GENETIC CONSEQUENCES OF RARITY IN ASTER FURCATUS (ASTERACEAE), A THREATENED,SELF-INCOMPATIBLE PLANT

Aster furcatus is a rare, self-incompatible plant with fewer than 50 known populations throughout its range. We verified self-incompatibility in A. furcatus by conducting experimental self- and cross-pollinations and by examining seed set in a small population comprised of a single clonal genet. We examined variation at 22 electrophoretic loci in 23 populations of A. furcatus from across its range in Wisconsin, Illinois, Indiana, and Missouri. Except for two rare alleles found in single individuals in three populations, all loci but one of those examined were fixed for single alleles. The only variable locus (triosephosphate isomerase, TPI-1) tended to exhibit genotype frequencies in Hardy-Weinberg equilibrium or with a slight excess of heterozygotes. Although overall gene diversity was extremely low, TPI genotype frequencies were indicative of an outcrossing plant. We examined the subpopulation genetic structure among clonal plants within one Wisconsin population in greater detail. F statistics indicated that much of the genetic variation at the polymorphic TPI locus was due to differentiation among populations. We discuss the implications of self-incompatibility and low levels of genetic variation for the evolution and conservation of Aster furcatus and other rare plants with similar breeding systems.     

Characterization of eight polymorphic microsatellite markers in the tarnished plant bug,Lygus lineolaris (Palisot de Beauvois)

A partial genomic library of the tarnished plant bug, Lygus lineolaris, enriched for microsatellite sequences was screened to identify marker loci. Eight polymorphic loci suitable for population genetic studies were identified by screening 192 field‐collected insects. The observed number of alleles ranged from four to 21 with an average of 12.25 (SE ± 1.94) while the effective number of alleles ranged from 1.23 to 11.05 with an average of 4.49 (SE ± 1.15). No linkage disequilibria or significant deviations from Hardy–Weinberg expectations were detected at any of the loci. Seven of the eight L. lineolaris microsatellite loci were transferable to Lygus hesperus.     

Genetic diversity and hierarchical population structure of a rare autotetraploid plant,Aster kantoensis (Asteraceae)

We sampled 17 populations of a rare autotetraploid Aster kantoensis (Asteraceae) from three river systems located in central Japan, and studied them for allelic variation at 22 enzyme loci. There was no significant correlation between the actual population size and three genetic diversity parameters, suggesting that the effective population size was very small even for the large populations, i.e., even large populations may still have a high probability of being of recent origin and remain influenced by the founder effect. Compared to other autotetraploid species, the total genetic variation of A. kantoensis is small. The number of alleles and gene diversity of a population were not significantly different among the river systems, although the percentage of polymorphic loci was different. Genetic differentiation among river systems was larger than between populations within the river systems, thereby indicating that gene flow between river systems is small, especially between the Kinu River system and Tama or Sagami River systems.     

Microsatellite loci in the house fly,Musca domestica L. (Diptera: Muscidae)

Genomic libraries from house flies enriched for (CA)₁₅ and (CAG)₁₀ repeats were constructed by using biotinylated probes. Twenty‐five loci were isolated and evaluated for polymorphisms in wild flies representing two geographically diverse populations. Fourteen of 19 dinucleotide loci, and one of six trinucleotide loci were polymorphic. One hundred and twenty‐seven alleles were detected, 39 of which were private. Average number of alleles per polymorphic locus was 8.4 ± 2.5 and average heterozygosity was 72 ± 4%. F_ST by the private allele method was 0.73. Three of 15 loci showed significant heterozygote deficiencies, attributed to null alleles. Five of 15 loci were amplified in the face fly, Musca autumnalis.     

Nine polymorphic microsatellite loci for the fennel Pondweed Potamogeton pectinatus L.

Fennel (= Sago) pondweed (Potamogeton pectinatus L.) is a submersed macrophyte of nearly cosmopolitan distribution. The plant is of worldwide ecological importance as structuring component of shallow lakes, and as food for waterfowl. We developed nine polymorphic microsatellite primers for the population genetic analysis of P. pectinatus. The loci were identified using a GA/CT‐enriched genomic library using subtractive hybridization with magnetic particles. All nine loci were highly polymorphic with 6–9 alleles and heterozygosities ranging from 0.23 to 0.80 in a subset of N = 40 genotypes from five locations.     

