A multi-genome analysis approach enables tracking of the invasion of a single Russian wheat aphid (Diuraphis noxia) clone throughout the New World

This study investigated the population genetics, demographic history and pathway of invasion of the Russian wheat aphid (RWA) from its native range in Central Asia, the Middle East and Europe to South Africa and the Americas. We screened microsatellite markers, mitochondrial DNA and endosymbiont genes in 504 RWA clones from nineteen populations worldwide. Following pathway analyses of microsatellite and endosymbiont data, we postulate that Turkey and Syria were the most likely sources of invasion to Kenya and South Africa, respectively. Furthermore, we found that one clone transferred between South Africa and the Americas was most likely responsible for the New World invasion. Finally, endosymbiont DNA was found to be a high resolution population genetic marker, extremely useful for studies of invasion over a relatively short evolutionary history time frame. This study has provided valuable insights into the factors that may have facilitated the recent global invasion by this damaging pest.

Development of polymorphic microsatellite markers suitable for genetic linkage mapping of olive flounder Paralichthys olivaceus

Microsatellite markers are important for gene mapping and for marker-assisted selection. Sixty-five polymorphic microsatellite markers were developed with an enriched partial genomic library from olive flounder Paralichthys olivaceus an important commercial fish species in Korea. The variability of these markers was tested in 30 individuals collected from the East Sea (Korea). The number of alleles for each locus ranged from 2 to 33 (mean, 17.1). Observed and expected heterozygosity as well as polymorphism information content varied from 0.313 to 1.000 (mean, 0.788), from 0.323 to 0.977 (mean, 0.820), and from 0.277 to 0.960 (mean, 0.787), respectively. Nine loci showed significant deviation from the Hardy-Weinberg equilibrium after sequential Bonferroni correction. Analysis with MICROCHECKER suggested the presence of null alleles at five of these loci with estimated null allele frequencies of 0.126-0.285. These new microsatellite markers from genomic libraries will be useful for constructing a P. olivaceus linkage map.

Whole-genome linkage analysis of rheumatoid arthritis susceptibility loci in 252 affected sibling pairs in the United Kingdom

Objective. To undertake a systematic wholegenome screen to identify regions exhibiting genetic linkage to rheumatoid arthritis (RA). Methods. Two hundred fifty-two RA-affected sibling pairs from 182 UK families were genotyped using 365 highly informative microsatellite markers. Microsatellite genotyping was performed using fluorescent polymerase chain reaction primers and semiautomated DNA sequencing technology. Linkage analysis was undertaken using MAPMAKER/SIBS for single-point and multipoint analysis. Results. Significant linkage (maximum logarithm of odds score 4.7 [P = 0.000003] at marker D6S276, 1 cM from HLA-DRB1) was identified around the major histocompatibility complex (MHC) region on chromosome 6. Suggestive linkage (P < 7.4 × 10-4) was identified on chromosome 6q by single- and multipoint analysis. Ten other sites of nominal linkage (P < 0.05) were identified on chromosomes 3p, 4q, 7p, 2 regions of 10q, 2 regions of 14q, 16p, 21q, and Xq by single-point analysis and on 3 sites (1q, 14q, and 14q) by multipoint analysis. Conclusion. Linkage to the MHC region was confirmed. Eleven non-HLA regions demonstrated evidence of suggestive or nominal linkage, but none reached the genome-wide threshold for significant linkage (P = 2.2 × 10-5). Results of previous genome screens have suggested that 6 of these regions may be involved in RA susceptibility.

Isolation and characterization of novel microsatellite markers and their application for diversity assessment in cultivated groundnut (Arachis hypogaea)

Background: Cultivated peanut or groundnut (Arachis hypogaea L.) is the fourth most important oilseed crop in the world, grown mainly in tropical, subtropical and warm temperate climates. Due to its origin through a single and recent polyploidization event, followed by successive selection during breeding efforts, cultivated groundnut has a limited genetic background. In such species, microsatellite or simple sequence repeat (SSR) markers are very informative and useful for breeding applications. The low level of polymorphism in cultivated germplasm, however, warrants a need of larger number of polymorphic microsatellite markers for cultivated groundnut. Results: A microsatellite- enriched library was constructed from the genotype TMV2. Sequencing of 720 putative SSR-positive clones from a total of 3,072 provided 490 SSRs. 71.2% of these SSRs were perfect type, 13.1% were imperfect and 15.7% were compound. Among these SSRs, the GT/CA repeat motifs were the most common (37.6%) followed by GA/CT repeat motifs (25.9%). The primer pairs could be designed for a total of 170 SSRs and were optimized initially on two genotypes. 104 (61.2%) primer pairs yielded scorable amplicon and 46 (44.2%) primers showed polymorphism among 32 cultivated groundnut genotypes. The polymorphic SSR markers detected 2 to 5 alleles with an average of 2.44 per locus. The polymorphic information content (PIC) value for these markers varied from 0.12 to 0.75 with an average of 0.46. Based on 112 alleles obtained by 46 markers, a phenogram was constructed to understand the relationships among the 32 genotypes. Majority of the genotypes representing subspecies hypogaea were grouped together in one cluster, while the genotypes belonging to subspecies fastigiata were grouped mainly under two clusters. Conclusion. Newly developed set of 104 markers extends the repertoire of SSR markers for cultivated groundnut. These markers showed a good level of PIC value in cultivated germplasm and therefore would be very useful for germplasm analysis, linkage mapping, diversity studies and phylogenetic relationships in cultivated groundnut as well as related Arachis species.

