The structure of interrupted human AC microsatellites

Microsatellite lengths change over evolutionary time through a process of replication slippage. A recently proposed model of this process holds that the expansionary tendencies of slippage mutation are balanced by point mutations breaking longer microsatellites into smaller units and that this process gives rise to the observed frequency distributions of uninterrupted microsatellite lengths. We refer to this as the slippage/point-mutation theory. Here we derive the theory's predictions for interrupted microsatellites comprising regions of perfect repeats, labeled segments, separated by dinucleotide interruptions containing point mutations. These predictions are tested by reference to the frequency distributions of segments of AC microsatellite in the human genome, and several predictions are shown not to be supported by the data, as follows. The estimated slippage rates are relatively low for the first four repeats, and then rise initially linearly with length, in accordance with previous work. However, contrary to expectation and the experimental evidence, the inferred slippage rates decline in segments above 10 repeats. Point mutation rates are also found to be higher within microsatellites than elsewhere. The theory provides an excellent fit to the frequency distribution of peripheral segment lengths but fails to explain why internal segments are shorter. Furthermore, there are fewer microsatellites with many segments than predicted. The frequencies of interrupted microsatellites decline geometrically with microsatellite size measured in number of segments, so that for each additional segment, the number of microsatellites is 33.6% less. Overall we conclude that the detailed structure of interrupted microsatellites cannot be reconciled with the existing slippage/point-mutation theory of microsatellite evolution, and we suggest that microsatellites are stabilized by processes acting on interior rather than on peripheral segments.

Microsatellites revealed no genetic differentiation between hatchery and contemporary wild populations of striped catfish, Pangasianodon hypophthalmus (Sauvage 1878) in Vietnam

Aquaculture of the striped catfish, Pangasianodon hypophthalmus (Sauvage 1878), in Vietnam has become one of the fastest growing primary food production sectors in the world. Although a demand on quantity of fingerlings is currently reached, it is likely that the long term quality of the stocks may be uncertain due to lacking of genetic broodstock management measures. The present study employed five microsatellite loci to investigate levels of genetic variation of the stripped catfish of the current wild stocks as well as of the selected hatcheries in Vietnam. The study included four hatchery populations and two wild populations spawned in 2005 in the Mekong and Bassac Rivers, and one wild population (spawned in 2006) in the Bassac River. The results showed no genetic differentiation among populations as revealed by F_ST and a model-based clustering method. AMOVA also showed no genetic differentiation between pooled wild and pooled hatchery populations while variation within groups was significant. Genetic variation of wild (mean number of alleles per locus, A = 4.80–6.20; allelic richness, A_r = 4.54–5.06; mean effective number of alleles per locus, A_e = 2.86–3.20; observed heterozygosity, H_o = 0.62–0.65; expected heterozygosity, H_e = 0.62–0.64) and hatchery populations (A = 4.60–5.20; A^_r = 4.10–4.83; A_e = 2.80–3.11; H_o = 0.61–0.66; H_e = 0.61–0.64) were not statistically different. There were no evidences for recent genetic bottleneck in all populations. Therefore it is implied that the hatchery stocks of striped catfish in Vietnam were founded from sufficient numbers of brooders and current population size is large. The domestication process is in an early stage.

Invasive species can't cover their tracks : using microsatellites to assist management of starling (Sturnus vulgaris) populations in Western Australia

Invasive species are known to cause environmental and economic damage, requiring management by control agencies worldwide. These species often become well established in new environments long before their detection, resulting in a lack of knowledge regarding their history and dynamics. When new invasions are discovered, information regarding the source and pathway of the invasion, and the degree of connectivity with other populations can greatly benefit management strategies. Here we use invasive common starling (Sturnus vulgaris) populations from Australia to demonstrate that genetic techniques can provide this information to aid management, even when applied to highly vagile species over continental scales. Analysis of data from 11 microsatellites in 662 individuals sampled at 17 localities across their introduced range in Australia revealed four populations. One population consisted of all sampling sites from the expansion front in Western Australia, where control efforts are focused. Despite evidence of genetic exchange over both contemporary and historical timescales, gene flow is low between this population and all three more easterly populations. This suggests that localized control of starlings on the expansion front may be an achievable goal and the long-standing practice of targeting select proximal eastern source populations may be ineffective on its own. However, even with low levels of gene flow, successful control of starlings on the expansion front will require vigilance, and genetic monitoring of this population can provide essential information to managers. The techniques used here are broadly applicable to invasive populations worldwide.

