A comparative genomics strategy for targeted discovery of single-nucleotide polymorphisms and conserved-noncoding sequences in orphan crops

Completed genome sequences provide templates for the design of genome analysis tools in orphan species lacking sequence information. To demonstrate this principle, we designed 384 PCR primer pairs to conserved exonic regions flanking introns, using Sorghum/Pennisetum expressed sequence tag alignments to the Oryza genome. Conserved-intron scanning primers (CISPs) amplified single-copy loci at 37% to 80% success rates in taxa that sample much of the approximately 50-million years of Poaceae divergence. While the conserved nature of exons fostered cross-taxon amplification, the lesser evolutionary constraints on introns enhanced single-nucleotide polymorphism detection. For example, in eight rice (Oryza sativa) genotypes, polymorphism averaged 12.1 per kb in introns but only 3.6 per kb in exons. Curiously, among 124 CISPs evaluated across Oryza, Sorghum, Pennisetum, Cynodon, Eragrostis, Zea, Triticum, and Hordeum, 23 (18.5%) seemed to be subject to rigid intron size constraints that were independent of per-nucleotide DNA sequence variation. Furthermore, we identified 487 conserved-noncoding sequence motifs in 129 CISP loci. A large CISP set (6,062 primer pairs, amplifying introns from 1,676 genes) designed using an automated pipeline showed generally higher abundance in recombinogenic than in nonrecombinogenic regions of the rice genome, thus providing relatively even distribution along genetic maps. CISPs are an effective means to explore poorly characterized genomes for both DNA polymorphism and noncoding sequence conservation on a genome-wide or candidate gene basis, and also provide anchor points for comparative genomics across a diverse range of species.     

General-use polymerase chain reaction primers for amplification and direct sequencing of enolase, a single-copy nuclear gene, from different animal phyla

In contrast to mitochondrial DNA, remarkably few general-use primer sets are available for single-copy nuclear genes across animal phyla. Here, we present a primer set that yields a c. 364-bp coding fragment of the metabolic gene enolase, which includes an intron in some taxa. In species where introns are absent or have few insertions/deletions, the amplified fragment can be sequenced directly for phylogenetic or population analysis. Between-species variation in the coding region occurs widely at third codon positions, even between closely related taxa, making the fragment useful for species-level systematics. In low gene-flow species, the primers may also be of use for population genetics, as intraspecific polymorphisms occur at several silent positions in the taxa examined.     

Conserved Microsatellites in Ants Enable Population Genetic and Colony Pedigree Studies across a Wide Range of Species

Broadly applicable polymorphic genetic markers are essential tools for population genetics, and different types of markers have been developed for this purpose. Microsatellites have been employed as particularly polymorphic markers for over 20 years. However, PCR primers for microsatellite loci are often not useful outside the species for which they were designed. This implies that a new set of loci has to be identified and primers developed for every new study species. To overcome this constraint, we identified 45 conserved microsatellite loci based on the eight currently available ant genomes and designed primers for PCR amplification. Among these loci, we chose 24 for in-depth study in six species covering six different ant subfamilies. On average, 11.16 of these 24 loci were polymorphic and in Hardy-Weinberg equilibrium in any given species. The average number of alleles for these polymorphic loci within single populations of the different species was 4.59. This set of genetic markers will thus be useful for population genetic and colony pedigree studies across a wide range of ant species, supplementing the markers available for previously studied species and greatly facilitating the study of the many ant species lacking genetic markers. Our study shows that it is possible to develop microsatellite loci that are both conserved over a broad range of taxa, yet polymorphic within species. This should encourage researchers to develop similar tools for other large taxonomic groups.     

