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.     

Isolation and characterization of microsatellite markers in the sacred lotus (Nelumbo nucifera Gaertn.)

The sacred lotus (Nelumbo nucifera Gaertn.) is an aquatic plant of economic and ornamental importance in China. From an (AG)_n‐enriched genomic library, 24 microsatellites were isolated and identified by using the (fast isolation by the AFLP of sequences containing repeats) FIASCO protocol. Eleven loci showed polymorphism with two to six alleles per locus. These markers yielded 42 alleles in a survey of 32 accessions of the sacred lotus. Eleven effective primer pairs of simple sequence repeats were designed and will be used as genetic markers to evaluate the fine‐scale population structure of the sacred lotus in the future.     

Identification of microsatellite loci in lake sturgeon,Acipenser fulvescens,and their variability in green sturgeon,A. medirostris

From (CATC)_n, (GATA)_n, (AAAC)_n, and (CA)_n–enriched libraries for the lake sturgeon Acipenser fulvescens, 254 primer pairs were developed. These primer pairs resulted in the identification of 128 microsatellite loci in either A. fulvescens or A. medirostris. Polymorphic loci were identified in both sturgeon species for 48 of the primer pairs and 14 of the primer pairs amplified polymorphic loci only in A. medirostris. Most of the identified loci appear to be tetrasomic (79.1% in A. fulvescens and 64.5% in A. medirostris). These results offer estimates of the degree of diploidization in each of these species.     

Tetranucleotide microsatellite loci from the black bear (Ursus americanus)

We describe primers and polymerase chain reaction conditions to amplify 21 tetranucleotide microsatellite DNA loci in black bears (Ursus americanus). We tested primers using individuals from two populations, one each in Georgia and Florida. Among individuals from Georgia (n = 29), primer pairs yielded an average of 2.9 alleles (range, one to four) and an average observed heterozygosity (H_O) of 0.50 (range, 0.00 to 0.79). Among individuals from Florida (n = 19), primer pairs yielded an average of 5.7 alleles (range, one to 14) and an H_O of 0.55 (range, 0.00 to 1.00). A comparison of previously developed markers with individuals from Georgia suggests that bear populations in Georgia and Florida have reduced allelic diversity relative to other populations.     

Ten polymorphic dinucleotide microsatellite markers of the noble scallop Chlamys nobilis

The primers flanking 22 microsatellites isolated from a genomic library enriched for (CA)_n and (GA)_n were designed in the noble scallop Chlamys nobilis. Ten primer pairs provided clear and polymorphic amplification products. Based on characterization with 48 individuals, the number of alleles ranged from three to six. The values of observed heterozygosity and expected heterozygosity varied from 0 to 0.88 and from 0.29 to 0.76, respectively. No significant linkage disequilibrium between pairs of loci was found and six of 10 loci conformed to the Hardy–Weinberg equilibrium. These markers are therefore potentially useful for studies of the population structure of the species.     

Characterization of 24 microsatellite loci in delta smelt, Hypomesus transpacificus, and their cross-species amplification in two other smelt species of the Osmeridae family

We characterized 24 polymorphic tetranucleotide microsatellite loci for delta smelt (Hypomesus transpacificus) endemic to the San Francisco Bay Estuary, CA, USA. Screening of samples (n = 30) yielded two to 26 alleles per locus with observed levels of heterozygosity ranging from 0.17 to 1.0. Only one locus deviated from Hardy–Weinberg equilibrium, suggesting these individuals originate from a single panmictic population. Linkage disequilibrium was found in two pairs of loci after excluding the locus out of Hardy–Weinberg equilibrium. Twenty‐two primer pairs cross‐amplified in wakasagi smelt (Hypomesus nipponensis), and 15 primer pairs cross‐amplified in longfin smelt (Spirinchus thaleichthys).     

Novel microsatellite DNA markers for the threatened African endemic tree species,Milicia excelsa (Moraceae), and cross‐species amplification in Milicia regia

Eleven microsatellite primer pairs were developed for the tropical African tree Milicia excelsa. Genomic DNA was enriched for dinucleotide (TC_n and TG_n) and tretranucleotide (GATA_n), and 188 random clones were sequenced from both orientations. We designed and tested 44 oligonucleotide primer pairs, which were evaluated using genomic DNA from 30 M. excelsa mature trees collected from a natural population in Benin. Eleven of the 44 markers showed good amplification and were polymorphic. The number of putative alleles for polymorphic primer pairs varied from three to seven, with expected and observed heterozygosities ranging from 0.10 to 0.64 and from 0.10 to 0.80, respectively. All 11 loci amplified the related species Milicia regia, indicating that these primers will be useful for population and ecology genetic studies in other species of the genus Milicia.     