Isolation and characterisation via 454 sequencing of microsatellites from the tawny frogmouth, Podargus strigoides (Class Aves, Family Podargidae)

We isolated 24 novel polymorphic microsatellite markers from the tawny frogmouth, a nocturnal bird endemic to Australia, which has successfully adapted to urban environments. Initially, 454 shotgun sequencing was used to identify 733 loci with primers designed. Of these, we trialled 30 in the target species of which all amplified a product of expected size. Subsequently, all 30 of these loci were screened for variation in 25 individuals, from a single population in Melbourne, Victoria, Australia. Twenty-eight loci were polymorphic with observed heterozygosity ranging from 0.03 to 0.96 (mean 0.58) and the number of alleles per locus ranged from 2 to 18 (average of 6.5); we confirmed that 24 loci conformed to Hardy–Weinberg expectations. The 24 loci identified here will be sufficient to unequivocally identify individuals and will be useful in understanding the reproductive ecology, population genetics and the gene flow amongst localities in urban environments where this bird thrives.

Satellyptus: analysis and database of microsatellites from ESTs of Eucalyptus

The main goal of our research was to search for SSRs in the Eucalyptus EST FORESTs database (using a software for mining SSR-motifs). With this objective, we created a database for cataloging Eucalyptus EST-derived SSRs, and developed a bioinformatics tool, named Satellyptus, for finding and analyzing microsatellites in the Eucalyptus EST database. The search for microsatellites in the FORESTs database containing 71,115 Eucalyptus EST sequences (52.09 Mb) revealed 20,530 SSRs in 15,621 ESTs. The SSR abundance detected on the Eucalyptus ESTs database (29% or one microsatellite every four sequences) is considered very high for plants. Amongst the categories of SSR motifs, the dimeric (37%) and trimeric ones (33%) predominated. The AG/CT motif was the most frequent (35.15%) followed by the trimeric CCG/CGG (12.81%). From a random sample of 1,217 sequences, 343 microsatellites in 265 SSR-containing sequences were identified. Approximately 48% of these ESTs containing microsatellites were homologous to proteins with known biological function. Most of the microsatellites detected in Eucalyptus ESTs were positioned at either the 5 or 3 end. Our next priority involves the design of flanking primers for codominant SSR loci, which could lead to the development of a set of microsatellite-based markers suitable for marker-assisted Eucalyptus breeding programs.

Effects of polymorphic microsatellites in the regulatory region of IGF1 and GHR on growth and carcass traits in beef cattle

Growth hormone (GH), insulin-like growth factors 1 and 2 (IGF1 and IGF2) and their associated binding proteins and transmembrane receptors (GHR, IGF1R and IGF2R) play an important role in the physiology of mammalian growth. The objectives of the present study were to estimate the allele and genotype frequencies of microsatellite markers located in the 5'-regulatory region of the IGF1 and GHR genes in beef cattle belonging to different genetic groups and to determine effects of these markers on growth and carcass traits in these animals under an intensive production system. For this purpose, genotyping was performed on 384 bulls including 79 Nellore, 30 Canchim (5/8 Charolais + 3/8 Zebu) and 275 crossbred animals originating from crosses of Simmental (1/2 Simmental, n = 30) and Angus (1/2 Angus, n = 245) sires with Nellore females. The effects of substituting L allele for S allele of GHR microsatellite across Nellore, Canchim and 1/2 Angus were significant for weight gain and body weight (P < 0.05). The IGF1 microsatellite allele substitutions of 229 for 225 within Nellore group and of 225 for 229 within 1/2 Angus were not significant for any of the traits.