微卫星位点筛选方法综述

微卫星标记因其丰富的多态性和共显性等特点,已得到了广泛的应用.应用微卫星标记首先需要获得微卫星位点的序列信息,用来设计引物.获得微卫星位点的方法有多种,本文综述了获得和富集微卫星位点的常用方法.最简便、最省时的方法是从公共数据库(如EMBL、Genbank、EST数据库等)或已发表的文献中查找到微卫星位点,但只限于已经有序列数据发布的物种.第二种方法是种间转移扩增,即从相近物种的数据库中查找微卫星位点,或使用已有数据发表的遗传距离相近物种的微卫星标记.第三种方法是从基因组DNA中筛选微卫星位点,其中用于富集微卫星的方法有引物法、磁珠杂交法、尼龙膜杂交法以及RAPD技术法.     

SSCP-SNP in pearl millet—a new marker system for comparative genetics

A considerable array of genomic resources are in place in pearl millet, and marker-aided selection is already in use in the public breeding programme at ICRISAT. This paper describes experiments to extend these publicly available resources to a single nucleotide polymorphism (SNP)-based marker system. A new marker system, single-strand conformational polymorphism (SSCP)-SNP, was developed using annotated rice genomic sequences to initially predict the intron-exon borders in millet expressed sequence tags (ESTs) and then to design primers that would amplify across the introns. An adequate supply of millet ESTs was available for us to identify 299 homologues of single-copy rice genes in which the intron positions could be precisely predicted. PCR primers were then designed to amplify approximately 500-bp genomic fragments containing introns. Analysis of these fragments on SSCP gels revealed considerable polymorphism. A detailed DNA sequence analysis of variation at four of the SSCP-SNP loci over a panel of eight inbred genotypes showed complex patterns of variation, with about one SNP or indel (insertion-deletion) every 59 bp in the introns, but considerably fewer in the exons. About two-thirds of the variation was derived from SNPs and one-third from indels. Most haplotypes were detected by SSCP. As a marker system, SSCP-SNP has lower development costs than simple sequence repeats (SSRs), because much of the work is in silico, and similar deployment costs and through-put potential. The rates of polymorphism were lower but useable, with a mean PIC of 0.49 relative to 0.72 for SSRs in our eight inbred genotype panel screen. The major advantage of the system is in comparative applications. Syntenic information can be used to target SSCP-SNP markers to specific chromosomal regions or, conversely, SSCP-SNP markers can be used to unravel detailed syntenic relationships in specific parts of the genome. Finally, a preliminary analysis showed that the millet SSCP-SNP primers amplified in other cereals with a success rate of about 50%. There is also considerable potential to promote SSCP-SNP to a COS (conserved orthologous set) marker system for application across species by more specifically designing primers to precisely match the model genome sequence.Electronic Supplementary Material Supplementary material is available for this article at     

Development of nine polymorphic microsatellite markers for the phytoparasitic nematode Xiphinema index, the vector of the grapevine fanleaf virus

We report isolation, characterization and cross-species amplification of nine microsatellite loci from the phytoparasitic nematode Xiphinema index, the vector of grapevine fanleaf virus. Levels of polymorphism were evaluated in 62 individuals from two X. index populations. The number of alleles varies between two and 10 depending on locus and population. Observed heterozygosity on loci across both populations varied from 0.32 to 0.857 (mean 0.545). The primers were tested for cross-species amplification in three other species of phytoparasitic nematodes of the Xiphinema genus. These nine microsatellite loci constitute valuable markers for population genetics and phylogeographical studies of X. index.     