Construction of microsatellite linkage maps for Corymbia

The genus Corymbia is closely related to the genus Eucalyptus, and like Eucalyptus contains tree species that are important for sub-tropical forestry. Corymbia's close relationship with Eucalyptus suggests genetic studies in Corymbia should benefit from transfer of genetic information from its more intensively studied relatives. Here we report a genetic map for Corymbia spp. based on microsatellite markers identified de novo in Corymbia sp or transferred from Eucalyptus. A framework consensus map was generated from an outbred F 2 population (n = 90) created by crossing two unrelated Corymbia torelliana x C. citriodora subsp. variegata F1 trees. The map had a total length of 367 cM (Kosambi) and was composed of 46 microsatellite markers distributed across 13 linkage groups (LOD 3). A high proportion of Eucalyptus microsatellites (90%) transferred to Corymbia. Comparative analysis between the Corymbia map and a published Eucalyptus map identified eight homeologous linkage groups in Corymbia with 13 markers mapping on one or both maps. Further comparative analysis was limited by low power to detect linkage due to low genome coverage in Corymbia, however, there was no convincing evidence for chromosomal structural differences because instances of non-synteny were associated with large distances on the Eucalyptus map. Segregation distortion was primarily restricted to a single linkage group and due to a deficit of hybrid genotypes, suggesting that hybrid inviability was one factor shaping the genetic composition of the F2 population in this inter-subgeneric hybrid. The conservation of microsatellite loci and synteny between Corymbia and Eucalyptus suggests there will be substantial value in exchanging information between the two groups.

Genetic alterations in microsatellite-unstable colorectal cancer

Colorectal cancer (CRC) is the third most common cancer in Finland. Of all CRC tumors, 15% display microsatellite-instability (MSI) caused by defective cellular mismatch repair. Cells displaying MSI accumulate a high number of mutations genome-wide, especially in short repeat areas, microsatellites. When targeting genes essential for cell growth or death, MSI can promote tumorigenesis. In non-coding areas, microsatellite mutations are generally considered as passenger events. Since the discovery of MSI and its linkage to cancer, more that 200 genes have been investigated for a role in MSI tumorigenesis. Although various criteria have been suggested for MSI target gene identification, the challenge has been to distinguish driver mutations from passenger mutations. This study aimed to clarify these key issues in the research field of MSI cancer. Prior to this, background mutation rate in MSI cancer has not been studied in a large-scale. We investigated the background mutation rate in MSI CRC by analyzing the spectrum of microsatellite mutations in non-coding areas. First, semenogelin I was studied for a possible role in MSI carcinogenesis. The intronic T9 repeat of semenogelin I was frequently mutated but no evidence for selection during tumorigenesis was obtained. Second, a sequencing approach was utilized to evaluate the general background mutation rate in MSI CRC. Both intronic and intergenic repeats harbored extremely high mutation rates of ≤ 87% and intergenic repeats were more unstable than the intronic repeats. As mutation rates of presumably neutral microsatellites can be high in MSI CRC in the absence of apparent selection pressure, high mutation frequency alone is not sufficient evidence for identification of driver MSI target genes. Next, an unbiased approach was designed to identify the mutatome of MSI CRC. By combining expression array data and a database search we identified novel genes possibly related to MSI CRC carcinogenesis. One of the genes was studied further. In the functional analysis this gene was observed to cause an abnormal cancer-prone cellular phenotype, possibly through altered responses to DNA damage. In our recent study, smooth muscle myosin heavy chain 11 (MYH11) was identified as a novel MSI CRC gene. Additionally, MYH11 has a well established role in acute myeloid leukemia (AML) through an oncogenic fusion protein CBFB-MYH11. We investigated further the role of MYH11 in AML by sequencing. Three novel missense variants of MYH11 were identified. None of the variants were present in the population-based control material. One of the identified variants, V71A, lies in the N-terminal SH3-like domain of MYH11 of unknown function. The other two variants, K1059E and R1792Q are located in the coil-coiled myosin rod essential for the regulation and filament formation of MYH11. The variant K1059E lies in the close proximity of the K1044N that has been functionally assessed in our earlier work of CRC and has been reported to cause total loss of MYH11 protein regulation. As the functional significance of the three novel variants examined in this work remains unknown, future studies should clarify the further role of MYH11 in AML leukaemogenesis and in other malignancies.

