Showing papers on "Microsatellite published in 2012"

Using next-generation sequencing approaches to isolate simple sequence repeat (SSR) loci in the plant sciences

Abstract: The application of next-generation sequencing (NGS) technologies for the development of simple sequence repeat (SSR) or microsatellite loci for genetic research in the botanical sciences is described. Microsatellite markers are one of the most informative and versatile DNA-based markers used in plant genetic research, but their development has traditionally been a diffi cult and costly process. NGS technologies allow the effi cient identifi cation of large numbers of microsatellites at a fraction of the cost and effort of traditional approaches. The major advantage of NGS methods is their ability to produce large amounts of sequence data from which to isolate and develop numerous genome-wide and gene-based microsatellite loci. The two major NGS technologies with emergent application in SSR isolation are 454 and Illumina. A review is provided of several recent studies demonstrating the effi cient use of 454 and Illumina technologies for the discovery of microsatellites in plants. Additionally, important aspects during NGS isolation and development of microsatellites are discussed, including the use of computational tools and high-throughput genotyping methods. A data set of microsatellite loci in the plastome and mitochondriome of cranberry ( Vaccinium macrocarpon Ait.) is provided to illustrate a successful application of 454 sequencing for SSR discovery. In the future, NGS technologies will massively increase the number of SSRs and other genetic markers available to conduct genetic research in understudied but economically important crops such as cranberry.

A direct characterization of human mutation based on microsatellites

Abstract: Mutations are the raw material of evolution but have been difficult to study directly. We report the largest study of new mutations to date, comprising 2,058 germline changes discovered by analyzing 85,289 Icelanders at 2,477 microsatellites. The paternal-to-maternal mutation rate ratio is 3.3, and the rate in fathers doubles from age 20 to 58, whereas there is no association with age in mothers. Longer microsatellite alleles are more mutagenic and tend to decrease in length, whereas the opposite is seen for shorter alleles. We use these empirical observations to build a model that we apply to individuals for whom we have both genome sequence and microsatellite data, allowing us to estimate key parameters of evolution without calibration to the fossil record. We infer that the sequence mutation rate is 1.4-2.3×10(-8) mutations per base pair per generation (90% credible interval) and that human-chimpanzee speciation occurred 3.7-6.6 million years ago.

De novo sequencing and a comprehensive analysis of purple sweet potato (Impomoea batatas L.) transcriptome

Abstract: High-throughput RNA sequencing was performed for comprehensively analyzing the transcriptome of the purple sweet potato A total of 58,800 unigenes were obtained and ranged from 200 nt to 10,380 nt with an average length of 476 nt The average expression of one unigene was 34 reads per kb per million reads (RPKM) with a maximum expression of 1,935 RPKM At least 40,280 (685%) unigenes were identified to be protein-coding genes, in which 11,978 and 5,184 genes were homologous to Arabidopsis and rice proteins, respectively Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) analysis showed that 19,707 (335%) unigenes were classified to 1,807 terms of GO including molecular functions, biological processes, and cellular components and 9,970 (170%) unigenes were enriched to 11,119 KEGG pathways We found that at least 3,553 genes may be involved in the biosynthesis pathways of starch, alkaloids, anthocyanin pigments, and vitamins Additionally, 851 potential simple sequence repeats (SSRs) were identified in all unigenes Transcriptome sequencing on tuberous roots of the sweet potato yielded substantial transcriptional sequences and potentially useful SSR markers which provide an important data source for sweet potato research Comparison of two RNA-sequence datasets from the purple and the yellow sweet potato showed that UDP-glucose-flavonoid 3-O-glucosyltransferase was one of the key enzymes in the pathway of anthocyanin biosynthesis and that anthocyanin-3-glucoside might be one of the major components for anthocyanin pigments in the purple sweet potato This study contributes to the molecular mechanisms of sweet potato development and metabolism and therefore that increases the potential utilization of the sweet potato in food nutrition and pharmacy

Biology and Genetics of New Autosomal STR Loci Useful for Forensic DNA Analysis.

Abstract: Short tandem repeats (STRs) are regions of tandemly repeated DNA segments found throughout the human genome that vary in length (through insertion, deletion, or mutation) with a core repeated DNA sequence. Forensic laboratories commonly use tetranucleotide repeats, containing a four base pair (4-bp) repeat structure such as GATA. In 1997, the Federal Bureau of Investigation (FBI) Laboratory selected 13 STR loci that form the backbone of the U.S. national DNA database. Building on the European expansion in 2009, the FBI announced plans in April 2011 to expand the U.S. core loci to as many as 20 STRs to enable more global DNA data sharing. Commercial STR kits enable consistency in marker use and allele nomenclature between laboratories and help improve quality control. The STRBase website, maintained by the U.S. National Institute of Standards and Technology (NIST), contains helpful information on STR markers used in human identity testing.