Generating single-copy nuclear gene data for a recent adaptive radiation

Recent adaptive radiations provide an exceptional opportunity to understand the processes of speciation and adaptation. However, reconstructing the phylogenetic history of recent and rapidly evolving clades often requires the use of multiple, independent gene genealogies. Nuclear introns are an obvious source of the necessary data but their use is often limited because degenerate primers can amplify paralogous loci. To identify PCR primers for a large number of loci in an especially rapid adaptive radiation, that of the flowering plant genus Aquilegia (Ranunculaceae), we developed an efficient method for amplifying multiple single-copy nuclear loci by sequencing a modest number of clones from a cDNA library and designing PCR primers; with one primer anchored in the 3' untranslated region (3'-UTR) and one primer in the coding region of each gene. Variation between paralogous loci evolves more quickly in 3'-UTR regions compared to adjacent exons, and therefore we achieved high specificity for isolating orthologous loci. Furthermore, we were able to identify genes containing large introns by amplifying genes from genomic DNA and comparing the PCR product size to that predicted from their cDNA sequence. In Aquilegia eight out of eleven loci were isolated with this method and six of these loci had introns. Among four genes sequenced for samples spanning the phylogenetic breadth of the genus, we found sequence variation at levels similar to that observed in ITS, further supporting the recent and rapid radiation in Aquilegia. We assessed the orthology of amplification products by phylogenetic congruence among loci, the presence of two well established phylogenetic relationships, and similarity among loci for levels of sequence variation. Higher levels of variation among samples for one locus suggest possible paralogy. Overall, this method provides an efficient means of isolating predominantly single-copy loci from both low and high-copy gene families, providing ample nuclear variation for reconstructing species-level phylogenies in non-model taxa.     

Rapid development of multiple nuclear loci for phylogenetic analysis using genomic resources: an example from squamate reptiles

Recently, as genome-scale data have become available for more organisms, the development of phylogenetic markers from nuclear protein-coding loci (NPCL) has become more tractable. However, new methods are needed to efficiently sort the large number of genes from genomic databases into more limited sets appropriate for particular phylogenetic questions, while avoiding introns and paralogs. Here we describe a general methodology for identifying candidate single-copy NPCL from genomic databases. Our method uses information from reference genomes to identify genes with relatively large continuous protein-coding regions (i.e., 700bp). BLAST comparisons are used to help avoid genes with paralogous copies or close relatives (i.e., gene families) that might confound phylogenetic analyses. Exon boundary information is used to identify appropriately spaced potential priming sites. Using this method, we have developed over 25 novel NPCL, which span a variety of desirable evolutionary rates for phylogenetic analyses. Although targeted for higher-level phylogenetics of squamate reptiles, many of these loci appear to be useful across and within other vertebrate clades (e.g., amphibians), and some are relatively rapidly evolving and may be useful for closely-related species (e.g., within genera). This general method can be used whenever large-scale genomic data are available for an appropriate reference species (not necessarily within the focal clade). The method is also well suited for the development of intron regions for lower-level phylogenetic and phylogeographic studies. We provide an online database of alignments and suggested primers for approximately 85 NPCL that should be useful across vertebrates.     

Widespread prevalence but contrasting patterns of intragenomic rRNA polymorphisms in nematodes: Implications for phylogeny,species delimitation and life history inference

Ribosomal RNA genes have long been a favoured locus in phylogenetic and metabarcoding studies. Within a genome, rRNA loci are organized as tandem repeated arrays and the copies are homogenized through the process of concerted evolution. However, some level of rRNA variation (intragenomic polymorphism) is known to persist and be maintained in the genomes of many species. In nematode worms, the extent of rRNA polymorphism (RP) across species and the evolutionary and life history factors that contribute to the maintenance of intragenomic RP is largely unknown. Here, we present an extensive analysis across 30 terrestrial nematode species representing a range of free‐living and parasitic taxa isolated worldwide. Our results indicate that RP is common and widespread, ribosome function appears to be maintained despite mutational changes, and intragenomic variants are stable in the genome and neutrally evolving. However, levels of variation were varied widely across rRNA locus and species, with some taxa observed to lack RP entirely. Higher levels of RP were significantly correlated with shorter generation time and high reproductive rates, and population‐level factors may play a role in the geographic and phylogenetic structuring of rRNA variants observed in genera such as Rotylenchulus and Pratylenchus. Although RP did not dramatically impact the clustering and recovery of taxa in mock metabarcoding analyses, the present study has significant implications for global biodiversity estimates of nematode species derived from environmental rRNA amplicon studies, as well as our understanding of the evolutionary and ecological factors shaping genetic diversity across the nematode Tree of Life.     

