Amplified fragment length polymorphism (AFLP) in soybean: species diversity, inheritance, and near-isogenic line analysis

Amplified fragment length polymorphism (AFLP) analysis is a PCR-based technique capable of detecting more than 50 independent loci in a single PCR reaction. The objectives of the present study were to: (1) assess the extent of AFLP variation in cultivated (Gycine max L. Merr.) and wild soybean (G. soja Siebold & Zucc.), (2) determine genetic relationships among soybean accessions using AFLP data, and (3) evaluate the usefulness of AFLPs as genetic markers. Fifteen AFLP primer pairs detected a total of 759 AFLP fragments in a sample of 23 accessions of wild and cultivated soybean, with an average of 51 fragments produced per primer pair per accession. Two-hundred and seventy four fragments (36% of the total observed) were polymorphic, among which 127 (17%) were polymorphic in G. max and 237 (31%) were polymorphic in G. soja. F₂ segregation analysis of six AFLP fragments indicated that they segregate as stable Mendelian loci. The number of polymorphic loci detected per AFLP primer pair in a sample of 23 accessions ranged from 9 to 27. The AFLP phenotypic diversity values were greater in wild than in cultivated soybean. Cluster and principal component analyses using AFLP data clearly separated G. max and G. soja accessions. Within the G. max group, adapted soybean cultivars were tightly clustered, illustrating the relatively low genetic diversity present in cultivated soybean. AFLP analysis of four soybean near-isogenic lines (NILs) identified three AFLP markers putatively linked to a virus resistance gene from two sources. The capacity of AFLP analysis to detect thousands of independent genetic loci with minimal cost and time requirements makes them an ideal marker for a wide array of genetic investigations.     

Isolation and characterization of microsatellite loci from Asparagus acutifolius (Liliaceae)

The isolation of molecular markers in Asparagus acutifolius, a wild edible plant species, is important to characterize local ecotypes that could be cultivated and preserved. We isolated and characterized polymorphic microsatellite loci from A. acutifolius by constructing and screening an enriched DNA library. Primer pairs were designed for 12 loci. Seven primer pairs worked well during amplification reactions and were tested on a wild population from Pontecagnano (SA), Italy. These loci showed a high level of genetic variability, with the numbers of alleles identified ranging from two to five and observed heterozygosity ranging from 0.20 to 0.73.     

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.     

Development and use of simple sequence repeat SSR markers in Rubus species

The isolation of polymorphic codominant microsatellite markers in Rubus and in particular red raspberry will provide a tool to investigate gene flow between cultivated and wild raspberries. Microsatellite loci were isolated by screening a PstI size selected genomic library with AC₍₁₃₎ and AG₍₁₃₎. Positive clones were sequenced and primer pairs designed to the sequences flanking identified SSRs. One primer of each pair was fluorescently labelled to facilitate polymerase chain reaction (PCR) product identification on an automated DNA sequencer. We describe 10 polymorphic microsatellite loci developed and demonstrate their usefulness in different Rubus species.     

Isolation and charaterization of 12 dinucleotide microsatellite loci from Belenger’s jewfish (Johnius belengerii Cuvier 1830)

We describe the first isolation of 12 polymorphic microsatellite markers from Belenger’s jewfish (Johnius belengnerii Cuvier 1830). From a (GT)n-enriched genomic library, 54 microsatellites were selected for designing microsatellite primers, of which 36 gave working primer pairs. 12 of these loci were polymorphic in a test population of 21 individuals with alleles ranging from 3 to 18, and expected and observed heterozygosities from 0.5772 to 0.9449 and from 0.4286 to 0.9231, respectively. No significant linkage disequilibrium between pairs of loci was found, however, loci Jobe24 significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction. These polymorphic microsatellite loci should provide sufficient level of genetic diversity to investigate population structure in Belenger’s jewfish.     

65份野生油茶种质遗传多样性的ISSR和RAPD标记分析

利用ISSR和RAPD标记,对名邛台地野生油茶种质进行遗传多样性分析。从60条简单重复序列引物中筛选出16条引物,在65份样品中共扩增出213条带,其中多态位点为203个,多态位点百分率为95.31%;从30条寡居核苷酸引物中筛选出8条引物,共扩增出105条带,其中多态性位点94个,多态位点百分率为89.52%。结果表明:名邛台地野生油茶种质具有较丰富的遗传多样性,ISSR和RAPD标记可以应用于油茶种质遗传多样性分析。     

Microsatellite markers for the palaeotropic pioneer tree genus Macaranga (Euphorbiaceae) and their cross‐species transferability

We developed primer sequences for five polymorphic microsatellite loci in the tropical ant‐plant genus Macaranga (Euphorbiaceae). Population genetic parameters were determined on the basis of 30 individuals from each of two Macaranga species in Borneo. Allele numbers per locus ranged from three to 13. Expected and observed heterozygosities ranged from 0.160 to 0.850 and from 0.130 to 0.700, respectively. Four of the five primer pairs cross‐amplify polymorphic PCR products in a wide range of Macaranga species.     