Genetic Mapping and QTL Analysis of Growth-Related Traits in the Pacific Oyster

Abstract: The Pacific oyster (Crassostrea gigas) is one of the most important oysters cultured worldwide. To analyze the oyster genome and dissect growth-related traits, we constructed a sex-averaged linkage map by combining 64 genomic simple sequence repeats, 42 expressed sequence tag-derived SSRs, and 320 amplified fragment length polymorphism markers in an F1 full-sib family. A total of 426 markers were assigned to 11 linkage groups, spanning 558.2 cM with an average interval of 1.3 cM and 94.7% of genome coverage. Segregation distortion was significant for 18.8% of the markers (P < 0.05), and distorted markers tended to occur on some genetic regions or linkage groups. Most growth-related quantitative traits were highly significantly (P < 0.01) correlated, and principal component analysis obtained four principal components. Quantitative trait locus (QTL) analysis identified three significant QTLs for two principal components, which explained 0.6–13.9% of the phenotypic variation. One QTL for sex was detected on linkage group 6, and the inheritabilities of sex for parental alleles and maternal alleles on that locus C15 are 39.8% and 0.01%, respectively. The constructed linkage map and determined QTLs can provide a tool for further genetic analysis of the traits and be potential for marker-assisted selection in C. gigas breeding.

SSR marker-based molecular characterization and genetic diversity analysis of aromatic landreces of rice (Oryza sativa L.)

Abstract: Molecular characterization of the genotypes gives precise information about the extent of genetic diversity which helps in the development of an appropriate breeding program. In the present study, a total of 24 SSR markers were used across 12 elite aromatic rice genotypes for their characterization and discrimination. Among these 24 markers 9 microsatellite markers were showed polymorphism. The number of alleles per locus ranged from 2 alleles (RM510, RM244, and RM277) to 6 alleles (RM 163), with an average of 3.33 alleles across 9 loci obtained in the study. The polymorphic information content values ranged from 0.14 (RM510) to 0.71 (RM163) in all 9 loci with an average of 0.48. RM163 was found the best marker for the identification of 12 genotypes as revealed by PIC values. The frequency of most common allele at each locus ranged from 41% (RM163, RM590, and RM413) to 91% (RM510). The pairwise genetic dissimilarity co-efficient indicated that the highest genetic distance was obtained between Basmati PNR 346 and Deepa; Basmati PNR 346 and Patnai-23; Dolargura and Sugandha; Bhogganijia and Sugandha; and finally between Dolargura and Chinikani (88.89%). Opchaya, Basmati PNR 346 and Sugandha had close similarity among them but showed wide dissimilarity with other genotypes. Being grouped into distant clusters Dolargura and Opchaya could be utilized as potential parents for the improvement of fine grain aromatic rice varieties. Genotypes Deepa and Patnai-23 (having zero dissimilarity) might have possessed somewhat similar genetic background and more markers are needed to discriminate them. The microsatellite marker based molecular fingerprinting could serve as a sound basis in the identification of genetically distant accessions as well as in the duplicate sorting of the morphologically close accessions.

An International Reference Consensus Genetic Map with 897 Marker Loci Based on 11 Mapping Populations for Tetraploid Groundnut (Arachis hypogaea L.)

Abstract: Only a few genetic maps based on recombinant inbred line (RIL) and backcross (BC) populations have been developed for tetraploid groundnut. The marker density, however, is not very satisfactory especially in the context of large genome size (2800 Mb/1C) and 20 linkage groups (LGs). Therefore, using marker segregation data for 10 RILs and one BC population from the international groundnut community, with the help of common markers across different populations, a reference consensus genetic map has been developed. This map is comprised of 897 marker loci including 895 simple sequence repeat (SSR) and 2 cleaved amplified polymorphic sequence (CAPS) loci distributed on 20 LGs (a01–a10 and b01–b10) spanning a map distance of 3, 863.6 cM with an average map density of 4.4 cM. The highest numbers of markers (70) were integrated on a01 and the least number of markers (21) on b09. The marker density, however, was lowest (6.4 cM) on a08 and highest (2.5 cM) on a01. The reference consensus map has been divided into 20 cM long 203 BINs. These BINs carry 1 (a10_02, a10_08 and a10_09) to 20 (a10_04) loci with an average of 4 marker loci per BIN. Although the polymorphism information content (PIC) value was available for 526 markers in 190 BINs, 36 and 111 BINs have at least one marker with >0.70 and >0.50 PIC values, respectively. This information will be useful for selecting highly informative and uniformly distributed markers for developing new genetic maps, background selection and diversity analysis. Most importantly, this reference consensus map will serve as a reliable reference for aligning new genetic and physical maps, performing QTL analysis in a multi-populations design, evaluating the genetic background effect on QTL expression, and serving other genetic and molecular breeding activities in groundnut.