Isolation and characterization of microsatellite loci in <Emphasis Type="Italic">Pedicularis verticillata</Emphasis> L. using PCR-based isolation of microsatellite arrays (PIMA)

Pedicularis verticillata L. is a highly valuable herb for traditional Chinese medical treatment. In this report, 11 microsatellite loci from P. verticillata were isolated. The simple sequence repeat (SSR) markers were screened in 23 samples of wild populations of P. verticillata, and eight samples from its sister P. ikomai. The number of alleles ranged from 4 to 13, and value of expected (H _E) and observed (H ₀) heterozygosity was 0.62609–0.89662 and 0–0.95652, respectively. All loci were significantly deviated from Hardy–Weinberg expectations due to the heterozygote deficiency, indicating a dramatic loss of genetic polymorphisms in the restrictedly distributed species. The markers amplified well in the two species are useful for examining genetic diversity and population genetic structure, which, in turn, can provide information for establishing conservation strategy for these endangered species.     

Isolation and characterization of 21 microsatellite loci in Lycium chinense and cross-amplification in Lycium barbarum

The present study reports isolation and characterization of 21 polymorphic microsatellite markers developed from Lycium chinense Mill. These markers produced a total of 86 alleles across 30 L. chinense accessions, with an average of 4.1 alleles per locus. Values for observed heterozygosity and polymorphism information content ranged from 0.03 to 0.81 (mean = 0.35) and from 0.03 to 0.78 (mean = 0.31), respectively. At the significance threshold (P < 0.05), 12 loci deviated from Hardy–Weinberg equilibrium, whereas significant linkage disequilibrium values were observed between 52 pairs of loci. All loci were successfully amplified for all L. barbarum accessions. These newly developed polymorphic microsatellite markers will be very useful for programming in the genetic conservation and classification of L. chinense and L. barbarum.     

Characterization of microsatellite loci in the western tarnished plant bug,Lygus hesperus Knight (Hemiptera: Miridae)

A microsatellite‐enriched partial genomic DNA library of Lygus hesperus was generated and screened by sequencing. Ten polymorphic microsatellite marker loci were characterized by genotyping 92 insect samples. The observed number of alleles ranged from three to seven with an average of 4.5 (SE ± 0.45), while the effective number of alleles ranged from 1.21 to 3.02 with an average of 2.14 (SE ± 0.20). Significant deviations from Hardy–Weinberg expectations were detected at three loci. Significant linkage disequilibrium was also detected between the loci LhMS2‐54 and LhMS3‐32. Seven of the L. hesperus markers could be transferred to Lygus lineolaris.     

Isolation and strategies of novel tetranucleotide microsatellites with polymorphisms from different chromosomes of the rhesus monkey (Macaca mulatta)

A total of 45 tetranucleotide chromosome-specific microsatellite markers with polymorphism were developed successfully based on three reference rhesus monkey genomes and on In-silico PCR prescreening. The polymorphic information content (PIC) values of 45 polymorphic microsatellite loci ranged from 0.487 to 0.879, with an average of 0.715, which were proven to be moderate to highly polymorphic. We detected 315 alleles on 45 microsatellite loci in 24 Rhesus monkeys. The number of alleles ranged from 3 to 15 and the mean number of alleles was 7 for each locus. Accordingly, the observed and expected heterozygosities obtained were between 0.417 and 1.0 and between 0.550 and 0.908, with an average value of 0.736 and 0.767, respectively. Genetic information demonstrated that 10 loci significantly deviated from Hardy–Weinberg equilibrium (P?<?0.05). All 45 primers were not significant with regard to linkage disequilibrium (P?>?0.001). Pearson correlation indicated that the PIC value exhibited a significant negative correlation with the loci number (r?=?? 0.741, P?=?0.022), whereas the positive correlation with the number of the samples (r?=?0.847, P?=?0.070) was not significant. This may be attributed to the presence of random particularities within the loci. The T test of the sample groups indicated that the PIC difference was not significant when the number of samples was set at 10 and/or?≥?15 (P?=?0.7472?~?0.8564). These polymorphic and valuable microsatellite loci will facilitate further conservation genetics studies for rhesus monkeys and can be further applied to develop novel genetic markers for other species.