甜瓜种质资源遗传多样性的SRAP分析

为研究甜瓜材料之间的亲缘关系及其分类, 更有效地利用种质资源, 为培育新品种提供依据, 文章采用 SRAP (Sequence-related amplified polymorphism)技术对61份甜瓜种质资源的遗传多样性进行研究。结果表明: 从42对引物组合中筛选出16对扩增条带清晰、多态性高的引物组合分析供试材料, 共检测出452个位点, 其中265个为多态性位点, 多态性比率达58.63％, 平均每对引物组合产生28.56个位点和16.56个多态性位点。61份材料间的相似系数为0.48～0.93, 平均为0.73。这些结果说明, 供试甜瓜材料具有较为丰富的遗传多样性。聚类分析结果表明, 61个甜瓜品种中首先可分为薄皮甜瓜与厚皮甜瓜两大类, 彼此亲缘关系最远。以遗传相似系数0.74为截值, 可把供试材料分为5个类群。在生态区域中, 新疆厚皮甜瓜的Nei’s基因多样性指数(0.2231)和Shannon’s信息指数(0.3422)最高。     

珊瑚菜居群遗传多样性的SRAP分析

利用SRAP对伞形科单种属珊瑚菜7个野生居群和1个栽培居群进行了研究。结果表明:共筛选出8个引物组合,在珊瑚菜8个居群中共扩增出168条条带,其中多态性条带为118条,多态性比率为70.23%;平均每对引物扩增的多态性条带为14.75。各居群之间珊瑚菜遗传相似性系数范围为0.8306~0.9836,遗传距离范围为0.0165~0.1856。聚类分析表明,以相似性系数大于0.8并结合地理分布来看,所研究的野生珊瑚菜居群可以大体分为3类,辽宁大连的野生居群为一类,山东威海—山东青岛的居群为一类,而山东日照—广州深圳之间的为一类。     

The development of eight microsatellite loci in the wild daffodil Narcissus triandrus (Amaryllidaceae)

We report microsatellite primer pairs for the wild tristylous daffodil, Narcissus triandrus (Amaryllidaceae). From enriched libraries, we identified 58 unique microsatellite loci. We designed primer pairs for 27 of these loci and screened genomic DNA from 38 to 40 adults from a single population. For eight polymorphic loci, the number of alleles per locus ranged from five to 17. As six primers also amplified loci in three other Narcissus species, including two horticultural varieties, we expect that some of these markers will be transferable to other Narcissus species.     

Development and characterization of 30 polymorphic microsatellite markers for the Atlantic surfclam, Spisula solidissima (Dillwyn, 1817)

Thirty polymorphic microsatellite markers were developed for the Atlantic surfclam, Spisula solidissima, from an enriched library and characterized in 24 clams from a wild population. The number of alleles ranged from 3 to 16 per locus. The expected and observed heterozygosities ranged from 0.1942 to 0.9238 and 0.0833 to 0.875 respectively. Six loci showed significant (P < 0.05 after Bonferroni correction) deviation from Hardy–Weinberg equilibrium, probably because of the presence of null alleles. Three primer pairs amplified duplicated loci with two involving tandem mini‐satellite repeats. Most of the microsatellite markers developed here should be useful for genetic studies in this species.     

Isolation and charaterization of polymorphic microsatellite loci from so-iuy mullet (Mugil soiuy Basilewsky 1855)

The first set of polymorphic microsatellite markers were developed from so-iuy mullet (Mugil soiuy Basilewsky 1855). From a (GT)n-enriched genomic library, 53 microsatellites were selected for designing microsatellite primers, of which 36 gave working primer pairs. Ten of these loci were polymorphic in a test population of 24 individuals with alleles ranging from 3 to 9, and observed and expected heterozygosities from 0.2083 to 0.9167 and from 0.2651 to 0.8812, respectively. No significant linkage disequilibrium between pairs of loci was found, but two loci significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction. These polymorphic microsatellite loci should provide sufficient level of genetic diversity to investigate the fine-scale population structure and evaluate the breeding strategy in Mullet. Genbo Xu and Changwei Shao Contributed equally.     

Development of microsatellite markers in the common fig,Ficus carica L.

We present a new set of 15 polymorphic microsatellite primer sequences developed from Ficus carica L. The variability of specific microsatellite regions was assessed in wild population of figs from the northern Adriatic coast and all 15 primer pairs showed single‐locus amplification with a total of 65 alleles and an observed heterozygosity ranging from 0.285 to 0.863. The 15 new microsatellite loci represent a significant tool for population genetic structure studies and will be further used to investigate the origin and maintenance of genetic variation within and between populations of figs along the Adriatic coastal region.     