Gene-Based Single Nucleotide Polymorphism Markers for Genetic and Association Mapping in Common Bean

Abstract: In common bean, expressed sequence tags (ESTs) are an underestimated source of gene-based markers such as insertion-deletions (Indels) or single-nucleotide polymorphisms (SNPs). However, due to the nature of these conserved sequences, detection of markers is difficult and portrays low levels of polymorphism. Therefore, development of intron-spanning EST-SNP markers can be a valuable resource for genetic experiments such as genetic mapping and association studies. In this study, a total of 313 new gene-based markers were developed at target genes. Intronic variation was deeply explored in order to capture more polymorphism. Introns were putatively identified after comparing the common bean ESTs with the soybean genome, and the primers were designed over intron-flanking regions. The intronic regions were evaluated for parental polymorphisms using the single strand conformational polymorphism (SSCP) technique and Sequenom MassARRAY system. A total of 53 new marker loci were placed on an integrated molecular map in the DOR364 × G19833 recombinant inbred line (RIL) population. The new linkage map was used to build a consensus map, merging the linkage maps of the BAT93 × JALO EEP558 and DOR364 × BAT477 populations. A total of 1,060 markers were mapped, with a total map length of 2,041 cM across 11 linkage groups. As a second application of the generated resource, a diversity panel with 93 genotypes was evaluated with 173 SNP markers using the MassARRAY-platform and KASPar technology. These results were coupled with previous SSR evaluations and drought tolerance assays carried out on the same individuals. This agglomerative dataset was examined, in order to discover marker-trait associations, using general linear model (GLM) and mixed linear model (MLM). Some significant associations with yield components were identified, and were consistent with previous findings. In short, this study illustrates the power of intron-based markers for linkage and association mapping in common bean. The utility of these markers is discussed in relation with the usefulness of microsatellites, the molecular markers by excellence in this crop.

Short-read, high-throughput sequencing technology for STR genotyping.

Abstract: DNA-based methods for human identification principally rely upon genotyping of short tandem repeat (STR) loci. Electrophoretic-based techniques for variable-length classification of STRs are universally utilized, but are limited in that they have relatively low throughput and do not yield nucleotide sequence information. High-throughput sequencing technology may provide a more powerful instrument for human identification, but is not currently validated for forensic casework. Here, we present a systematic method to perform high-throughput genotyping analysis of the Combined DNA Index System (CODIS) STR loci using short-read (150 bp) massively parallel sequencing technology. Open source reference alignment tools were optimized to evaluate PCR-amplified STR loci using a custom designed STR genome reference. Evaluation of this approach demonstrated that the 13 CODIS STR loci and amelogenin (AMEL) locus could be accurately called from individual and mixture samples. Sensitivity analysis showed that as few as 18,500 reads, aligned to an in silico referenced genome, were required to genotype an individual (>99% confidence) for the CODIS loci. The power of this technology was further demonstrated by identification of variant alleles containing single nucleotide polymorphisms (SNPs) and the development of quantitative measurements (reads) for resolving mixed samples.

Forensic performance of two insertion–deletion marker assays

Abstract: Improving the amplification and analysis of highly degraded DNA extracts has been a longstanding area of research in forensic genetics. One of the most promising recent developments in analysis of degraded DNA is the availability of short, biallelic insertion-deletion length polymorphisms (InDels) in highly multiplexed assays. InDels share many of the favourable characteristics of singlenucleotide polymorphisms (SNPs) that make them ideal markers for analysis of degraded DNA, including: analysis in short amplicon size ranges, high multiplexing capability and low mutation rates. In addition, as length-based polymorphisms, InDels can be analysed with the same simple dye-labelled PCR primer methods as standard forensic short tandem repeats. Separation and detection of fluorescently dye-labelled PCR products by capillary electrophoresis eliminate the multiple step protocols required by SNP typing with single-base extension assays and provide a closer relationship between the input DNA and the profile peak height ratios. Therefore InDel genotyping represents an effective new approach for human identification that adds informative new loci to the existing battery of forensic markers. To assess the utility of InDels for forensic analysis, we characterised population variation with two InDel identification assays: the 30-plex Qiagen DIPplex panel and a 38-plex panel developed by Pereira et al. in 2009 [1]. Allele frequencies were generated for the 68 markers in US African American, Caucasian, East Asian and Hispanic samples. We made a thorough assessment of the individual and combined performance of the InDel sets, as well as characterising profile artifacts and other issues related to the routine use of these newly developed forensic assays based on artificially degraded DNA and mixed source samples. © 2012 Springer-Verlag.

A protocol for direct and rapid multiplex PCR amplification on forensically relevant samples

Abstract: Forensic DNA typing involves a multi-step workflow. Steps include DNA isolation, quantification, amplification of a set of short tandem repeat (STR) markers, separation of polymerase chain reaction (PCR) products by capillary electrophoresis (CE) and DNA profile analysis and interpretation. With that, the process takes around 10-12h. For several scenarios it may be very valuable to speed up this process and obtain an interpretable DNA profile, suited to search a DNA database, within a few hours. For instance in cases of national security, abduction with danger of life, risk of repetition by a serial perpetrator or when custody time of suspects is limited. By a direct and rapid PCR approach we reduced the total DNA profiling time to 2-3h after which genotyping information for the 10 STR markers plus the amelogenin (AMEL) marker present in the commercially available AmpFlSTR(®) SGM Plus™ (SGM+) profiling kit is obtained. This reduction in time is achieved by using the following elements: (1) the inhibitor tolerant, highly processive Phusion(®) Flash DNA polymerase; (2) a modified, non-adenylated allelic ladder; (3) the quick PIKO(®) thermal cycler system with ultra-thin walled reaction tubes; (4) profile interpretation guidelines with an increased allele calling threshold, modified stutter ratios and marked low-level artefact peaks and (5) regulation of sample input by the use of mini-tapes that lift a limited amount of cell material from swabs or fabrics. The procedure is specifically effective for high level DNA, single source samples such as samples containing saliva, blood, semen and hair roots. Success rates, defined as a complete DNA profile, depend on stain type and surface. Due to the use of tape lifting as the sampling technique, the swab or fabric remains dry and intact and can be analyzed at a later stage using regular procedures. Validation experiments were performed which showed that the protocol effectively instructs researchers unfamiliar with the procedure. We have incorporated direct and rapid PCR in a "DNA-6h" service that can assist police investigations by rapidly deriving DNA information from trace evidence left by a perpetrator, searching the STR profile against a DNA database and reporting the outcomes to police or prosecution.

