Isolation and characteristics of eight novel polymorphic microsatellite loci in Lippia alba (Verbenaceae)

? Premise of the study: A set of eight microsatellite (simple sequence repeat [SSR]) markers for Lippia alba, an important medicinal and cosmetic plant, was developed to aid studies of genetic diversity and to define efficient strategies for breeding programs. ? Methods and Results: Using a (CT)(8)- and (GT)(8)-enriched library, a total of 11 SSR loci were developed and optimized in L. alba. Of the 11 loci, eight were found to be polymorphic after screening 61 accessions from two populations. The parameters used to characterize loci were expected heterozygosity (H(e)) and number of alleles. A total of 44 alleles were identified, with an average of 5.5 alleles per loci, which were moderately to highly informative according to H(e). ? Conclusions: These new SSR markers have potential for informing genetic diversity, allele mining, and mapping studies and will be used to generate information for breeding programs of L. alba.     

Microsatellite development for the endangered Yangtze sturgeon (Acipenser dabryanus Duméril, 1869) using 454 sequencing

The Yangtze sturgeon (Acipenser dabryanus) is an endemic species in China. Using 454 sequencing, eight polymorphic tri‐, tetra‐, penta‐, and hexanucleotide microsatellite loci were isolated in this study. The raw sequence data from a one‐eighth run of 454 sequencings were 38.0 Mbp containing 94 222 reads/sequences. Of 80 microsatellite loci, only eight loci were polymorphic in a population of 30 individuals. The number of alleles per locus ranged from 4 to 14 (mean 7.62), and the observed heterozygosities varied between 0.46 and 0.88 (mean 0.74). Cross amplification was tested in congeneric species Acipenser sturio and Acipenser sinensis. These new microsatellite markers will be useful for further studies on genetic variation, parentage analysis, and conservation management for this critically endangered species.     

Isolation and characterization of microsatellite loci in Santalum lanceolatum and Santalum leptocladum (Santalaceae)

? Premise of the study: Microsatellite primers were developed for the first time in the native Australian sandalwood species Santalum lanceolatum. ? Methods and Results: Using an enrichment cloning protocol, five novel polymorphic codominant loci were developed and characterized in S. lanceolatum and S. leptocladum. In addition to these, three existing microsatellite loci from other sandalwood species were successfully amplified and characterized for S. lanceolatum and S. leptocladum. Among the eight loci, allelic diversity ranged from 4 to 29. ? Conclusions: Primers will be useful for studies of clonality, genetic diversity and spatial genetic structure in wild populations. When coupled with other molecular techniques will help investigate the relationship between S. lanceolatum and S. leptocladum, species of commercial and conservation interest.     

New polymorphic microsatellite loci,cross‐species amplification and PCR multiplexing in the black aphid,Aphis fabae Scopoli

Aphis fabae includes four morphological cryptic subspecies, which are mostly identified by their partially distinct secondary host range. To determine the extent of gene flow and isolation between these four taxa, we isolated and characterized 12 microsatellite loci from Aphis fabae fabae and tested cross‐species amplification of eight loci from the closely related species Aphis gossypii. Using eight previously described microsatellite loci, we have developed the polymerase chain reaction (PCR) multiplexing of 24 loci, which were separated in tree sets and five PCRs. These sets of microsatellite loci provide high throughput capacity for large‐scale population genetic studies at a minimum cost.     

Evaluation of factors affecting individual assignment precision using microsatellite data from horse breeds and simulated breed crosses

Assignment tests have been utilized to investigate population classification, measure genetic diversity and to solve forensic questions. Using microsatellite data from 26 loci genotyped in eight horse breeds we examined how population differentiation, number of scored loci, number of scored animals per breed and loci variability affected individual assignment precision applying log likelihood methods. We found that both genetic differentiation and number of scored loci were highly important for recognizing the breed of origin. When comparing two and two breeds, a proportion of 95% of the most differentiated breeds (0.200 < or = FST < or = 0.259) could be identified scoring only three loci, while the corresponding number was six for the least differentiated breeds (0.080 < or = FST < or = 0.139). An identical proportion of simulated breed crosses, differentiated from their parental breeds by FST estimates in the range 0.050-0.069, was identified when scoring 12 loci. This level of source identification was not obtained for the less differentiated breed crosses. The current data further suggested that population sample size and locus variability were not critical for the assignment precision as long as moderately large sample sizes (> or = 20 animals per population) and fairly variable loci were used.     