Characterization of microsatellite loci from Litsea hypophaea (Lauraceae), a tree endemic to Taiwan

? Premise of the study: Microsatellite primers were developed for the endemic tree Litsea hypophaea (Lauraceae) in Taiwan to investigate its genetic diversity and population genetic structure and to investigate species delimitation within Litsea. ? Methods and Results: Fifteen new simple sequence repeat markers were developed from L. hypophaea with a magnetic bead enrichment method. Most loci were also amplified from three closely related species, L. coreana, L. lii, and L. acutivena. The number of alleles and observed and expected heterozygosities across loci varied with a range of 1-25, 0.000-1.000, and 0.000-0.956, respectively. ? Conclusions: The application of these microsatellite markers of L. hypophaea provides a tool for understanding genetic diversity and population differentiation. In addition, interspecific amplification suggests that these markers will also be useful for species identification of related taxa within Litsea in Taiwan.     

Molecular signatures of divergence and selection in closely related pine taxa

Efforts to detect loci under selection in plants have mostly focussed on single species. However, assuming that intraspecific divergence may lead to speciation, comparisons of genetic variation within and among recently diverged taxa can help to locate such genes. In this study, coalescent and outlier detection methods were used to assess nucleotide polymorphism and divergence at 79 nuclear gene fragments (1212 SNPs) in 16 populations (153 individuals) of the closely related, but phenotypically and ecologically distinct, pine taxa Pinus mugo, P. uliginosa and P. uncinata across their European distributions. Simultaneously, mitochondrial DNA markers, which are maternally inherited in pines and distributed by seeds at short geographic distance, were used to assess genetic relationships of the focal populations and taxa. The majority of nuclear loci showed homogenous patterns of variation between the taxa due to a high number of shared SNPs and haplotypes, similar levels of polymorphism, and low net divergence. However, against this common genetic background and an overall low population structure within taxa at mitochondrial markers, we identified several genes showing signatures of selection, accompanied by significant intra- and interspecific divergence. Our results indicate that loci involved in species divergence may be involved in intraspecific local adaptation.     

Microsatellite loci for studies of population differentiation and range expansion in Solidago sempervirens L. (Asteraceae)

For studies of population differentiation and range expansion in the seaside goldenrod, Solidago sempervirens, we identified hypervariable molecular markers by screening genomic libraries enriched for microsatellite motifs. We designed primers that reliably amplified nine polymorphic loci. High polymorphism in a population from Delaware Bay, USA suggests that the loci will be useful in population studies. The success of cross‐amplifications in 11 species of Asteraceae varied among loci and did not appear to reflect phylogenetic relationships within Solidago.     

New genes for phylogenetic studies of lichenized fungi: glyceraldehyde-3-phosphate dehydrogenase and beta-tubulin genes

Abstract:Primers for amplification and sequencing of partial glyceraldehyde-3-phosphate dehydrogenase (gpd) gene were designed for lichenized fungi. The 5′ gpd primer is most probably fungal specific, since a BLAST search in GenBank found identical sequences only from ascomycetous taxa, whereas the 3′ gpd primer was more universal. Utility of the gpd primers and previously designed beta-tubulin primers was tested in nine lichen taxa. Both the gpd and beta-tubulin primer pairs amplified in most of the taxa examined: the gpd primers generated a c. 1100 nucleotide fragment, whereas the PCR product obtained from the beta-tubulin primers was c. 900 nucleotides long. The gpd amplification products of Cladonia arbuscula and C. rangiferina were sequenced and both were found to contain three introns, the length of which varied between 49 to 83 nucleotides. To examine the applicability of gpd sequences in resolving relationships within Ascomycota, trees were calculated from 22 fungal gpd sequences obtained from GenBank together with the twoCladonia sequences using parsimony jackknifing. The gpd tree was compared with the SSU rDNA tree of the respective species (or genera). A similar analysis of the beta-tubulin gene was not performed, because only a few beta-tublin sequences from the same taxa were available in GenBank. The gpd tree was well resolved but in conflict with the SSU rDNA tree. In contrast to the SSU rDNA tree, the gpd tree did not support the monophyly of the Ascomycota. Analysis of the combined data set produced a tree very similar to that of the SSU rDNA data. However, the relationship of Lecanorales to the other orders remained unresolved. Even though gpd and beta-tubulin are highly conserved proteins, the third codon positions and introns are variable and both genes have the potential for inferring phylogenetic relationships at the lower taxonomic levels in the lichenized fungi. The two genes may be useful even below species level, depending on the species investigated.     