High-throughput novel microsatellite marker of faba bean via next generation sequencing.

Abstract: Faba bean (Vicia faba L.) is an important food legume crop, grown for human consumption globally including in China, Turkey, Egypt and Ethiopia. Although genetic gain has been made through conventional selection and breeding efforts, this could be substantially improved through the application of molecular methods. For this, a set of reliable molecular markers representative of the entire genome is required. A library with 125,559 putative SSR sequences was constructed and characterized for repeat type and length from a mixed genome of 247 spring and winter sown faba bean genotypes using 454 sequencing. A suit of 28,503 primer pair sequences were designed and 150 were randomly selected for validation. Of these, 94 produced reproducible amplicons that were polymorphic among 32 faba bean genotypes selected from diverse geographical locations. The number of alleles per locus ranged from 2 to 8, the expected heterozygocities ranged from 0.0000 to 1.0000, and the observed heterozygosities ranged from 0.0908 to 0.8410. The validation by UPGMA cluster analysis of 32 genotypes based on Nei's genetic distance, showed high quality and effectiveness of those novel SSR markers developed via next generation sequencing technology. Large scale SSR marker development was successfully achieved using next generation sequencing of the V. faba genome. These novel markers are valuable for constructing genetic linkage maps, future QTL mapping, and marker-assisted trait selection in faba bean breeding efforts.

Mining and validation of pyrosequenced simple sequence repeats (SSRs) from American cranberry (Vaccinium macrocarpon Ait.)

Abstract: The American cranberry (Vaccinium macrocarpon Ait.) is a major commercial fruit crop in North America, but limited genetic resources have been developed for the species. Furthermore, the paucity of codominant DNA markers has hampered the advance of genetic research in cranberry and the Ericaceae family in general. Therefore, we used Roche 454 sequencing technology to perform low-coverage whole genome shotgun sequencing of the cranberry cultivar ‘HyRed’. After de novo assembly, the obtained sequence covered 266.3 Mb of the estimated 540–590 Mb in cranberry genome. A total of 107,244 SSR loci were detected with an overall density across the genome of 403 SSR/Mb. The AG repeat was the most frequent motif in cranberry accounting for 35% of all SSRs and together with AAG and AAAT accounted for 46% of all loci discovered. To validate the SSR loci, we designed 96 primer-pairs using contig sequence data containing perfect SSR repeats, and studied the genetic diversity of 25 cranberry genotypes. We identified 48 polymorphic SSR loci with 2–15 alleles per locus for a total of 323 alleles in the 25 cranberry genotypes. Genetic clustering by principal coordinates and genetic structure analyzes confirmed the heterogeneous nature of cranberries. The parentage composition of several hybrid cultivars was evident from the structure analyzes. Whole genome shotgun 454 sequencing was a cost-effective and efficient way to identify numerous SSR repeats in the cranberry sequence for marker development.

Sequence-based novel genomic microsatellite markers for robust genotyping purposes in foxtail millet [Setaria italica (L.) P. Beauv.].

Abstract: The unavailability of microsatellite markers and saturated genetic linkage map has restricted the genetic improvement of foxtail millet [Setaria italica (L.) P. Beauv.], despite the fact that in recent times it has been documented as a new model species for biofuel grasses. With the objective to generate a good number of microsatellite markers in foxtail millet cultivar ‘Prasad’, 690 clones were sequenced which generated 112.95 kb high quality sequences obtained from three genomic libraries each enriched with different microsatellite repeat motifs. Microsatellites were identified in 512 (74.2%) of the 690 positive clones and 172 primer pairs (pp) were successfully designed from 249 (48.6%) unique SSR-containing clones. The efficacies of the microsatellite containing genomic sequences were established by superior primer designing ability (69%), PCR amplification efficiency (85.5%) and polymorphic potential (52%) in the parents of F2 mapping population. Out of 172 pp, functional 147 markers showed high level of cross-species amplification (~74%) in six grass species. Higher polymorphism rate and broad range of genetic diversity (0.30–0.69 averaging 0.58) obtained in constructed phylogenetic tree using 52 microsatellite markers, demonstrated the utility of markers in germplasm characterizations. In silico comparative mapping of 147 foxtail millet microsatellite containing sequences against the mapping data of sorghum (~18%), maize (~16%) and rice (~5%) indicated the presence of orthologous sequences of the foxtail millet in the respective species. The result thus demonstrates the applicability of microsatellite markers in various genotyping applications, determining phylogenetic relationships and comparative mapping in several important grass species.