Characterization of eight VNTR loci by agarose gel electrophoresis

Allelic frequencies and their confidence intervals were obtained for eight independent VNTR loci from a sample of more than 75 Utah Caucasians. Using high-resolution agarose gel electrophoresis, we were able to resolve alleles at the D17S5 locus that differed by only one repeating unit; it was therefore possible to name the alleles according to the number of repeating units each contained. Two a priori probabilities were calculated for each VNTR locus separately and for all eight loci jointly: (i) the "power of exclusion" for an alleged father/mother/child trio and for an alleged parent/child duo, and (ii) the "probability of matching" when two unrelated individuals or two siblings are genotyped.     

Polymorphisms at VNTR loci suggest homogeneity of the white population of Utah

Apparent departure from equilibrium of genetic parameters measured for multiallelic single-locus markers such as VNTR (variable number of tandem repeat) loci has been suggested as evidence of underlying heterogeneity of the tested population. Using allele frequency distributions at eight VNTR loci from the white population of Utah, we show that the observed number of alleles and the gene diversity at each locus are congruent according to expectations of the neutral mutation model. This demonstrates the genetic homogeneity of the white population of Utah with reference to the allele (total and rare) frequency distribution at eight VNTR loci. The importance of such procedures is discussed in the context of using VNTR polymorphism data for forensic and medicolegal applications. Recommendations for reporting population data for hypervariable loci are also made to aid potential users in conducting similar analyses.     

Isolation and characterization of microsatellite loci for alligator gar (Atractosteus spatula) and their variability in two other species (Lepisosteus oculatus and L. osseus) of Lepisosteidae

We report on the isolation of 17 polymorphic microsatellite loci from alligator gar (Atractosteus spatula), a large-bodied species that has experienced population declines across much of its range. These loci possessed 2-19 alleles and observed heterozygosities of 0-0.974. All loci conformed to Hardy-Weinberg equilibrium expectations, and none exhibited linkage disequilibrium. Nine and eight of these loci were found to be polymorphic in the related species Lepisosteus oculatus and L. osseus, respectively. These microsatellite loci should prove useful in conservation efforts of A. spatula through the study of population structure and hatchery broodstock management.     

Chromosomal localization of seven members of the murine TGF-beta superfamily suggests close linkage to several morphogenetic mutant loci

Chromosomal locations have been assigned to seven members of the TGF-beta superfamily using an interspecific mouse backcross. Probes for the Tgfb-1, -2, and -3, Bmp-2a and -3, and Vgr-1 genes recognized only single loci, whereas the Bmp-2b probe recognized two independently segregating loci (designated Bmp-2b1 and Bmp-2b2). The results show that the seven members of the TGF-beta superfamily map to eight different chromosomes, indicating that the TGF-beta family has become widely dispersed during evolution. Five of the eight loci (Tgfb-1, Bmp-2a, Bmp-2b1, Bmp-2b2, Vgr-1) mapped near mutant loci associated with connective tissue and skeletal disorders, raising the possibility that at least some of these mutations result from defects in TGF-beta-related genes.     

Analysis of genetic diversity and population structure within Florida coconut (<Emphasis Type="Italic">Cocos nucifera</Emphasis> L.) germplasm using microsatellite DNA,with special emphasis on the Fiji Dwarf cultivar

