SSCP markers provide a useful alternative to microsatellites in genotyping and estimating genetic diversity in populations and germplasm collections of plant specialty crops

For well-studied plant species with whole genome sequence or extensive EST data, SNP markers are the logical choice for both genotyping and whole genome association studies. However, SNP markers may not address the needs of researchers working on specialty crops with limited available genomic information. Microsatellite markers have been frequently employed due to their robustness, but marker development can be difficult and may result in few polymorphic markers. SSCP markers, such as microsatellites, are PCR-based and scored by electrophoretic mobility but, because they are based on SNPs rather than length differences, occur more frequently and are easier to develop than microsatellites. We have examined how well correlated the estimation of genetic diversity and genetic distance are in a population or germplasm collection when measured by 13 highly polymorphic microsatellite markers or 20 SSCP markers. We observed a significant correlation in pairwise genetic distances of 82 individuals in an international cacao germplasm collection (Mantel test Rxy=0.59, p<0.0001 for 10 000 permutations). Both sets of markers could distinguish each individual in the population. These data provide strong support for the use of SSCP markers in the genotyping of plant species where development of microsatellites would be difficult or expensive.     

Comparison of three DNA marker systems for assessing genetic diversity in Asian arowana (Scleropages formosus)

Three DNA marker systems -- random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP) and microsatellites -- were used to estimate the genetic diversity in Asian arowana (Scleropages formosus) by genotyping fish individuals from three different sources. Parallel application of the three DNA marker systems allowed us to compare their utility for the analysis of genetic diversity. Microsatellites displayed the highest expected heterozygosity, whereas the values obtained by RAPD and AFLP were much lower. Multiplex ratio and marker index were higher for AFLP than for RAPD or microsatellites. Weak correlation was detected between genetic similarity estimated from data obtained with the three DNA marker systems: estimates from RAPD and AFLP data turned out to be higher than those from microsatellites. On the other hand genetic similarity was higher in the red variety than in the green one, especially when tested with microsatellites. Based on the genetic distance matrices calculated from microsatellite analysis, all red individuals were clustered into one group, whereas only a subset of them was clustered when either RAPD or AFLP was used. This indicated that the microsatellite system detected population subdivision more efficiently than either RAPD or AFLP.     

虾夷马粪海胆微卫星标记制备及对3个养殖群体的遗传多样性分析

通过磁珠富集法分离获得了虾夷马粪海胆微卫星DNA序列160个.其中完美型108个(67.50%),非完美型33个(20.63%),复合型19个(11.88%),微卫星DNA序列最多重复次数达到107次.根据这些微卫星DNA序列的两翼序列,采用Primer 5软件设计出虾夷马粪海胆的微卫星引物,通过筛选,采用其中的12对微卫星DNA标记对大连凌水群体(DL)、大连獐子岛群体(DZ)、山东荣城群体(SR)3个虾夷马粪海胆养殖群体的遗传多样性进行分析,共获得73个等位基因,不同引物获得的等位基因数为1~11个,片段大小为84~362 bp.除SUX001位点外,其余位点的PIC值在0.264 0~0.670 5之间,3个群体的平均观测杂合度分别为0.4618(DL),0.437 5(SR)和0.434 0(DZ),平均期望杂合度分别为0.502 6(DL),0.507 9(SR)和0.449 4(DZ).Hardy-Weinberg平衡分析显示,73%的被检测位点显著偏离平衡,F-检验显示4个位点的Fst值低于0.05,表明群体间存在一定程度的分化.群体间遗传相似性系数、遗传距离及UPGMA聚类分析表明大连獐子岛群体(DZ)与大连凌水群体(DL)群体亲缘关系较近,二者与山东荣成群体(SR)亲缘关系较远.对深入了解我国虾夷马粪海胆养殖群体遗传结构特征及其种质资源状况具有重要意义.     

