in U. pumila and eight in U. rubra, each with two to eight alleles per locus. In addition, five primer pairs previously developed in U. laevis and U. carpinifolia (syn. U. minor) cross-amplified and showed polymorphic loci in U. pumila and/or U. rubra. These markers will facilitate the study of genetic structure and gene flow between U. rubra and exotic, invasive U. pumila.     

Development of microsatellites from the rare ironstone endemic, Tetratheca paynterae ssp. paynterae and cross-species amplification

Nineteen microsatellite markers were developed from Tetratheca paynterae ssp. paynterae, a rare and endangered, leafless, perennial shrub. Twelve loci were polymorphic in T. paynterae ssp. paynterae with two to 14 alleles per locus and mean expected heterozygosity of 0.62. Primer pairs were tested on four other Tetratheca species from ironstone ranges in southern Western Australia. Ten loci were polymorphic in T. paynterae ssp. cremnobata and T. aphylla ssp. aphylla, three in T. harperi and four in T. erubescens. The level of polymorphism was adequate for studies of genetic structure and mating systems in three of the five taxa.     

Development of novel polymerase chain reaction (PCR) based microsatellite markers in Armillaria gallica by cross‐species amplification and species‐specific cloning

Cross‐species PCR amplification of Armillaria mellea group taxa with previously reported A. ostoyae microsatellite markers, indicative of flanking sequence conservation, was exploited for the species‐specific isolation of simple sequence repeat (SSR) motifs from A. gallica. Six SSR motifs were sequence characterized from cloned PCR fragments generated with primers previously developed from A. ostoyae. Five novel primer pairs, designed from motif flanking regions, allowed for improved, efficient amplification in this species. One original A. ostoyae primer pair was used directly. Polymorphims were observed at wide geographical levels only. Relative cross‐species amplification intensities generally supported the currently accepted molecular phylogeny of this group.     

Screening of ESTs from Mytilus for the detection of SSR markers in Mytilus californianus

A set of expressed sequence tag‐simple sequence repeat (EST‐SSR) markers for the genus Mytilus was developed through bioinformatic mining of the GenBank public database. A total of 33 782 EST sequences from GenBank were downloaded and screened for di‐, tri‐ and tetranucleotide, with 1274 EST containing SSR markers. Nine microsatellite markers were characterized in Mytilus californianus with a number of alleles per locus ranging from two to six, and total observed and expected heterozygosities ranging from 0.490 to 0.730 and from 0.510 to 0.860 respectively. Cross‐species amplification was achieved in several other species, confirming the usefulness of these markers in Mytilus genetics.     

Identification and characterization of nuclear microsatellite loci in Abies alba Mill.

Eleven polymorphic nuclear microsatellite markers for Abies alba Mill. were developed from an enriched genomic library. An average of 5.2 alleles per locus and a mean expected heterozygosity of 0.532 were found in a sample of 24 Abies alba individuals from different populations within Europe. These loci can be used in studies of genetic diversity for parentage analysis and for estimation of gene flow in silver fir populations. Moreover, successful amplifications were obtained for eight other Mediterranean Abies species, suggesting that these loci may be useful for similar applications in other fir species.     

Development and characterization of 60 novel EST-SSR markers in half-smooth tongue sole Cynoglossus semilaevis

Sixty novel simple sequence repeat (SSR) markers were developed from expressed sequence tags (EST) of half-smooth tongue sole Cynoglossus semilaevis exploited in the laboratory. The number of alleles, observed and expected heterozygosity per locus ranged from two to 16, from 0·0833 to 1·0000 and from 0·0816 to 0·913, respectively. Of these SSRs, 20 had significant homology to known genes by BLASTx (basic local alignment search tool x) search. For cross-species amplification, there are 53 positive amplifications in Japanese flounder Paralichthys olivaceus with 12 polymorphic loci and 51 positive amplifications in Senegalese sole Solea senegalensis with 11 polymorphic loci. These new EST-SSR markers will be useful for genetic studies and genome mapping of C. semilaevis and its closely related fishes.     