Using 15 simple sequence repeat (SSR) microsatellite DNA loci, we analyzed genetic variation within Cocos nucifera germplasm collections at two locations in south Florida, representing eight cultivars. The loci were also used in a parentage analysis of progeny of the 'Fiji Dwarf' variety at both locations. A total of 67 alleles were detected, with eight the highest number at any one locus. These loci identified 83 of the 110 individual palms. Gene diversity of the 15 loci ranged from 0.778 to 0.223, with a mean of 0.574. 'Fiji Dwarf', 'Malayan Dwarf', 'Green Ni?o' and 'Red Spicata' cultivars resolve as distinct clusters in a neighbor joining tree using modified Rogers distance, while the tall varieties form two aggregates. The highest gene diversity was found in the tall cultivars (H = 0.583 cumulatively), and the lowest in the 'Malayan Dwarf' (H = 0.202). After the tall coconuts, the 'Fiji Dwarf' was most genetically diverse (H = 0.436), and had the largest number of unique alleles. Genetic identity is highest among the 'Malayan Dwarf' phenotypes, and between the tall varieties. The 'Red Malayan Dwarf' is genetically distinct from the 'Green' and 'Yellow Malayan Dwarf' phenotypes, which cannot be distinguished with the SSR loci used. Off-type 'Malayan Dwarf' phenotypes (putative hybrids with talls) can be identified genotypically. Parentage analyses of 30 'Fiji Dwarf' progeny propagated from five adults surrounded by other cultivars estimate that only 20% of the progeny were out-crossed to the other varieties, while 40-46% were possible selfs. This suggests that a seed-production orchard of the variety maintained at reasonable distance from other varieties, will likely yield only 'Fiji Dwarf' genotypes. Our data are discussed in the context of hypotheses of coconut dissemination around the world.     

Microsatellite markers for eastern hemlock (Tsuga canadensis)

We describe polymerase chain reaction primer pairs and reaction conditions for amplification of 15 microsatellite loci from eastern hemlock (Tsuga canadensis). The primers were tested on 23 individuals from a natural population in southwestern North Carolina, USA. These primers yielded an average of 5.9 alleles per locus (range of 2-14), an average observed heterozygosity of 0.45 (range 0.14-0.73), and an average polymorphic information content of 0.54 (range 0.28-0.86). In addition, eight of the primer pairs were found to amplify microsatellite loci in one or more additional species of Tsuga.     

Isolation and characterization of microsatellite loci in Psilopeganum sinense Hemsl (Rutaceae), an endangered herb endemic to Yangtze River valley

Twenty-nine primer pairs flanking microsatellite repeats were designed from an AC-enriched genomic library of Psilopeganum sinense and tested using 24 individuals derived from a natural population. A total of 11 microsatellite loci were found polymorphic. The number of alleles per locus varied from two to eight, with an average value of 3.7. The ranges of observed and expected heterozygosities were 0.000-0.750 and 0.365-0.800, respectively. These microsatellite loci have been directly applied to the ongoing conservation genetic studies of P. sinense.     

Rapid microsatellite marker development in the endangered neotropical freshwater turtle Podocnemis lewyana (Testudines: Podocnemididae) using 454 sequencing

Using high throughput sequencing we obtained a large number of microsatellites from Podocnemis lewyana, an endemic turtle from northwestern South America. We used 454 Genome Sequence FLX platform of sheared genomic DNA from randomly sampling approximately 17% of the haploid genome. We identified 86,501 reads (8.1% of all reads) that contained our definition of microsatellite loci. AC and TC were the most abundant motifs in the P. lewyana genome. TGC and AAAC were most abundant tri and tetra-nucleotide motifs respectively. 72.7% of microsatellite reads had flanking sequence regions suitable for primer design and PCR amplification. We validated the identified potentially amplifiable loci (PAL) and tested for polymorphism by selecting 15 loci corresponding to tetranucleotides. Twelve loci showed polymorphism in eight individuals. These findings demonstrates that microsatellite detection using next-generation sequencing is an efficient way of getting a lot of loci for listed taxa and in turn will have a large impact on future genetic studies aiming to understand and implement conservation plans for this highly threatened freshwater turtle.     

Isolation and characterization of microsatellite loci in Portunus pelagicus (Crustacea: Portunidae)

Seven dinucleotide and one tetranucleotide microsatellite loci were isolated and characterized, using routine protocols, from the blue swimmer crab, Portunus pelagicus. These loci will be useful for investigating the population structure of this broadly distributed and economically important portunid. In particular, all eight loci were polymorphic in P. pelagicus collected from throughout Australia. In addition, the genotype frequencies at each locus in each population sample were usually not significantly different from those expected under Hardy–Weinberg equilibrium conditions. Six of the eight loci also showed moderate to high levels of polymorphism in an undescribed species of Portunus from northern Australia.     