A set of novel multi-allelic SNPs for forensic application developed through massively parallel sequencing and its examples of population genetic studies

Massively parallel sequencing (MPS) technology allows to simultaneously type multitudinous molecular genetic markers for many samples in one run with the feature of high detection resolution, and thereby arouses the increasing attention from forensic science. Herein, multiple allelic single nucleotide polymorphisms (multi-allelic SNPs) were screened for personal identification and parentage testing, and then were genotyped using MPS platform. Unrelated individuals of Chinese Mongolian and Kazakh groups were investigated to further estimate forensic effectiveness and applicability of these multi-allelic SNPs. The results of sequencing efficiency estimations and forensic genetic statistical parameters demonstrated that this MPS panel of multi-allelic SNPs was expected to be work for forensic applications. Subsequently, the exploration of population genetic variation patterns among the two investigated groups and other 26 reference populations revealed that these Chinese Mongolian and Kazakh groups had the similar population genetic patterns with the populations from East Asian, but European ancestral composition in the Kazakh group was higher than that in the Mongolian group. Currently, the present results were the preliminary research to scrutinize genetic information of these two ethnic minority groups employing multi-allelic SNPs.     

Developing a new nonbinary SNP fluorescent multiplex detection system for forensic application in China

Nonbinary single‐nucleotide polymorphisms (SNPs) are potential forensic genetic markers because their discrimination power is greater than that of normal binary SNPs, and that they can detect highly degraded samples. We previously developed a nonbinary SNP multiplex typing assay. In this study, we selected additional 20 nonbinary SNPs from the NCBI SNP database and verified them through pyrosequencing. These 20 nonbinary SNPs were analyzed using the fluorescent‐labeled SNaPshot multiplex SNP typing method. The allele frequencies and genetic parameters of these 20 nonbinary SNPs were determined among 314 unrelated individuals from Han populations from China. The total power of discrimination was 0.9999999999994, and the cumulative probability of exclusion was 0.9986. Moreover, the result of the combination of this 20 nonbinary SNP assay with the 20 nonbinary SNP assay we previously developed demonstrated that the cumulative probability of exclusion of the 40 nonbinary SNPs was 0.999991 and that no significant linkage disequilibrium was observed in all 40 nonbinary SNPs. Thus, we concluded that this new system consisting of new 20 nonbinary SNPs could provide highly informative polymorphic data which would be further used in forensic application and would serve as a potentially valuable supplement to forensic DNA analysis.     

Validation of a tentative microsatellite marker for the dopamine D4 receptor gene by capillary gel electrophoresis

Two to four-basepair-short tandem repeats (i.e. microsatellites) are broadly utilized as genetic markers for mapping disease loci in whole genome search analyses. Based on their close vicinity on chromosome 11, the D11S1984 microsatellite was anticipated as a tentative marker for the dopamine D4 receptor gene. A capillary gel electrophoresis based genotype analysis method and an in-house made computational tool was developed for the analysis of the D11S1984 microsatellite marker to examine a healthy Hungarian population of n=106. The data obtained did not suggest significant linkage between the D11S1984 marker and the DRD4 gene.     

Development of microsatellite markers in<Emphasis Type="Italic"> Cannabis sativa</Emphasis> for DNA typing and genetic relatedness analyses

Microsatellite markers were developed for Cannabis sativa L. (marijuana) to be used for DNA typing (genotype identification) and to measure the genetic relationships between the different plants. Twelve different oligonucleotide probes were used to screen an enriched microsatellite library of Cannabis sativa in which 49% of the clones contained microsatellite sequences. Characterization of microsatellite loci in Cannabis revealed that GA/CT was the most abundant class of the isolated microsatellites representing 50% overall followed by GTT/CAA, AAG/TTC, and GAT/CTA representing 16%, 15%, and 10%, respectively. Eleven polymorphic STR markers were developed, three derived from dinucleotide motifs and eight from trinucleotide motifs. A total of 52 alleles were detected averaging 4.7 alleles/locus. The expected heterozygosity of the eleven loci ranged between 0.368 and 0.710 and the common probability of identical genotypes was 1.8×10^–7. The loci identified 27 unique profiles of the 41 Cannabis samples. The 11 microsatellite markers developed in this study were found to be useful for DNA typing and for assessing genetic relatedness in Cannabis.     