Evidence of reproductive isolation among closely related sympatric species of Serrasalmus (Ostariophysii, Characidae) from the Upper Madeira River, Amazon, Bolivia

The delimitation of the Serrasalmus species (Characiformes, Serrasalminae) from the Bolivian Amazon (Amazonas, Madeira) was examined using intron length polymorphism assessed by EPIC-PCR. The six pairs of primers provided 16 polymorphic loci across the species of the region and the allelic diversity ranged between two and 10 alleles per locus. For each locus, the lack of departure from Hardy-Weinberg expectations in a large number of the populations analysed and the homogenous distribution of linkage disequilibrium between paralogous loci and loci belonging to different intronic systems provided strong lines of evidence that the 16 amplified loci constituted independent neutral markers. Furthermore, allelic diversity was size-dependent, thereby indicating that insertion-deletions occurred frequently but randomly in introns, and that intron length polymorphism was a valid marker for investigating the systematics of piranhas. EPIC-PCR demonstrated that eight of the nine nominal species of piranha of the Upper Madeira were reproductively isolated and identified a new species that differed from its closest morphological and genetic relatives by seven diagnostic or semi-diagnostic loci. By contrast, no diagnostic or semi-diagnostic locus was found between S. spilopleura and S. eigenmanni , nor were their allelic frequencies different, thereby questioning the validity of their biological species status, at least in the Upper Madeira. This study, which was one of the first applications of EPIC-PCR to a large-scale molecular systematic purpose, demonstrates that it is a rapid, reliable and cost-effective tool for elucidating issues pertaining to fish systematics.     

Three nuclear genes for phylogenetic, SNP and population genetic studies of molluscs and other invertebrates

The study reports new primers capable of amplifying fragments from three nuclear protein-coding genes in a variety of deep-sea molluscs and annelids - adenine nucleotide translocase (Ant), calmodulin (Cal) and cyclophilin A (CycA). The Ant primers appear to be restricted to bivalve molluscs, whereas the Cal and CycA primers also amplified appropriate gene fragments from Lepetodrilus gastropod molluscs and Osedax polychaete worms. The amplified fragment of Cal contains an intron in the molluscs, but no intron was detected in the Ant and CycA fragments from any of the tested animals. DNA sequences generated by the three primer sets exhibited one to 15 single nucleotide polymorphism sites in deep-sea vesicomyid clams and Osedax boneworms. The observed levels of polymorphism indicate that the genes are likely to be useful in both population genetic and phylogenetic analyses of different invertebrate taxa.     

Universal primers for the amplification of chloroplast microsatellites in grasses (Poaceae)

Attempts to design truly universal primers to amplify chloroplast microsatellites have met with limited success due to nonconservation of repeat loci across widely divergent taxa. We have used the complete chloroplast genome sequences of rice, maize and wheat to design five pairs of primers that amplify homologous mononucleotide repeats across the Poaceae (grasses). Sequencing confirmed conservation of repeat motifs across subfamilies and a preliminary study in Anthoxanthum odoratum revealed polymorphism at two loci with a haplotype diversity value of 0.495. These primers provide a valuable tool to study cytoplasmic diversity in this extensively studied and economically important range of taxa.     

Polymorphic microsatellite markers for the striped skunk, Mephitis mephitis, and other mephitids

We report 10 polymorphic microsatellite loci primers developed for striped skunks (Mephitis mephitis), a widespread mesocarnivore in North America. Numbers of alleles in these loci ranged from seven to 14 and the observed heterozygosity ranged from 0.76 to 1.0. These primers will be useful for studying population dynamics of skunks where rabies is endemic and will be useful to estimate genetic relatedness among females sharing winter dens. Most of these primers amplify across species within the Mephitidae.