What phylogeny and gene genealogy analyses reveal about homoplasy in citrus microsatellite alleles (original) (raw)
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Hereditas, 2004
One of the key issues concerning the application of microsatellite DNA data in evolutionary studies is how the number of loci applied may influence the stability of genetic distances and corresponding phylograms. While computer simulations have suggested that over 30 microsatellites are required for accurate evolutionary inference, we show that a median of only six loci have been generally applied in studies of wild populations. Factors contributing to this contrast include: i) uncertainty regarding the potential benefits that can be gained from a realistic increase in the number of loci used; and ii) the lack of empirical studies assessing the influence of the number of microsatellites on the reliability of genetic distance estimation and phylogeny construction. In order to address these issues, we applied resampling techniques to microsatellite data in widely distributed populations of European grayling (Thymallus thymallus, Salmonidae). In agreement with expectations based on simulated data, we demonstrate empirically that the stability of commonly used genetic distances (D CE , D A and (dm) 2) and the corresponding neighbor-joining phylograms is positively associated with the number of microsatellites utilized. For instance, increasing the number of loci from six to 17 resulted in a striking 75% increase in the proportion of D CE phylogram nodes supported by a bootstrap estimate of over 70%. Our results demonstrate that even moderately increasing the number of loci can be very beneficial Á/ a finding extremely relevant for studies of natural populations for which optimally high microsatellite numbers are out of reach. Furthermore, the number of loci most commonly used to date may lead to erroneous inference of the evolutionary relationships between populations.
Frequency and distribution of microsatellites from ESTs of citrus
Genetics and Molecular Biology, 2007
Nearly 65,000 citrus EST (Expressed Sequence Tags) have been investigated using the CitEST project database. Microsatellites were investigated in the unigene sequences from Citrus spp. and Poncirus trifoliata. From these sequences, approximately 35% of the non-redundant ESTs contained SSRs. The frequencies of different SSR motifs were similar between Citrus spp and trifoliate orange. In general, mononucleotide repeats appeared to be the most abundant SSRs in the CitEST database, but we also identify di-, tri-, tetra-, penta-and hexanucleotide repeats. The AG/CT and AAG/CTT were the most common dinucleotide and trinucleotide motifs, with frequencies of 54.4% and 25.2%, respectively. Primer sequences flanking SSR motifs were successfully designed and synthesized. After in silico polymorphism analysis, a subset of sixty-eight primers was validated in different Citrus spp. and Poncirus trifoliata. PCR-amplification revealed polymorphism in citrus with all tested primer pairs and showed the potential of these markers for linkage mapping. Our study showed that the CitEST database can be exploited for the development of SSR markers that can amplify Citrus spp. and related genus for comparative mapping and other genetic analyses.
Standardizing for microsatellite length in comparisons of genetic diversity
Molecular Ecology, 2005
Mutation rates at microsatellites tend to increase with the number of repeats of the motif, leading to higher levels of polymorphism at long microsatellites. To standardize levels of diversity when microsatellites differ in size, we investigate the relationship between tract length and variation and provide a formula to adjust allelic richness to a fixed mean number of repeats in the specific case of chloroplast microsatellites. A comparison between 39 loci from eight species of conifers (where chloroplast DNA is paternally inherited) and 64 loci from 12 species of angiosperms (where chloroplast DNA is generally predominantly maternally inherited) indicates that the greater allelic richness found in conifers remains significant after controlling for number of repeats. The approach stresses the advantage of reporting variation in number of repeats instead of relative fragment sizes.
Transferability and Level of Heterozygosity of Microsatellite Markers in Citrus Species
Plant Molecular Biology Reporter, 2011
Microsatellite markers are a powerful tool for genetic studies, including germplasm conservation, cultivar identification, and integration of linkage maps. Several works have shown that primer pairs designed for one species can be used in related species to facilitate wider application because it reduces the costs for primer development. The objective of this study was to evaluate the transferability of microsatellite primers which was previously developed from the genomic library of Pêra sweet orange (Citrus sinensis L. Osbeck) and to determine the level of heterozygosity between citrus accessions and related genera. Twenty-four microsatellite loci were evaluated on 12 genotypes of Citrus, Poncirus, and an intergeneric hybrid. All analyzed markers were transferable across all genotypes. Seventeen loci were polymorphic, and the number of alleles per loci ranged from one to six. The lowest level of heterozygosity was observed for Poncirus trifoliata (L.) Raf. cultivars while the highest level was for Swingle citrumelo. In general, microsatellite markers showed wide genetic variation and demonstrated that they can be useful in citrus breeding programs.