Inference of population structure and admixture proportion from Y chromosomal data of Chinese population

In the past two decades, Y chromosome data has been generated for human population genetic studies. These Y chromosome datasets were produced with various testing methods and markers, thus difficult to combine them for a comprehensive analysis. In this study, we combine four human Y chromosomal datasets of Han, Tibetan, Hui, and Li ethnic groups. The dataset contains 27 microsatellites and 137 single nucleotide polymorphisms these populations share in common. We assembled a single dataset containing 2439 individuals from 25 nationwide populations in China. A systematic analysis of genetic distance and clustering was performed. To determine the gene flow of the studied population with worldwide populations, we modeled the ancestry informative markers. The reference panel was regarded as a mixture of South Asian (SAS), East Asian (EAS), European (EUR), African (AFR), and American (AMR) populations from 1000 Genomes data of Y chromosome using nonlinear data-fitting. We then calculated the admixture proportion of these four studied populations with 26 worldwide populations. The results showed that the Han and Hui have great genetic affinity, and Hui is the most admixed ethnic group, with 61.53% EAS, 34.65% SAS, 1.91% AFR, 1.56% AMR, and 0.04% EUR ancestry component (the AMR is highly admixed and thus should be ignored). All the other three ethnic groups contained more than 97% EAS ancestry component. The Li is the least admixed population in this study. The combined dataset in this study is the largest of this kind reported to date and proposes reference population data for use in future paternal genetic studies and forensic genealogical identification.     

Identification of novel microsatellite markers <1 Mb from the HBB gene and development of a single‐tube pentadecaplex PCR panel of highly polymorphic markers for preimplantation genetic diagnosis of beta‐thalassemia

Beta (β)‐thalassemia is one of the most common monogenic diseases worldwide. Affected pregnancies can be avoided through preimplantation genetic diagnosis (PGD), which commonly involves customized assays to detect the different combinations of β‐globin (HBB) gene mutations present in couples, in conjunction with linkage analysis of flanking microsatellite markers. Currently, the limited number of reported closely linked markers hampers their utility in indirect linkage‐based PGD for this disorder. To increase the available markers closely flanking the HBB gene, an in silico search was performed to identify all markers within 1 Mb flanking the HBB gene. Fifteen markers with potentially high polymorphism information content (PIC) and heterozygosity values were selected and optimized into a single‐tube pentadecaplex PCR panel. Allele frequencies and polymorphism and heterozygosity indices of each marker were assessed in five populations. A total of 238 alleles were observed from the 15 markers. PIC was >0.7 for all markers, with expected heterozygosity and observed heterozygosity values ranging from 0.74 to 0.90 and 0.72 to 0.88, respectively. Greater than 99% of individuals were heterozygous for at least seven markers, with at least two heterozygous markers on either side of the HBB gene. The pentadecaplex marker assay also performed reliably on single cells either directly or after whole genome amplification, thus validating its use in standalone linkage‐based β‐thalassemia PGD or in conjunction with HBB mutation detection.     

Refining the results of a whole-genome screen based on 4666 microsatellite markers for defining predisposition factors for multiple sclerosis

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system with a complex genetic background. In order to identify loci associated with the disease, we had performed a genome screen initially using 6000 microsatellite markers in pooled DNA samples of 198 MS patients and 198 controls. Here, we report on the detailed reanalysis of this set of data. Distinctive features of microsatellites genotyped in pooled DNA causing false-positive association or masking existing association were met by improved evaluation and refined correction factors in the statistical analyses. In order to assess potential errors introduced by DNA pooling and genotyping, we resurveyed the experiment in a subset of microsatellite markers using de novo-composed DNA pools. True MS associations of markers were verified via genotyping all individual DNA samples comprised in the pools. Microsatellites share characteristically superb information content but they do not lend themselves to automation in very large scale formats. Especially after DNA pooling many artifacts of individual marker systems require special attention and treatment. Therefore, in the near future comprehensive whole-genome screens may rather be performed by typing single nucleotide polymorphisms on chip-based platforms.     