Isolation and characterization of eight highly variable microsatellite markers in the Pacific jumping mouse (Zapus trinotatus)

In patchily distributed populations it is possible to investigate gene flow and relatedness at multiple scales. Using an enrichment procedure, I developed a suite of eight microsatellite markers in such a heterogeneously distributed species, the Pacific jumping mouse (Zapus trinotatus). The identified loci were highly polymorphic (10–36 alleles per locus) and will be valuable for investigating this species at multiple levels, from that of parentage and breeding structure within a subpopulation, to gene‐flow and population structure across populations.     

Autosomal and sex-linked microsatellite loci in the green oak leaf roller Tortrix viridana L. (Lepidoptera, Tortricidae)

Eight microsatellite markers were developed for the lepidopteran species Tortrix viridana using an enrichment protocol. The loci were highly variable with number of alleles ranging from four to 38. Six of the eight loci were in Hardy-Weinberg equilibrium. The other two were linked to the Z-chromosome. Values of observed heterozygosity ranged for the autosomal loci from 0.510 to 0.957. All loci will be useful to study dispersal and the autosomal loci, as well for phylogeographical studies.     

Development and characterization of eight polymorphic microsatellite loci from Pistacia lentiscus L. (Anacardiaceae)

We have developed a set of eight polymorphic nuclear microsatellite markers for the Mediterranean shrub Pistacia lentiscus by means of an enriched library method. Characterization for the eight loci was carried out on 42 individuals from two populations sampled in southern Spain. The overall number of alleles detected was 59, ranging from three to 13 per locus. Expected heterozygosity per locus and population ranged from 0.139 to 0.895. Two loci albeit only in one population (Seville) departed significantly from Hardy-Weinberg equilibrium expectations and no linkage disequilibrium between pairs of loci was detected. These markers will be used in studies of gene flow across a fragmented landscape.     

Isolation and characterization of microsatellite loci in the entomopathogenic nematode Heterorhabditis bacteriophora

Twenty microsatellite loci were identified from genomic DNA enrichment and expressed sequence tags of entomopathogenic nematode Heterorhabditis bacteriophora. Eight loci were found to be polymorphic in a Northeast Ohio H. bacteriophora population. Levels of polymorphism were evaluated in 31-56 individuals and the number of alleles ranged from two to three. The values of observed and expected heterozygosities ranged from 0 to 0.536 and from 0.223 to 0.616, respectively. All eight loci showed heterozygote deficiencies, but three conformed to Hardy-Weinberg equilibrium at the subpopulation level. This is the first set of microsatellite markers in entomopathogenic nematodes.     

Microsatellite loci for the Australian field cricket Teleogryllus oceanicus and their cross‐utility in Teleogryllus commodus

Microsatellite loci were isolated from the Australian field cricket Teleogryllus oceanicus. Seven polymorphic loci were found with an observed number of alleles ranging from eight to 17 and observed heterozygosities between 0.26 and 0.94. One locus was found to be X‐linked. These seven loci were also tested for amplification and polymorphism in the congeneric species Teleogryllus commodus. The loci will be used for paternity studies in these species.     

Isolation and characterization of eight polymorphic microsatellite loci for the coconut pest, Brontispa longissima (Coleoptera: Hispidae)

Brontispa longissima is one of the most serious insect pests of coconut in Southeast Asia; it was first discovered on Hainan Island in June 2002. Despite the economic risk associated with this pest, genetic aspects of the invasion process have remained relatively unexplored. Using microsatellite markers, we investigated the population structure, genetic variability and pattern of invasion in various geographic populations. The methodology was based on a modified biotin-capture method. Eight polymorphic microsatellite loci were isolated and characterized for the pest. The allele number per locus varied from 2 to 3 (N = 30). The expected and observed heterozygosities of the eight loci ranged from 0.042 to 0.509 and from 0.042 to 0.963, respectively. Although the frequency of polymorphisms was not very high in this population, the microsatellite loci that were isolated will be useful for investigating the genetic diversity and migration routes of B. longissima populations.