Development of new microsatellite loci for the genus <Emphasis Type="Italic">Polistes</Emphasis> from publicly available expressed sequence tag sequences

Over the last 20 years, microsatellites have revolutionized the study of cooperation in the social insects. The Polistes paper wasps have been an important model system for investigations of cooperative behavior. Recently, an expressed sequence tag (EST) library has been developed for P. metricus, allowing researchers to investigate the genetic basis of cooperative behavior in primitive social insect societies for the first time. We searched these freely available EST sequences for microsatellite motifs. This represents a relatively new approach to the development of microsatellite loci that allows for the development of a greater number of loci at less expense. We designed 32 PCR primer pairs, of which 23 amplified PCR products and 18 were polymorphic. These loci exhibited high levels of polymorphism, comparable to anonymous loci isolated via screens of partial genomic libraries. Thus, they are appropriate for population genetic studies as well as the reconstruction of colony genetic structure. A screen of the entire EST database found a total of 708 di-, tri-, tetra- and penta-nucleotide repeats with large repeat units typical of polymorphic loci and at least 30 bp of flanking sequence for primer design. This pool of potential loci represents a new genetic tool for P. metricus, as well as Polistes more generally, as there is great promise for cross amplification in other species.     

Isolation and characterization of microsatellite markers in the East African tree,Acacia brevispica (Fabaceae: Mimosoideae)

We have developed primer pairs for 24 microsatellite loci isolated from Acacia brevispica, which is widespread in woodland savannas of East Africa. The loci were screened for levels of variation using 16–52 individuals from Mpala Research Centre, Kenya. Number of alleles per locus ranged from two to 17, and polymorphic information content ranged from 0.248 to 0.864. Several loci showed the presence null alleles, but many loci will be useful in ongoing studies of genetic structure, gene flow, breeding systems, and natural selection.     

Characterization of microsatellite loci in village indigobirds Vidua chalybeata and cross‐species amplification in estrildid and ploceid finches

We characterized 11 microsatellite primer pairs for the village indigobird Vidua chalybeata. The loci were highly polymorphic, with 7–13 alleles per locus. Gene diversity, estimated as expected heterozygosity, ranged from 0.52 to 0.86, and was generally matched by levels of observed heterozygosity (0.49–0.91). Many of these primer pairs amplified polymorphic loci in cross‐species amplification trials with a variety of estrildid and ploceid finches and a sparrow, Passer griseus. These primers will be valuable for genetic analyses of the brood parasitic indigobirds and whydahs (genus Vidua) as well as other Old World finches.     

Rapid isolation and characterization of microsatellites from the genome of pearl oyster (Pinctada martensi Dunker)

A new set of 34 microsatellite markers was isolated and characterized in pearl oyster Pincada martensi from a CA₁₆-enriched genomic library. Of 60 microsatellite markers screened in a wild population which contained 30 individuals, 34 markers occurred unambiguous bands, including seven monomorphic loci, 5 primer pairs (HHM844, HHM852, HHM888, HHM891, HHM899) were duplicated. These polymorphic markers showed high genetic variability represented mainly by the number of alleles, which ranged from 2 to 6, the expect heterozygosity which ranged from 0.1653 to 0.7622, and the polymorphic information content which ranged from 0.1516 to 0.7279. These markers could increase the available molecular resources that can be used to analyze genetic diversity and structure of pearl oyster.     

Isolation and characterization of polymorphic nuclear microsatellite loci in <Emphasis Type="Italic">Taxus baccata</Emphasis> L.

Seven polymorphic nuclear microsatellite markers for Taxus baccata L. (English yew) were developed using an enriched-library method. An additional polymorphic SSR was obtained by testing eight primer pairs from the congeneric species Taxus sumatrana. Mendelian inheritance for the seven Taxus baccata SSRs was proved by genotyping 17 individuals and 124 megagametophytes (conifer seed haploid tissue). A total of 96 individuals from 5 different populations (10–26 samples per population) were used to estimate genetic diversity parameters. High levels of genetic diversity, with values ranging from 0.533 to 0.929 (6–28 alleles per SSR) were found. No linkage disequilibrium between pairs of loci was detected. All loci but one showed significant departures from Hardy–Weinberg equilibrium. Excess of homozygosity was probably due to high inbreeding in English yew populations, an outcome of low effective population size and/or fragmented distribution. Highly polymorphic SSRs will be used to conduct population genetic studies at different geographical scales and to monitor gene flow.     

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.     

Development and genetic mapping of 127 new microsatellite markers in barley

To enhance the marker density of existing genetic maps of barley (Hordeum vulgare L.), a new set of microsatellite markers containing dinucleotide motifs was developed from genomic clones. Out of 254 primer pairs tested, a total of 167 primer pairs were classifed as functional in a panel of six barley cultivars and three H. spontaneum accessions, and of those, 127 primer pairs resulting in 133 loci were either mapped or located onto chromosomes. The polymorphism information content (PIC) ranged from 0.05 to 0.94 with an average of 0.60. The number of alleles per locus varied from 1 to 9. On average, 3.9 alleles per primer pair were observed. The RFLP frameworks of two previously published linkage maps were used to locate a total of 115 new microsatellite loci on at least one mapping population. The chromosomal assignment of 48 mapped loci was corroborated on a set of wheat-barley chromosome addition lines; 18 additional loci which were not polymorphic in the mapping populations were assigned to chromosomes by this method. The microsatellites were located on all seven linkage groups with four significant clusters in the centromeric regions of 2H, 3H, 6H and 7H. These newly developed microsatellites improve the density of existing barley microsatellite maps and can be used in genetic studies and breeding research.Communicated by G. Wenzel