Which genetic marker for which conservation genetics issue?

Conservation genetics focuses on the effects of contemporary genetic structuring on long-term survival of a species. It helps wildlife managers protect biodiversity by identifying a series of conservation units, which include species, evolutionarily significant units (ESUs), management units (MUs), action units (AUs), and family nets (FNs). Although mitochondrial DNA (mtDNA) evolves 5-10 times faster than single-copy nuclear DNA (scnDNA), it records few traces of contemporary events. Thus, mtDNA can be used to resolve taxonomic uncertainties and ESUs. Variable number of tandem repeats (VNTRs) evolve 100-1000 times faster than scnDNA and provide a powerful tool for analyzing recent and contemporary events. VNTR analysis techniques include polymerase chain reaction (PCR)-based microsatellite assays and oligonucleotide probing. Size homoplasy problems in PCR-based microsatellite assays can strongly affect the inference of recent population history. The high homozygosity in endangered species is reflected in a relatively low number and level of variability in microsatellite loci. This combined with "allelic dropout" and "misprinting" errors contributes to the generation of highly biased genetic data following analyses of natural populations. Thus, in conservation genetics, microsatellites are of limited use for identifying ESUs, MUs, and AUs. In contrast to PCR-based microsatellite analysis, oligonucleotide probing avoids errors resulting from PCR amplification. It is particularly suitable for inferring recent population history and contemporary gene flow between fragmented subpopulations. Oligonucleotide fingerprinting generates individual-specific DNA banding patterns and thus provides a highly precise tool for monitoring demography of natural populations. Hence, DNA fingerprinting is powerful for distinguishing ESUs, MUs, AUs, and FNs. The use of oligonucleotide fingerprinting and fecal DNA is opening new areas for conservation genetics.     

Analysis of the variations in IL-28RA gene and their association with allergic rhinitis

IL-28RA is one of the important candidate genes for complex trait of genetic diseases, but there is no published information of the genetic variation in this gene. We scanned the seven exons and their boundary introns sequence of IL-28RA including the promoter regions to analyze genetic variation sites, and identified eighteen single nucleotide polymorphisms (SNPs) and two variation sites. We chose seven SNPs (g.-1193 A>C, g.-30 C>T, g.17654 C>T, g.27798 A>G, g.31265 C>T, g.31911 C>T and g.32349 G>A) of them for large sample size genotyping, and assessed the association of genotype and allele frequencies of these SNPs between allergic rhinitis patients and non-allergic rhinitis controls. We also compared the genotype frequencies between Korean controls and Han Chinese control or Korean Chinese control. We investigated the frequencies of haplotype constructed by these SNPs between allergic rhinitis patients and non-allergic rhinitis controls. Our results suggested that the g.32349 G>A polymorphism of IL-28RA might be associated with susceptibility to allergic rhinitis (P=0.032), but seems to have no relationship with serum total IgE levels. The haplotype frequencies by these SNPs also show significant association between controls and allergic rhinitis patients.     

Development and Characterization of Microsatellite Markers (SSR) in Sesamum (Sesamum indicum L.) Species

Microsatellites, also known as simple sequence repeats (SSRs), are the class of repetitive DNA sequences present throughout the genome of many plant and animal species. Recent advances in molecular genetics had been the introduction of microsatellite markers to investigate the genetic structuring of natural plant populations. We have employed an enrichment strategy for microsatellite isolation by using multi-enzymes digestion, microsatellite oligoprobes, and streptavidin magnetic beads in Sesamum (Sesamum indicum L.). More than 200 SSR motifs were detected (SSR motifs ??2 repeat units or 6?bp); 80?% of the clones contained SSR motifs. When regarding SSRs with four or more repeat units and a minimum length of 10?bp, 132 of them showed repeats. Eighteen SSR markers were initially characterized for optimum annealing temperature using a gradient PCR technique. Among the 18 SSR markers characterized, five were found to be polymorphic and used to analyze 60 Sesamum germplasm accessions. The maximum number of alleles detected was four with a single primer and the least number of two alleles with three primers with an average PIC value of 0.77. SSRs are a valuable tool for estimating genetic diversity and analyzing the evolutionary and historical development of cultivars at the genomic level in sesame breeding programs.     