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 11 microsatellite loci from Camellia sinensis in Taiwan using PCR-based isolation of microsatellite arrays (PIMA)

We report 11 novel microsatellite primer pairs for the wild tea, Camellia sinensis (L.) O. Kuntze forma formosensis Kitamura. These simple sequence repeat markers were tested in 24 samples collected from wild tea populations, and in cultivars and C. japonica. The number of alleles ranged from 4 to18. The expected (H _E) and observed (H _O) heterozygosity were 0.687–0.946 and 0.042–0.792, respectively. All loci were significantly deviated from Hardy-Weinberg expectations due to the heterozygote deficiency, indicating a dramatic loss of genetic polymorphisms in the rare species. Significant LD was discovered in most loci. These primers may provide a tool for understanding demography and population structure in wild tea.     

Isolation and characterization of microsatellites in Chinese soft‐shelled turtle,Pelodiscus sinensis

Fifteen polymorphic microsatellite loci were developed for the Chinese soft‐shelled turtle (Pelodiscus sinensis) from the (GT)_n microsatellite‐enriched genomic library, using the fast isolation by amplified fragment length polymorphism of sequences containing repeats protocol. The polymorphism of all 15 loci ranged from two to seven alleles with observed heterozygosities ranging from 0.03 to 0.98 (mean 0.43) in one population of 40 individuals. These novel loci will be helpful for understanding the population structure at genetic level and marker‐assisted breeding of this vulnerable species.     

Identification and characterization of microsatellite loci in the common fig (Ficus carica L.) and representative species of the genus Ficus

We developed microsatellites in fig (Ficus carica L.). A TC and TG‐enriched genomic library was screened, and after sequencing, primers were designed for 20 microsatellites. Eight primer pairs produced amplification products that were both interpretable and polymorphic in 14 fig cultivars and two French wild‐growing populations of F. carica (n₁ = 9 and n₂ = 10). Number of alleles per locus ranged from three to six. Except for one microsatellite locus, the observed heterozygosity was higher than the expected value. The F. carica microsatellites gave amplification products in 17 other Ficus species in 86% of the cases.     

Microsatellites for Lindera species

Microsatellite markers were developed for conservation genetic studies of Lindera melissifolia (pondberry), a federally endangered shrub of southern bottomland ecosystems. Microsatellite sequences were obtained from DNA libraries that were enriched for the (AC)_n simple sequence repeat motif. From 35 clone sequences, 20 primer pairs were designed and evaluated. Eleven primer pairs amplified polymorphic marker loci in pondberry while two did so in Lindera benzoin (spicebush). In 46 samples from a single pondberry site the number of microsatellite alleles ranged from two to 11 per locus with observed heterozygosity values of 0.07–0.91.     

Isolation and characterization of microsatellite loci in the Pacific pleasure oyster,Crassostrea corteziensis,and their cross‐species amplification in four other oyster species

Microsatellite loci were isolated in Crassostrea corteziensis using (GT)_n, (CT)_n and (CTGT)_n‐enriched genomic libraries. Within each of 45 sequenced clones, an average of three microsatellite regions (156 total) were observed. Thirty‐three primers were designed, from which 11 microsatellite loci amplified. Ten of those were polymorphic, with a range of two to 30 alleles. Three loci were not in Hardy–Weinberg equilibrium, and linkage disequilibrium was found for six pairs of loci. These microsatellite loci will be further tested for segregation distortions and null alleles to establish a set for population genetic studies of the species in the Northwest coasts of Mexico, and for optimization of aquaculture development. Seven of the microsatellite loci cross‐amplified in Crassostrea palmula, a sympatric species, and will be useful in further genetic studies.     

Polymorphic microsatellite primers developed from DNA of the endangered Mekong giant catfish,Pangasianodon gigas (Chevey) and cross‐species amplification in three species of Pangasius

The critically endangered Pangasianodon gigas is endemic to the Mekong River. Despite its importance, little is known about its genetic diversity and conservation efforts are hampered. Ten polymorphic dinucleotide microsatellite primer pairs were developed from DNA of P. gigas. The analysis of 20 individuals from hatchery stocks using these primers resulted in two to six alleles/locus; H_O = 0.05–0.95; H_E = 0.05–0.81. All but one locus (Pg‐3) conformed to Hardy–Weinberg expectation. Eight, six and seven primer pairs were amplified with the DNA from Pangasianodon hypophthalmus, Pangasius larnaudii and Pangasius sanitwongsei, respectively. These markers will be useful for genetic monitoring of wild and hatchery stocks of these pangasiids.     

Development of microsatellite markers and analysis of intraspecific genetic variability in chickpea (Cicer arietinum L.)