Genetic structure and linkage disequilibrium pattern of a rapeseed (Brassica napus L.) association mapping panel revealed by microsatellites

Abstract: Understanding the population structure and linkage disequilibrium (LD) is a prerequisite for association mapping of complex traits in a target population. In this study, we assessed the genetic diversity, population structure and the extent of LD in a panel of 192 inbred lines of Brassica napus from all over the world using 451 single-locus microsatellite markers. The inbred lines could be divided into P1 and P2 groups by a model-based population structure analysis. Out of the 142 inbred lines in the P1 group, 126 lines were from China and Japan, and the remaining 16 lines were from Europe, Canada and Australia. In the P2 group, 33 out of the 50 lines were from Europe, Canada, and Australia, and the remaining 17 lines were from China. Structure analysis further divided each group into two subgroups. AMOVA, pairwise FST and neutrality analyses confirmed the differentiation between groups and subgroups. More than 80 % of the pairwise kinship estimates between inbred lines were <0.05, indicating that relative kinship is weak in our panel. Only 6 % linked marker pairs showed LD, suggesting the low level of LD in this association panel. The LD decayed within 0.5–1 cM at the genome level, and varied considerably across each group and subgroup, due to the population size, genetic background and genetic drift. The characterization of the population structure and LD patterns would be useful for performing association studies for complex agronomic traits in rapeseed.

A microsatellite linkage map for the cultivated strawberry ( Fragaria × ananassa ) suggests extensive regions of homozygosity in the genome that may have resulted from breeding and selection

Abstract: The linkage maps of the cultivated strawberry, Fragaria × ananassa (2n = 8x = 56) that have been reported to date have been developed predominantly from AFLPs, along with supplementation with transferrable microsatellite (SSR) markers. For the investigation of the inheritance of morphological characters in the cultivated strawberry and for the development of tools for marker-assisted breeding and selection, it is desirable to populate maps of the genome with an abundance of transferrable molecular markers such as microsatellites (SSRs) and gene-specific markers. Exploiting the recent release of the genome sequence of the diploid F. vesca, and the publication of an extensive number of polymorphic SSR markers for the genus Fragaria, we have extended the linkage map of the ‘Redgauntlet’ × ‘Hapil’ (RG × H) mapping population to include a further 330 loci, generated from 160 primer pairs, to create a linkage map for F. × ananassa containing 549 loci, 490 of which are transferrable SSR or gene-specific markers. The map covers 2140.3 cM in the expected 28 linkage groups for an integrated map (where one group is composed of two separate male and female maps), which represents an estimated 91% of the cultivated strawberry genome. Despite the relative saturation of the linkage map on the majority of linkage groups, regions of apparent extensive homozygosity were identified in the genomes of ‘Redgauntlet’ and ‘Hapil’ which may be indicative of allele fixation during the breeding and selection of modern F. × ananassa cultivars. The genomes of the octoploid and diploid Fragaria are largely collinear, but through comparison of mapped markers on the RG × H linkage map to their positions on the genome sequence of F. vesca, a number of inversions were identified that may have occurred before the polyploidisation event that led to the evolution of the modern octoploid strawberry species.

Polymorphic chloroplast microsatellite loci in Nelumbo (Nelumbonaceae).

Abstract: PREMISE OF THE STUDY To study population genetics, phylogeography, and hybridization of Nelumbo (Nelumbonaceae), chloroplast microsatellite markers were developed. METHODS AND RESULTS Seventeen microsatellite loci were identified from the chloroplast genomes of N. nucifera and N. lutea. Polymorphisms were assessed in three populations of N. nucifera and one population of N. lutea. Nine loci were found to be polymorphic in N. nucifera, and all 17 loci were found to be polymorphic in N. lutea. In N. nucifera, the number of alleles per locus ranged from two to six, and the unbiased haploid diversity per locus ranged from 0.198 to 0.790. In N. lutea, the number of alleles ranged from two to four, and the unbiased haploid diversity per locus ranged from 0.245 to 0.694. CONCLUSIONS The identified chloroplast simple sequence repeat markers will be useful for the study of genetic diversity, phylogeography, and identification of Nelumbo cultivars.