Genetic variability of Herpailurus yagouaroundi, Puma concolor and Panthera onca (Mammalia, Felidae) studied using Felis catus microsatellites

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Structure and genetic relationships between Brazilian naturalized and exotic purebred goat domestic goat (Capra hircus) breeds based on microsatellites

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Isolation and characterization of polymorphic microsatellites for the natural populations of barker frog Physalaemus cuvieri

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Isolation and characterization of 15 polymorphic microsatellites in the Plethodontid salamander Ensatina eschscholtzii

We developed 15 new polymorphic microsatellites for the plethodontid salamander Ensatina eschscholtzii. Loci were isolated from a genomic library from Ensatina eschscholtzii xanthoptica enriched for (AAAG)(n) repetitive elements. The number of alleles per locus ranged from 4 to 20 (mean 9) in the sampled population. Observed heterozygosity ranged from 0.37 to 1. None of the loci deviated from Hardy-Weinberg equilibrium or showed significant linkage disequilibrium after a Bonferroni correction for multiple comparisons. All loci amplified in the six other subspecies of the Ensatina eschscholtzii complex. These new markers will prove useful in measuring gene flow and population structure as well as patterns of mating and sperm use in Ensatina.

Identification of microsatellites markers associated with yield components and quality parameters in sugarcane

Marker assisted selection depends on the identification of tightly linked association between marker and the trait of interest. In the present work, functional (EST-SSRs) and genomic (gSSRs) microsatellite markers were used to detect putative QTLs for sugarcane yield components (stalk number, diameter and height) and as well as for quality parameters (Brix, Pol and fibre) in plant cane. The mapping population (200 individuals) was derived from a bi-parental cross (IACSP95-3018 x IACSP93-3046) from the IAC Sugarcane Breeding Program. As the map is under construction, single marker trait association analysis based on the likelihood ratio test was undertaken to detect the QTLs. Of the 215 single dose markers evaluated (1:1 and 3:1), 90 (42%) were associated with putative QTLs involving 43 microsatellite primers (18 gSSRs and 25 EST-SSRs). For the yield components, 41 marker/trait associations were found: 20 for height, 6 for diameter and 15 for stalk number. An EST-SSRs marker with homology to non-phototropic hypocotyls 4 (NPH4) protein was associated with a putative QTL with positive effect for diameter as also with a negative effect for stalk number. In relation to the quality parameters, 18 marker trait associations were found for Brix, 19 for Pol, and 12 for fibre. For fibre, 58% of the QTLs detected showed a negative effect on this trait. Some makers associated with QTLs with a negative effect for fibre showed a positive effect for Pol, reflecting the negative correlation generally observed between these traits.

Development and characterization of 16 microsatellites for the Neotropical catfish Pseudoplatystoma reticulatum and cross species analysis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Isolation and characterization of microsatellites markers for two South American frogs (Leptodactylus bufonius and L. chaquensis) using next generation sequencing

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

SEQUENTIAL SAMPLING AND MICROSATELLITES FOR EVALUATION OF THE GENETIC QUALITY IN CORN SEEDS LOTS

In the seed production system, genetic purity is one of the fundamental requirements for its commercialization. The present work had the goal of determined the sample size for genetic purity evaluation, in order to protect the seed consumer and the producer and to evaluate the sensitivity of microsatellite technique for discriminating hybrids from their respective relatives and for detecting mixtures when they are present in small amounts in the samples. For the sequential sampling, hybrid seeds were marked and mixed in with the seed lots, simulating the following levels of contamination: 0.25, 0.5, 1.0, 2.0, 4.0, and 6.0%. After this, groups of 40 seeds were taken in sequence, up to a maximum of 400 seeds, with the objective of determining the quantity of seeds necessary to detect the percentage of mixture mentioned above. The sensitivity of microsatellite technique was evaluated by mixing different proportions of DNA from the hybrids with their respective seed lines. For the level of mixture was higher than 1:8 (1P1:8P2; 8P1:1P2), the sensitivity of the marker in detecting different proportions of the mixture varied according to the primer used. In terms of the sequential sampling, it was verified that in order to detect mixture levels higher than 1% within the seed lot- with a risk level for both the producer and the consumer of 0.05- the size of the necessary sample was smaller than the size needed for the fixed sample size. This also made it possible to reduce costs, making it possible to use microsatellites to certify the genetic purity of corn seeds lots.