Rapid microsatellite marker development for African mahogany ( Khaya senegalensis, Meliaceae) using next-generation sequencing and assessment of its intra-specific genetic diversity.

Khaya senegalensis (African mahogany or dry-zone mahogany) is a high-value hardwood timber species with great potential for forest plantations in northern Australia. The species is distributed across the sub-Saharan belt from Senegal to Sudan and Uganda. Because of heavy exploitation and constraints on natural regeneration and sustainable planting, it is now classified as a vulnerable species. Here, we describe the development of microsatellite markers for K. senegalensis using next-generation sequencing to assess its intra-specific diversity across its natural range, which is a key for successful breeding programs and effective conservation management of the species. Next-generation sequencing yielded 93943 sequences with an average read length of 234bp. The assembled sequences contained 1030 simple sequence repeats, with primers designed for 522 microsatellite loci. Twenty-one microsatellite loci were tested with 11 showing reliable amplification and polymorphism in K. senegalensis. The 11 novel microsatellites, together with one previously published, were used to assess 73 accessions belonging to the Australian K. senegalensis domestication program, sampled from across the natural range of the species. STRUCTURE analysis shows two major clusters, one comprising mainly accessions from west Africa (Senegal to Benin) and the second based in the far eastern limits of the range in Sudan and Uganda. Higher levels of genetic diversity were found in material from western Africa. This suggests that new seed collections from this region may yield more diverse genotypes than those originating from Sudan and Uganda in eastern Africa.

Genome-wide linkage analysis of multiple measures of neuroticism of 2 large cohorts from Australia and the Netherlands

CONTEXT People meeting diagnostic criteria for anxiety or depressive disorders tend to score high on the personality scale of neuroticism. Studying this personality dimension can give insights into the etiology of these important psychiatric disorders. OBJECTIVES To undertake a comprehensive genome-wide linkage study of neuroticism using large study samples that have been measured multiple times and to compare the results between countries for replication and across time within countries for consistency. DESIGN Genome-wide linkage scan. SETTING Twin individuals and their family members from Australia and the Netherlands. PARTICIPANTS Nineteen thousand six hundred thirty-five sibling pairs completed self-report questionnaires for neuroticism up to 5 times over a period of up to 22 years. Five thousand sixty-nine sibling pairs were genotyped with microsatellite markers. METHODS Nonparametric linkage analyses were conducted in MERLIN-REGRESS for the mean neuroticism scores averaged across time. Additional analyses were conducted for the time-specific measures of neuroticism from each country to investigate consistency of linkage results. RESULTS Three chromosomal regions exceeded empirically derived thresholds for suggestive linkage using mean neuroticism scores: 10p 5 Kosambi cM (cM) (Dutch study sample), 14q 103 cM (Dutch study sample), and 18q 117 cM (combined Australian and Dutch study sample), but only 14q retained significance after correction for multiple testing. These regions all showed evidence for linkage in individual time-specific measures of neuroticism and 1 (18q) showed some evidence for replication between countries. Linkage intervals for these regions all overlap with regions identified in other studies of neuroticism or related traits and/or in studies of anxiety in mice. CONCLUSIONS Our results demonstrate the value of the availability of multiple measures over time and add to the optimism reported in recent reviews for replication of linkage regions for neuroticism. These regions are likely to harbor causal variants for neuroticism and its related psychiatric disorders and can inform prioritization of results from genome-wide association studies.