Identification, characterization, and utilization of genome-wide simple sequence repeats to identify a QTL for acidity in apple

Abstract: Apple is an economically important fruit crop worldwide. Developing a genetic linkage map is a critical step towards mapping and cloning of genes responsible for important horticultural traits in apple. To facilitate linkage map construction, we surveyed and characterized the distribution and frequency of perfect microsatellites in assembled contig sequences of the apple genome. A total of 28,538 SSRs have been identified in the apple genome, with an overall density of 40.8 SSRs per Mb. Di-nucleotide repeats are the most frequent microsatellites in the apple genome, accounting for 71.9% of all microsatellites. AT/TA repeats are the most frequent in genomic regions, accounting for 38.3% of all the G-SSRs, while AG/GA dimers prevail in transcribed sequences, and account for 59.4% of all EST-SSRs. A total set of 310 SSRs is selected to amplify eight apple genotypes. Of these, 245 (79.0%) are found to be polymorphic among cultivars and wild species tested. AG/GA motifs in genomic regions have detected more alleles and higher PIC values than AT/TA or AC/CA motifs. Moreover, AG/GA repeats are more variable than any other dimers in apple, and should be preferentially selected for studies, such as genetic diversity and linkage map construction. A total of 54 newly developed apple SSRs have been genetically mapped. Interestingly, clustering of markers with distorted segregation is observed on linkage groups 1, 2, 10, 15, and 16. A QTL responsible for malic acid content of apple fruits is detected on linkage group 8, and accounts for ~13.5% of the observed phenotypic variation. This study demonstrates that di-nucleotide repeats are prevalent in the apple genome and that AT/TA and AG/GA repeats are the most frequent in genomic and transcribed sequences of apple, respectively. All SSR motifs identified in this study as well as those newly mapped SSRs will serve as valuable resources for pursuing apple genetic studies, aiding the apple breeding community in marker-assisted breeding, and for performing comparative genomic studies in Rosaceae.

Genetic Diversity and Population Structure of Ethiopian Chickpea (Cicer arietinum L.) Germplasm Accessions from Different Geographical Origins as Revealed by Microsatellite Markers

Abstract: Genetic diversity and population structure were studied in 155 chickpea (Cicer arietinum L.) entries using 33 polymorphic microsatellite (SSR) markers. Molecular analysis of variance showed variations of 73% within and 27% among populations. Introduced genotypes exhibited highest polymorphism (70.27%) than the landraces (36–57%). Collections from Shewa, Harerge, W. Gojam and S. Gonder regions also showed higher polymorphism (50–57%) than the rest of the local accessions (36–45%). Analyses of pairwise population Nei genetic distance and PhiPT coefficients, expected heterozygosity (He) and unbiased expected heterozygosity (UHe), Shannon’s information index (I) and percent polymorphism (% P) showed existence of high genetic variation between geographical regions. Accessions from adjoining geographical regions mostly showed more genetic similarities than those from origins far isolated apart. This could be associated with the ease and likelihood of inter-regional gene flow and seed movement particularly during times of drought. The 155 entries were grouped into five clusters following analysis of population structure. The first cluster (C1) constituted accessions from Arsi; the second (C2) from Gojam and Gonder; the third (C3) from Harerge and E. and N. Shewa; and the fourth (C4) from W. Shewa, Tigray, and Wello regions. The fifth cluster (C5) was entirely consisted of improved genotypes. Improved genotypes of both Kabuli and Desi types distinctly fell into cluster five (C5) regardless of their difference in seed types. The result has firmly established that introduction of genetic materials from exotic sources has broadened the genetic base of the national chickpea breeding program. Further implications of the findings as regards to chickpea germplasm management and its utilization in breeding program are also discussed.

Microsatellite markers for linkage and association studies

Abstract: During the 1990s and the first several years of this century, microsatellites or short tandem repeats were the workhorse genetic markers for hypothesis-independent studies in human genetics, facilitating genome-wide linkage studies and allelic imbalance studies. However, the rise of higher throughput and cost-effective single-nucleotide polymorphism (SNP) platforms led to the era of the SNP for genome scans. Nevertheless, it is important to note that microsatellites remain highly informative and useful measures of genomic variation for linkage and association studies. Their continued advantage in complementing SNPs lies in their greater allelic diversity than biallelic SNPs as well as in their population history, in which single-step expansion or contraction of the tandem repeat on the background of ancestral SNP haplotypes can break up common haplotypes, leading to greater haplotype diversity within the linkage disequilibrium block of interest. In fact, microsatellites have starred in association studies leading to widely replicated discoveries of type 2 diabetes (TCF7L2) and prostate cancer genes (the 8q21 region). At the end of the day, it will be important to catalog all variation, including SNPs, microsatellites, copy number variations, and polymorphic inversions in human genetic studies. This article describes the utilities of microsatellites and experimental approaches in their use.

Fine mapping of Rf3 and Rf4 fertility restorer loci of WA-CMS of rice Oryza sativa L and validation of the developed marker system for identification of restorer lines