Paucity of polymorphic molecular markers in chickpea (Cicer arietinum L.) has been a major limitation in the improvement of this important legume. Hence, in an attempt to develop sequence-tagged microsatellite sites (STMS) markers from chickpea, a microsatellite enriched library from the C. arietinum cv. Pusa362 nuclear genome was constructed for the identification of (CA/GT) _n and (CT/GA) _n microsatellite motifs. A total of 92 new microsatellites were identified, of which 74 functional STMS primer pairs were developed. These markers were validated using 9 chickpea and one C. reticulatum accession. Of the STMS markers developed, 25 polymorphic markers were used to analyze the intraspecific genetic diversity within 36 geographically diverse chickpea accessions. The 25 primer pairs amplified single loci producing a minimum of 2 and maximum of 11 alleles. A total of 159 alleles were detected with an average of 6.4 alleles per locus. The observed and expected heterozygosity values averaged 0.32 (0.08–0.91) and 0.74 (0.23–0.89) respectively. The UPGMA based dendrogram was able to distinguish all the accessions except two accessions from Afghanistan establishing that microsatellites could successfully detect intraspecific genetic diversity in chickpea. Further, cloning and sequencing of size variant alleles at two microsatellite loci revealed that the variable numbers of AG repeats in different alleles were the major source of polymorphism. Point mutations were found to occur both within and immediately upstream of the long tracts of perfect repeats, thereby bringing about a conversion of perfect motifs into imperfect or compound motifs. Such events possibly occurred in order to limit the expansion of microsatellites and also lead to the birth of new microsatellites. The microsatellite markers developed in this study will be useful for genetic diversity analysis, linkage map construction as well as for depicting intraspecific microsatellite evolution.     

云南莲瓣兰主栽品种SSR指纹图谱的构建和遗传差异分析

为了解云南莲瓣兰(Cymbidium tortisepalum)的遗传多样性,利用SSR技术对32个莲瓣兰主栽品种进行遗传变异分析,并构建莲瓣兰栽培品种的指纹图谱。结果表明,筛选出的12对多态性高、稳定性好的引物共检测到95个等位基因,每对引物检测到4~18个等位基因,有效等位基因数(N E)为61.489,平均有效等位基因数(NA)为5.124,Shannon信息指数(I)和多态性信息含量(PIC)分别为0.806~2.624和0.789~0.953。12对引物中,以引物SSR03的等位基因数、NE、观测杂合度、I和PIC最高。32个品种在12对引物上都具有不同的特异性条带,可以彼此区别。从12对引物中筛选出3对核心引物SSR02、SSR03和SSR12构建了莲瓣兰主栽品种SSR分子指纹图谱,这3对核心引物组合即可鉴定32个莲瓣兰栽培品种。这为莲瓣兰的品种鉴定、遗传多样性分析和分子育种研究提供理论基础和技术支持。     

Characterization of 35 microsatellite loci in the Pacific lion‐paw scallop (Nodipecten subnodosus) and their cross‐species amplification in four other scallops of the Pectinidae family

Four microsatellite‐enriched DNA libraries yielded 35 microsatellite loci from 100 primer pairs designed for Pacific lion‐paw scallop, Nodipecten subnodosus. The number of alleles ranged from four to 28. Three of the 35 loci were not in Hardy–Weinberg equilibrium and linkage disequilibrium was found for one pair of loci. These microsatellites will be used to analyse the population structure of the species in Mexico's Baja Peninsula to propose management strategies for scallop aquaculture development. Twenty‐six primer pairs cross‐amplified in Nodipecten nodosus, whereas none (Argopecten ventricosus) or few cross‐amplified in the Argopecten species.     

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.     

Abundance, variability and chromosomal location of microsatellites in wheat

The potential of microsatellite sequences as genetic markers in hexaploid wheat (Triticum aestivum) was investigated with respect to their abundance, variability, chromosomal location and usefulness in related species. By screening a lambda phage library, the total number of (GA)_n blocks was estimated to be 3.6 x 10⁴ and the number of (GT)_n blocks to be 2.3 x 10⁴ per haploid wheat genome. This results in an average distance of approximately 270 kb between these two microsatellite types combined. Based on sequence analysis data from 70 isolated microsatellites, it was found that wheat microsatellites are relatively long containing up to 40 dinucleotide repeats. Of the tested primer pairs, 36% resulted in fragments with a size corresponding to the expected length of the sequenced microsatellite clone. The variability of 15 microsatellite markers was investigated on 18 wheat accessions. Significantly, more variation was detected with the microsatellite markers than with RFLP markers with, on average, 4.6 different alleles per microsatellite. The 15 PCR-amplified microsatellites were further localized on chromosome arms using cytogenetic stocks of Chinese Spring. Finally, the primers for the 15 wheat microsatellites were used for PCR amplification with rye (Secale cereale) and barley accessions (Hordeum vulgare, H. spontaneum). Amplified fragments were observed for ten primer pairs with barley DNA and for nine primer pairs with rye DNA as template. A microsatellite was found by dot blot analysis in the PCR products of barley and rye DNA for only one primer pair.     

Development and characterization of polymorphic microsatellite markers from Fragaria viridis,a wild diploid strawberry

To date, the development of microsatellite (SSR) markers in the genus Fragaria has focused on F. vesca. However, further species are thought to have contributed to the complex allo‐octoploid genome of the cultivated strawberry, F.×ananassa. Here, we present 22 new SSR markers developed from the diploid species F. viridis. Twenty‐one of the primer pairs amplified polymorphisms in six F. viridis accessions, with an average of 4.95 alleles per primer pair and an average expected heterozygosity of 0.68. Fourteen of these primer pairs, and a locus monomorphic in F. viridis, amplified polymorphic alleles in the parents of a F. vesca mapping population.