     

Characteristics of 13 polymorphic microsatellite markers in the corn earworm,Helicoverpa zea (Lepidoptera: Noctuidae)

Thirteen polymorphic microsatellite loci suitable for population genetic studies of Helicoverpa zea were discovered by screening partial genomic libraries enriched for microsatellite sequences. Insects collected (N = 96) in Stoneville, Mississippi were used to characterize these markers. The observed and effective number of alleles per locus ranged from two to nine (average 4.46) and from 1.07 to 2.45 (average 1.81), respectively. Fisher exact tests detected significant deviations from Hardy–Weinberg equilibrium at three loci, probably due to inbreeding, null alleles, or Wahlund's effect. Significant genotypic disequilibrium was not detected between any pair of loci.     

Development and characterization of microsatellite markers in the Colorado potato beetle,Leptinotarsa decemlineata

The characterization of 11 Leptinotarsa decemlineata microsatellite loci isolated using the fast isolation by AFLP of sequences containing repeats (FIASCO) protocol is reported. Three to 15 alleles per locus were detected in 54 beetles collected from four populations of L. decemlineata. The mean number of alleles was 7.4 ± 3.2 and the level of expected heterozygosity ranged from 0.451 to 0.798. The total exclusionary probabilities using these loci for the first and the second parent were 0.982 and 0.999, respectively. These are the first microsatellite loci characterized from the Colorado potato beetles that can be used for estimating genetic diversity, population structure and parentage analysis.     

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.     

Variable microsatellite loci isolated from the azure damselfly,Coenagrion puella (L.) (Zygoptera; Coenagrionidae)

We isolated and characterized 10 polymorphic microsatellite loci from the azure damselfly Coenagrion puella (Zygoptera; Coenagrionidae) as part of a study assessing reproductive success and genetic structure in an isolated population of this species. Levels of genetic diversity were assessed in 50 individuals collected from Queen Elizabeth Country Park, Hampshire, UK. The number of alleles per microsatellite loci ranged from three to 22 and the observed and expected heterozygosities varied between 0.26 and 0.84 and between 0.23 and 0.91, respectively. Two loci showed significant (P < 0.05) heterozygote deficits, likely because of null (non‐amplifying) alleles; one pair of loci was in linkage disequilibrium.     

Isolation and characterization of microsatellites in yellow perch (Perca flavescens)

A total of 45 microsatellite loci from yellow perch, Perca flavescens, were isolated and characterized. Among the 45 microsatellite loci, 32 had more than two alleles. A wild population of P. flavescens (n = 48) was used to examine the allele range of the microsatellite loci. Mendelian inheritance of alleles was confirmed by examining the amplified products in pair‐mated families. The number of alleles for the 32 polymorphic loci varied from two to 16, and observed heterozygosity ranged between 0.024 (YP79) and 0.979 (YP60). Cross‐species polymorphic amplification in four other Percidae species was successful for 22 loci.     

Nitrate accumulation and growth of Aster tripolium L. with a continuous and intermittent nitrogen supply*

Abstract The ‘tidal salt marsh’ ecotype of the halophyte Aster tripolium L. was grown in a nutrient solution with either a continuous or an intermittent NO₃^? supply with either Cl^? or SO₄^2? as the alternative anion. With increasing periods of NO₃^? supply per week the rate of the dry weight increment increased. When NO₃^? was supplied for longer than 48 h per week, the dry weight and the organic-N content in the shoots hardly increased, whereas the NO₃^? content in shoots and roots increased further. With alternated supply of a nutrient solution containing NO₃^? with one containing Cl^?, the internal NO₃^? content in the shoot was lower than in shoots grown in solutions in which NO₃^? alternated with SO₄^2?. It is concluded, that NO₃^? does not have a specific function in osmoregulation.     