Abstract: The Wild Abortive (WA) system is the major cytoplasmic male sterility (CMS) source for hybrid rice production in indica rice and its fertility restoration is reported to be controlled by two major loci viz. Rf3 on chromosome 1 and Rf4 on chromosome 10. With the availability of the rice genome sequence, an attempt was made to fine map, develop candidate gene based markers for Rf3 and Rf4 and validate the developed marker system in a set of known restorer lines. Using polymorphic markers developed from microsatellite markers and candidate gene based markers from Rf3 and Rf4 loci, local linkage maps were constructed in two mapping populations of ~1,500 F2 progeny from KRH2 (IR58025A/KMR3R) and DRRH2 (IR68897A/DR714-1-2R) hybrids. QTLs and their interactions for fertility restoration in Rf3 and Rf4 loci were identified. The identified QTL in both mapping populations together explained 66–72 % of the phenotypic variance of the trait suggesting their utility in developing a marker system for identification of fertility restorers for WA-CMS. Sequence comparison of the two candidate genes from the Rf3 and Rf4 regions in male sterile (A) and restorer (R) lines showed 2–3 bp indels and a few substitutions in the Rf3 region and indels of 327 and 106 bp in the Rf4 region respectively. The marker system identified in the present study was validated in 212 restorers and 34 maintainers along with earlier reported markers for fertility restoration of WA-CMS. Together DRCG-RF4-14 and DRCG-RF4-8 for the Rf4 locus and DRRM-RF3-5/DRRM-RF3-10 for the Rf3 locus showed a maximum efficiency of 92 % for identification of restorers.

Genetic diversity, population structure and genome-wide marker-trait association analysis emphasizing seed nutrients of the USDA pea (Pisum sativum L.) core collection

Abstract: Genetic diversity, population structure and genome-wide marker-trait association analysis was conducted for the USDA pea (Pisum sativum L.) core collection. The core collection contained 285 accessions with diverse phenotypes and geographic origins. The 137 DNA markers included 102 polymorphic fragments amplified by 15 microsatellite primer pairs, 36 RAPD loci and one SCAR (sequence characterized amplified region) marker. The 49 phenotypic traits fall into the categories of seed macro- and micro-nutrients, disease resistance, agronomic traits and seed characteristics. Genetic diversity, population structure and marker-trait association were analyzed with the software packages PowerMarker, STUCTURE and TASSEL, respectively. A great amount of variation was revealed by the DNA markers at the molecular level. Identified were three sub-populations that constituted 56.1%, 13.0% and 30.9%, respectively, of the USDA Pisum core collection. The first sub-population is comprised of all cultivated pea varieties and landraces; the second of wild P. sativum ssp. elatius and abyssinicum and the accessions from the Asian highland (Afghanistan, India, Pakistan, China and Nepal); while the third is an admixture containing alleles from the first and second sub-populations. This structure was achieved using a stringent cutoff point of 15% admixture (q-value 85%) of the collection. Significant marker-trait associations were identified among certain markers with eight mineral nutrient concentrations in seed and other important phenotypic traits. Fifteen pairs of associations were at the significant levels of P ≤ 0.01 when tested using the three statistical models. These markers will be useful in marker-assisted selection to breed pea cultivars with desirable agronomic traits and end-user qualities.

Identification of Novel Single Nucleotide Polymorphisms (SNPs) in Deer (Odocoileus spp.) Using the BovineSNP50 BeadChip

Abstract: Single nucleotide polymorphisms (SNPs) are growing in popularity as a genetic marker for investigating evolutionary processes. A panel of SNPs is often developed by comparing large quantities of DNA sequence data across multiple individuals to identify polymorphic sites. For non-model species, this is particularly difficult, as performing the necessary large-scale genomic sequencing often exceeds the resources available for the project. In this study, we trial the Bovine SNP50 BeadChip developed in cattle (Bos taurus) for identifying polymorphic SNPs in cervids Odocoileus hemionus (mule deer and black-tailed deer) and O. virginianus (white-tailed deer) in the Pacific Northwest. We found that 38.7% of loci could be genotyped, of which 5% (n = 1068) were polymorphic. Of these 1068 polymorphic SNPs, a mixture of putatively neutral loci (n = 878) and loci under selection (n = 190) were identified with the F(ST)-outlier method. A range of population genetic analyses were implemented using these SNPs and a panel of 10 microsatellite loci. The three types of deer could readily be distinguished with both the SNP and microsatellite datasets. This study demonstrates that commercially developed SNP chips are a viable means of SNP discovery for non-model organisms, even when used between very distantly related species (the Bovidae and Cervidae families diverged some 25.1-30.1 million years before present).

Microsatellites as Tools for Genetic Diversity Analysis

Abstract: Powerful tools for the analysis of genetic biodiversity are molecular markers, which are based on DNA sequence polymorphisms. Indeed, DNA sequences determine the diversity of organisms, and therefore, the techniques used to evaluate DNA polymorphisms directly measure the genetic diversity. Because molecular markers show Mendelian inheritance, it is possible to trace the fingerprint of each organism and determine the evolutionary history of the species by phylogenetic analysis, studies of genetic relationship, population genetic structures and genetic mapping.

Whole genome sequencing of mutation accumulation lines reveals a low mutation rate in the social amoeba Dictyostelium discoideum.