Correlation between the genetic diversity and variation of total phenolic acids contents in Fructus Xanthii from different populations in China

Fructus Xanthii (Cang-Er-Zi) is a traditional Chinese medicine that is used in curing nasal diseases and headache according to the Chinese Pharmacopoeia. For the effective quality control of its medicinal values, reflected by chemical variation patterns, in addition to the relationship with genetic diversity, analyses based on UV spectrophotometry, HPLC fingerprinting and inter-simple sequence repeat (ISSR) molecular markers were carried out, involving 16 Xanthium populations from different locations in China. The HPLC data showed considerable variation of chemical constituents among the 16 Xanthium populations, and they were classified to three chemotypes by hierarchical clustering analysis. Abundant genetic diversity was detected among the Xanthium populations, which were also clustered into three groups based on their ISSR data and varied according to different species. Combining the genetic divergence and chemical differences showed an important result that, in the two chemotypes, the higher contents of total phenolic acids (TPA) in Fructus Xanthii showed greater genetic diversity (I). We suggest that genetic diversity affects the contents of TPA. Since variable phenolic acid contents may affect therapeutic efficacy, it is important to point out that combining the use of genetic base with chemotype will help control the favourable chemotypes and breed new cultivars with more desirable chemical constituents.     

Evaluation of the microhaplotype markers in kinship analysis

Microhaplotype markers are emerging forensic genetic markers, which may supplement existing markers. Consisting of two to four SNPs with an extent of <200 bp, microhaplotype can be genotyped through massively parallel sequencing technology. Articles that have been published suggested that microhaplotype markers have good application prospect in forensics. Multiallelic haplotype loci are potentially important in certain forensic works, as the stutter and high mutation rate of short tandem repeats and the low polymorphism of single nucleotide polymorphisms may limit the power of these two kind of regular markers. In this study, we explored the potential of 11 new microhaplotype loci in kinship analysis. The results suggested that these loci have relatively high polymorphic information in different populations worldwide and relatively high system effectiveness in the kinship analysis. Microhaplotypes have potential for forensic kinshipg analysis.     

Rapid and precise genotyping of porcine microsatellites

Microsatellites are useful markers for genetic mapping and linkage analysis because they are highly polymorphic, abundant in genomes and relatively easily scored with polymerase chain reaction (PCR). A rapid genotyping system for microsatellites was developed, which included multiplex PCRs, multiple use of Hydrolink gels, automated fluorescent detection of fragments on an A.L.F. DNA sequencer, automatic assignment of alleles to each locus and verification of genotypes with a self-developed computer program "Fragtest". Eight multiplex PCRs have been developed to genotype 29 microsatellites for genetic and quantitative trait loci (QTL) mapping on pig chromosomes 6, 7, 12 and 13. Three to six microsatellites could be amplified in one multiplex PCR. Each multiplex reaction required only different concentrations of each pair of primers and a low concentration of dNTP (100 microM). A dNTP concentration of 100 microM proved to be optimal for the coamplification of microsatellites under the concentration of 1.5 mM MgCl2. Using four internal size standards added in each sample, the 5% Hydrolink gel could subsequently be used up to five times (total running time of 500 min) on the A.L.F. automated sequencer without significant loss of resolution and precision of fragment length analysis. Automatic assignment of alleles on each locus using "Fragtest" significantly increased the efficiency and precision of the genotyping. This system is thus a rapid, cheap, and highly discriminating genotyping system.     