Origin, evolution and genome distribution of microsatellites
Genetics and Molecular Biology, 2006
Microsatellites, or simple sequence repeats (SSRs), have been the most widely applied class of molecular markers used in genetic studies, with applications in many fields of genetics including genetic conservation, population genetics, molecular breeding, and paternity testing. This range of applications is due to the fact that microsatellite markers are co-dominant and multi-allelic, are highly reproducible, have high-resolution and are based on the polymerase chain reaction (PCR). When first introduced, the development of microsatellite markers was expensive but now new and efficient methods of repetitive sequence isolation have been reported, which have led to reduced costs and microsatellite-technology has been increasingly applied to several species, including non-model organisms. The advent of microsatellite markers revolutionized the use of molecular markers but the development of biometric methods for analyzing microsatellite data has not accompanied the progress in the application of these markers, with more effort being need to obtain information on the evolution of the repetitive sequences, which constitute microsatellites in order to formulate models that fit the characteristics of such markers. Our review describes the genetic nature of microsatellites, the mechanisms and models of mutation that control their evolution and aspects related to their genesis, distribution and transferability between taxa. The implications of the use of microsatellites as a tool for estimating genetic parameters are also discussed.
Molecular Ecology, 2002
Homoplasy has recently attracted the attention of population geneticists, as a consequence of the popularity of highly variable stepwise mutating markers such as microsatellites. Microsatellite alleles generally refer to DNA fragments of different size (electromorphs). Electromorphs are identical in state (i.e. have identical size), but are not necessarily identical by descent due to convergent mutation(s). Homoplasy occurring at microsatellites is thus referred to as size homoplasy. Using new analytical developments and computer simulations, we first evaluate the effect of the mutation rate, the mutation model, the effective population size and the time of divergence between populations on size homoplasy at the within and between population levels. We then review the few experimental studies that used various molecular techniques to detect size homoplasious events at some microsatellite loci. The relationship between this molecularly accessible size homoplasy size and the actual amount of size homoplasy is not trivial, the former being considerably influenced by the molecular structure of microsatellite core sequences. In a third section, we show that homoplasy at microsatellite electromorphs does not represent a significant problem for many types of population genetics analyses realized by molecular ecologists, the large amount of variability at microsatellite loci often compensating for their homoplasious evolution. The situations where size homoplasy may be more problematic involve high mutation rates and large population sizes together with strong allele size constraints.
Microsatellite allele sizes: a simple test to assess their significance on genetic differentiation
2003
The mutation process at microsatellite loci typically occurs at high rates and with stepwise changes in allele sizes, features that may introduce bias when using classical measures of population differentiation based on allele identity (e.g., F ST , Nei's Ds genetic distance). Allele size-based measures of differentiation, assuming a stepwise mutation process [e.g., Slatkin's R ST , Goldstein et al.'s (␦) 2 ], may better reflect differentiation at microsatellite loci, but they suffer high sampling variance. The relative efficiency of allele size-vs. allele identity-based statistics depends on the relative contributions of mutations vs. drift to population differentiation. We present a simple test based on a randomization procedure of allele sizes to determine whether stepwise-like mutations contributed to genetic differentiation. This test can be applied to any microsatellite data set designed to assess population differentiation and can be interpreted as testing whether F ST ϭ R ST . Computer simulations show that the test efficiently identifies which of F ST or R ST estimates has the lowest mean square error. A significant test, implying that R ST performs better than F ST , is obtained when the mutation rate, , for a stepwise mutation process is (a) Ն m in an island model (m being the migration rate among populations) or (b) Ն 1/t in the case of isolated populations (t being the number of generations since population divergence). The test also informs on the efficiency of other statistics used in phylogenetical reconstruction [e.g., Ds and (␦) 2 ], a nonsignificant test meaning that allele identity-based statistics perform better than allele size-based ones. This test can also provide insights into the evolutionary history of populations, revealing, for example, phylogeographic patterns, as illustrated by applying it on three published data sets.
Development of Microsatellites: A Powerful Genetic Marker
The tandem repeats, conserved short segments of DNA, which are found in all prokaryotic and eukaryotic genomes, are called microsatellites. It is also known as variable number tandem repeats (VNTRs), simple sequence repeats (SSRs) and short tandem repeats (STRs). Microsatellites present in both coding and non-coding regions of a genome. The high polymorphism of microsatellites makes them powerful genetic markers for genome mapping of many organisms. It is also suitable for ancient and forensic DNA studies for population genetics and conservation of biological resources. The major disadvantage of microsatellites is that for the first time they need to be isolated de novo from most species being examined. The task of microsatellite isolation is quite cumbersome involving in terms of effort and time, because it traditionally involves screening of genomic libraries. Cross-species amplification, Mining microsatellites from nucleotide sequenced data and Genomic library-based method are general methods of microsatellite isolation. Cross-species method may not effective for all species, Data mining is not applicable if there is no or limited data of DNA sequence. Genomic library based method is the best choice. Traditional protocol, primer extension protocol, selective hybridization, and Fast Isolation by AFLP of Sequences containing repeats (FIASCO) are the protocols of microsatellite development based on genomic library. FIASCO is the best protocol ever developed.