Cross-species amplification of primers developed from Plantago major and P. intermedia in two Hawaiian Plantago species from the section Plantago

Thirty-nine primers, developed from the sister species Plantago major and P. intermedia, were tested in two Hawaiian Plantago species from the section Plantago. Eight primers were polymorphic, of which three were published earlier, and five are new ones presented here. Amplification and polymorphism levels appeared to be high in these Hawaiian species. These markers will be valuable for further mating system and evolutionary studies in species from the section Plantago that are closely related to P. major and P. intermedia.     

Isolation and characterization of microsatellite markers in the gecko Gekko swinhonis and cross‐species amplification in other gekkonid species

Gekko swinhonis is a gekkonid lizard endemic to China, inhabiting Loess Plateau, Huabei Plain, Huanghuai Plain and areas north of the Yangtze River. We characterized 21 dinucleotide microsatellite loci from an AC/AG‐enriched genomic library of G. swinhonis. The number of alleles per locus ranged from eight to 24 and the observed and expected heterozygosities ranged from 0.160 to 0.834 and from 0.584 to 0.917, respectively. We also tested the utility of these loci in other Gekko and Hemidactylus species; many of these loci can be cross‐amplified.     

Development and characterization of microsatellite markers for Prosopis chilensis and Prosopis flexuosa and cross‐species amplification

Prosopis chilensis and Prosopis flexuosa (Fabaceae) are closely related hardwood arboreal species that are widely distributed in the arid regions of Argentina. The development of highly polymorphic markers, such as microsatellites, is desirable for genetic studies of these species. Here, we present the development and characterization of six polymorphic microsatellite markers in P. chilensis and P. flexuosa. These markers showed a polymorphism information content between 0.14 and 0.85 and the number of alleles varied from two to 13 considering both species. All markers revealed a broad cross‐species affinity when tested in seven other Prosopis species. All primers amplified in at least five species.     

Characterization of microsatellite markers in sycamore (Acer pseudoplatanus L.)

Sycamore (Acer pseudoplatanus L.) is a tetraploid European hardwood tree species. The reproduction system of the insect‐pollinated trees and patterns of genetic variation are largely unknown. We isolated and characterized eight polymorphic microsatellite markers for Acer pseudoplatanus L. The high degree of polymorphism observed at these markers makes them useful to observe genetic variation patterns at various spatial scales and to analyse gene flow and the mating system. Primers developed for the amplification of microsatellites in A. pseudoplatanus were tested for 21 different species of genus Acer. Amplification products of the expected size were obtained in most cases.     

Development of microsatellite markers for the European white elm (Ulmus laevis Pall.) and cross‐species amplification within the genus Ulmus

Ulmus laevis Pall. is a broad‐leaved deciduous tree with a central and eastern European distribution. We describe the development of six polymorphic microsatellite markers for this species. These markers were also tested for utility in U. americana, U. glabra, U. minor and U. pumila. One additional marker gave ambiguous results in U. laevis but amplified clearly in three other species. In U. laevis, the number of alleles observed per locus ranged from two to nine. Five loci showed polymorphism in at least one of the nontarget species tested.     

Identification of polymorphic microsatellite markers from RAPD product in turbot (Scophthalmus maximus) and a test of cross‐species amplification

Five polymorphic microsatellite loci have been isolated and characterized from random amplified polymorphic DNA product in turbot, Scophthalmus maximus. Twelve microsatellites were selected for designing microsatellite primers, of which five gave working primer pairs. They had between four and nine alleles. Observed and expected heterozygosities varied from 0.76 to 0.90 and from 0.63 to 0.83, respectively. Five additional fish species assessed for cross‐species amplification revealed between one and three positive amplifications and between zero and two polymorphic loci per species.