A multiplex SNP genotyping by allele‐specificspecific PCR based on stem‐loop and universal fluorescent primers of Chr1daxin mice

Single nucleotide polymorphisms (SNPs) are one of the most common markers in mammals. Rapid, accurate, and multiplex typing of SNPs is critical for subsequent biological and genetic research. In this study, we have developed a novel method for multiplex genotyping SNPs in mice. The method involves allele‐specific PCR amplification of genomic DNA with two stem‐loop primers accompanied by two different universal fluorescent primers. Blue and green fluorescent signals were conveniently detected on a DNA sequencer. We verified four SNPs of 65 mice based on the novel method, and it is well suited for multiplex genotyping as it requires only one reaction per sample in a single tube with multiplex PCR. The use of universal fluorescent primers greatly reduces the cost of designing different fluorescent probes for each SNP. Therefore, this method can be applied to many biological and genetic studies, such as multiple candidate gene testing, genome‐wide association study, pharmacogenetics, and medical diagnostics.     

Five non‐synonymous SNPs in the gene encoding human deoxyribonuclease I‐like 2 implicated in terminal differentiation of keratinocytes reduce or abolish its activity

Several non‐synonymous SNPs in the human deoxyribonuclease I‐like 2 (DNase 1L2) gene responsible for DNA degradation during terminal differentiation of epidermal keratinocytes have been identified. However, only limited population data are available, and furthermore the effect of these SNPs on the DNase 1L2 activity remains unknown. Genotyping of all of the 17 SNPs was performed using the PCR‐RFLP method in three ethnic groups including 14 different populations. A series of amino acid‐substituted DNase 1L2 corresponding to each SNP was expressed, and its activity was measured. All of the six non‐synonymous SNPs exhibited a mono‐allelic distribution, whereas the distribution of some SNPs other than exonic ones was ethnicity‐dependent. Each of the minor alleles in SNPs, p.Ala20Asp, p.Val104Leu, p.Asp197Ala, p.Glu274Lys and p.Asp287Asn, among the non‐synonymous SNPs produced low or no activity‐harbouring DNase 1L2. DNase 1L2 is well conserved, retaining full levels of enzymatic activity, with regard to these exonic SNPs in human populations. It seems plausible to assume that these SNPs affecting the activity may be one of the factors responsible for a genetic pre‐disposition for failure of differentiation‐associated cell death in various keratinocyte lineages, thereby leading to the development of parakeratosis. Our results may have clinical implications in relation to the pathogenesis of parakeratosis.     

Gene mapping study for constitutive skin color in an isolated Mongolian population

To elucidate the genes responsible for constitutive human skin color, we measured the extent of skin pigmentation in the buttock, representative of lifelong non-sun-exposed skin, and conducted a gene mapping study on skin color in an isolated Mongolian population composed of 344 individuals from 59 families who lived in Dashbalbar, Mongolia. The heritability of constitutive skin color was 0.82, indicating significant genetic association on this trait. Through the linkage analysis using 1,039 short tandem repeat (STR) microsatellite markers, we identified a novel genomic region regulating constitutive skin color on 11q24.2 with an logarithm of odds (LOD) score of 3.39. In addition, we also found other candidate regions on 17q23.2, 6q25.1, and 13q33.2 (LOD ≥ 2). Family-based association tests on these regions with suggestive linkage peaks revealed ten and two significant single nucleotide polymorphisms (SNPs) on the linkage regions of chromosome 11 and 17, respectively. We were able to discover four possible candidate genes that would be implicated to regulate human skin color: ETS1, UBASH3B, ASAM, and CLTC.     

Genetic polymorphisms of the A and B subunits of human coagulation factor XIII in mainland Italy and Sardinia: description of a new FXIIIA variant allele.

The distribution of the two alleles of FXIIIA and the three alleles of FXIIIB were studied in populations from mainland Italy and from Sardinia. The frequencies of the FXIIIA*2 allele were 0.266 and 0.265. The frequencies of FXIIIB*1 were 0.787 and 0.765; of B*2, 0.070 and 0.094; of B*3, 0.143 and 0.141. A new cathodal FXIIIA allele (A*7) was described in the Rome sample. No significant difference in the distribution of allele frequencies for either system was found between the two populations studied. For typing both markers, good results were also obtained by using whole blood instead of plasma.