Polymorphic microsatellite loci isolated from the great scallop,Pecten maximus (Bivalvia: Pectinidae)

We isolated and characterized nine polymorphic microsatellite loci from the great scallop (Pecten maximus) as part of an attempt to better understand the population–genetic structure of this species. Levels of genetic diversity were assessed in 32 scallops collected from the coastal waters of Port Erin Bay, Isle of Man. The number of alleles ranged from two to 29 and observed and expected heterozygosities varied between 0.031 to 0.906 and 0.062 to 0.973, respectively. Two loci showed significant (P < 0.05) heterozygote deficits, likely because of null (nonamplifying) alleles; one pair of loci was in linkage disequilibrium.     

Isolation and characterization of ten novel microsatellite loci in the red-winged tinamou (<Emphasis Type="Italic">Rhynchotus rufescens</Emphasis>, Tinamiformes,Aves) and cross-amplification in other tinamous

We describe the isolation and characterization of ten microsatellite loci from the red-winged tinamou (Rhynchotus rufescens) and also evaluated the cross-amplification of these loci and other ten loci previously developed for the great tinamou (Tinamus major) in other tinamous. Genetic variability was assessed using 24 individuals. Six loci were polymorphic with moderate to high number of alleles per locus (2–12 alleles) and showed expected heterozygosity (HE) ranging from 0.267 to 0.860. All loci conformed to the Hardy–Weinberg expectation and linkage disequilibrium was not significant for any pair of loci. This battery of polymorphic loci showed high paternity exclusion probability (0.986) and low genetic identity probability (4.95 × 10⁻⁵), proving to be helpful for parentage tests and population analyses in the red-winged tinamou. The cross-amplification was moderate where of the 160 locus/taxon combinations, 46 (28.75%) successfully amplified.     

Development of microsatellite markers for Polemonium kiushianum (Polemoniaceae), a critically endangered grassland plant species in Japan

Eight microsatellite loci were isolated and characterized for Polemonium kiushianum (Polemoniaceae), an endangered perennial herb endemic to the Kyushu region in Japan. The number of individuals of this species is presumed to be about 400 individuals. The loci showed polymorphisms with two to ten alleles per locus. The expected and observed heterozygositis ranged from 0.039 to 0.744 and 0.039 to 0.784, respectively. Although significant deviations (P < 0.05) from Hardy–Weinberg equilibrium were detected for one locus, no significant deviations were detected for other seven loci. There was no evidence of significant linkage disequilibrium between any two of the loci. The markers described here will be useful to establish conservation programs based on genetic traits of P. kiushianum.     

Characterization of microsatellite loci in polyploid Lavatera assurgentiflora ssp. assurgentiflora and ssp. glabra (Malvaceae)

Lavatera assurgentiflora (Malvaceae) is one of four species of the genus Lavatera native to California and Baja California. Two geographically defined subspecies are recognized: L. a. assurgentiflora on the northern islands and L. a. glabra on the southern islands. We isolated nine polymorphic microsatellite loci that amplify in both subspecies of L. assurgentiflora. Substantial levels of polymorphism were observed at many of the loci. Four loci exhibited more than 10 alleles, polymorphism information content ranged from 0.4 to 0.8, and up to six alleles were found in some individuals, supporting reports that these taxa are hexaploid. All loci also amplified in Lavatera lindsayi from Guadalupe Island, and we anticipate that they will cross‐amplify in other California Lavatera species as well.     

Development and characterization of microsatellite markers in the fungus Antrodia cinnamomea from dbEST database

We developed and characterized 8 polymorphic microsatellite loci or simple sequence repeats from the expressed sequence tags database of the medicinal fungus, Antrodia cinnamomea. Variability was assessed in a panel of 23 strains collected from various regions of Taiwan. The number of alleles per polymorphic locus ranged from two to seven loci with an average of 4.375 alleles per locus. The observed and expected heterozygosity values ranged from 0.130 to 0.783 and from 0.122 to 0.809, respectively. No significant Hardy–Weinberg equilibrium was detected (P < 0.01) at all loci. The EST-SSR markers developed in the present study can be used for strain identification and population genetic studies in A. cinnamomea.