Abstract: Spontaneous mutations play a central role in evolution. Despite their importance, mutation rates are some of the most elusive parameters to measure in evolutionary biology. The combination of mutation accumulation (MA) experiments and whole-genome sequencing now makes it possible to estimate mutation rates by directly observing new mutations at the molecular level across the whole genome. We performed an MA experiment with the social amoeba Dictyostelium discoideum and sequenced the genomes of three randomly chosen lines using high-throughput sequencing to estimate the spontaneous mutation rate in this model organism. The mitochondrial mutation rate of 6.76×10−9, with a Poisson confidence interval of 4.1×10−9 − 9.5×10−9, per nucleotide per generation is slightly lower than estimates for other taxa. The mutation rate estimate for the nuclear DNA of 2.9×10−11, with a Poisson confidence interval ranging from 7.4×10−13 to 1.6×10−10, is the lowest reported for any eukaryote. These results are consistent with low microsatellite mutation rates previously observed in D. discoideum and low levels of genetic variation observed in wild D. discoideum populations. In addition, D. discoideum has been shown to be quite resistant to DNA damage, which suggests an efficient DNA-repair mechanism that could be an adaptation to life in soil and frequent exposure to intracellular and extracellular mutagenic compounds. The social aspect of the life cycle of D. discoideum and a large portion of the genome under relaxed selection during vegetative growth could also select for a low mutation rate. This hypothesis is supported by a significantly lower mutation rate per cell division in multicellular eukaryotes compared with unicellular eukaryotes.

Tools for the management and conservation of genetic diversity in brook trout (Salvelinus fontinalis): tri- and tetranucleotide microsatellite markers for the assessment of genetic diversity, phylogeography, and historical demographics

Abstract: We document isolation and characterization of 13 tri- and tetranucleotide microsatellite DNA markers in brook trout (Salvelinus fontinalis). These markers displayed moderate to high levels of allelic diversity (averaging 20.5 alleles/locus) and heterozygosity (averaging 53.5%) in a range-wide survey of more than 13,000 fish. A comparison of two geographically proximal populations located on opposite sides of the eastern continental divide in Maryland, USA, found no deviations from Hardy–Weinberg equilibrium and minimal linkage disequilibrium. Microsatellite markers developed for S. fontinalis yielded sufficient genetic diversity to: (1) produce unique multilocus genotypes; (2) elucidate phylogeographic structure; and (3) provide unique demographic perspectives of population sizes and historical demographics. This suite of markers also provided considerable cross-species amplification utility among related salmonids.

Development of gene-based markers and construction of an integrated linkage map in eggplant by using Solanum orthologous (SOL) gene sets

Abstract: We constructed an integrated DNA marker linkage map of eggplant (Solanum melongena L.) using DNA marker segregation data sets obtained from two independent intraspecific F2 populations. The linkage map consisted of 12 linkage groups and encompassed 1,285.5 cM in total. We mapped 952 DNA markers, including 313 genomic SSR markers developed by random sequencing of simple sequence repeat (SSR)-enriched genomic libraries, and 623 single-nucleotide polymorphisms (SNP) and insertion/deletion polymorphisms (InDels) found in eggplant-expressed sequence tags (ESTs) and related genomic sequences [introns and untranslated regions (UTRs)]. Because of their co-dominant inheritance and their highly polymorphic and multi-allelic nature, the SSR markers may be more versatile than the SNP and InDel markers for map-based genetic analysis of any traits of interest using segregating populations derived from any intraspecific crosses of practical breeding materials. However, we found that the distribution of microsatellites in the genome was biased to some extent, and therefore a considerable part of the eggplant genome was first detected when gene-derived SNP and InDel markers were mapped. Of the 623 SNP and InDel markers mapped onto the eggplant integrated map, 469 were derived from eggplant unigenes contained within Solanum orthologous (SOL) gene sets (i.e., sets of orthologous unigenes from eggplant, tomato, and potato). Out of the 469 markers, 326 could also be mapped onto the tomato map. These common markers will be informative landmarks for the transfer of tomato’s more saturated genomic information to eggplant and will also provide comparative information on the genome organization of the two solanaceous species. The data are available from the DNA marker database of vegetables, VegMarks (http://vegmarks.nivot.affrc.go.jp).

Microsatellite resources for Passeridae species: a predicted microsatellite map of the house sparrow Passer domesticus

Abstract: We identified microsatellite sequences of potential utility in the house sparrow (Passer domesticus) and assigned their predicted genome locations. These sequences included newly isolated house sparrow loci, which we fully characterized. Many of the newly isolated loci were polymorphic in two other species of Passeridae: Berthelot’s pipit Anthus berthelotii and zebra finch Taeniopygia guttata. In total, we identified 179 microsatellite markers that were either isolated directly from, or are of known utility in, the house sparrow. Sixty-seven of these markers were designed from unique sequences that we isolated from a house sparrow genomic library. These new markers were combined with 36 house sparrow markers isolated by other studies and 76 markers isolated from other passerine species but known to be polymorphic in the house sparrow. We utilized sequence homology to assign chromosomal locations for these loci in the assembled zebra finch genome. One hundred and thirty-four loci were assigned to 25 different autosomes and eight loci to the Z chromosome. Examination of the genotypes of known-sex house sparrows for 37 of the new loci revealed a W-linked locus and an additional Z-linked locus. Locus Pdol2, previously reported as autosomal, was found to be Z-linked. These loci enable the creation of powerful and cost-effective house sparrow multiplex primer sets for population and parentage studies. They can be used to create a house sparrow linkage map and will aid the identification of quantitative trait loci in passerine species.