Within-family outliers: Segregating alleles or environmental effects? A linkage analysis of height from 5815 sibling pairs

Most information in linkage analysis for quantitative traits comes from pairs of relatives that are phenotypically most discordant or concordant. Confounding this, within-family outliers from non-genetic causes may create false positives and negatives. We investigated the influence of within-family outliers empirically, using one of the largest genome-wide linkage scans for height. The subjects were drawn from Australian twin cohorts consisting of 8447 individuals in 2861 families, providing a total of 5815 possible pairs of siblings in sibships. A variance component linkage analysis was performed, either including or excluding the within-family outliers. Using the entire dataset, the largest LOD scores were on chromosome 15q (LOD 2.3) and 11q (1.5). Excluding within-family outliers increased the LOD score for most regions, but the LOD score on chromosome 15 decreased from 2.3 to 1.2, suggesting that the outliers may create false negatives and false positives, although rare alleles of large effect may also be an explanation. Several regions suggestive of linkage to height were found after removing the outliers, including 1q23.1 (2.0), 3q22.1 (1.9) and 5q32 (2.3). We conclude that the investigation of the effect of within-family outliers, which is usually neglected, should be a standard quality control measure in linkage analysis for complex traits and may reduce the noise for the search of common variants of modest effect size as well as help identify rare variants of large effect and clinical significance. We suggest that the effect of within-family outliers deserves further investigation via theoretical and simulation studies.

Association of a low density lipoprotein receptor microsatellite variant with obesity

OBJECTIVE: To determine whether a microsatellite polymorphism located towards the 3' end of the low density lipoprotein receptor gene (LDLR) is associated with obesity. DESIGN: A cross-sectional case-control study. SUBJECTS: One hundred and seven obese individuals, defined as a body mass index (BMI) > or = 26 kg/m2, and 163 lean individuals, defined as a BMI < 26 kg/m2. MEASUREMENTS: BMI, blood pressure, serum lipids, alleles of LDLR microsatellite (106 bp, 108 bp and 112 bp). RESULTS: There was a significant association between variants of the LDLR microsatellite and obesity, in the overall tested population, due to a contributing effect in females (chi 2 = 12.3, P = 0.002), but not in males (chi 2 = 0.3, P = 0.87). In females, individuals with the 106 bp allele were more likely to be lean, while individuals with the 112 bp and/or 108 bp alleles tended to be obese. CONCLUSIONS: These results suggest that in females, LDLR may play a role in the development of obesity.

DNA analysis indicates that Asian elephants are native to Borneo and are therefore a high priority for conservation

The origin of Borneo's elephants is controversial. Two competing hypotheses argue that they are either indigenous, tracing back to the Pleistocene, or were introduced, descending from elephants imported in the 16th-18th centuries. Taxonomically, they have either been classified as a unique subspecies or placed under the Indian or Sumatran subspecies. If shown to be a unique indigenous population, this would extend the natural species range of the Asian elephant by 1300 km, and therefore Borneo elephants would have much greater conservation importance than if they were a feral population. We compared DNA of Borneo elephants to that of elephants from across the range of the Asian elephant, using a fragment of mitochondrial DNA, including part of the hypervariable d-loop, and five autosomal microsatellite loci. We find that Borneo's elephants are genetically distinct, with molecular divergence indicative of a Pleistocene colonisation of Borneo and subsequent isolation. We reject the hypothesis that Borneo's elephants were introduced. The genetic divergence of Borneo elephants warrants their recognition as a separate evolutionary significant unit. Thus, interbreeding Borneo elephants with those from other populations would be contraindicated in ex situ conservation, and their genetic distinctiveness makes them one of the highest priority populations for Asian elephant conservation.

Genetic analysis of an Indian family with members affected with Waardenburg syndrome and Duchenne muscular dystrophy

Purpose: Waardenburg syndrome (WS) is characterized by sensorineural hearing loss and pigmentation defects of the eye, skin, and hair. It is caused by mutations in one of the following genes: PAX3 (paired box 3), MITF (microphthalmia-associated transcription factor), EDNRB (endothelin receptor type B), EDN3 (endothelin 3), SNAI2 (snail homolog 2, Drosophila) and SOX10 (SRY-box containing gene 10). Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder caused by mutations in the DMD gene. The purpose of this study was to identify the genetic causes of WS and DMD in an Indian family with two patients: one affected with WS and DMD, and another one affected with only WS. Methods: Blood samples were collected from individuals for genomic DNA isolation. To determine the linkage of this family to the eight known WS loci, microsatellite markers were selected from the candidate regions and used to genotype the family. Exon-specific intronic primers for EDN3 were used to amplify and sequence DNA samples from affected individuals to detect mutations. A mutation in DMD was identified by multiplex PCR and multiplex ligation-dependent probe amplification method using exon-specific probes. Results: Pedigree analysis suggested segregation of WS as an autosomal recessive trait in the family. Haplotype analysis suggested linkage of the family to the WS4B (EDN3) locus. DNA sequencing identified a novel missense mutation p.T98M in EDN3. A deletion mutation was identified in DMD. Conclusions: This study reports a novel missense mutation in EDN3 and a deletion mutation in DMD in the same Indian family. The present study will be helpful in genetic diagnosis of this family and increases the mutation spectrum of EDN3.

Genetic analysis of Indian tasar silkmoth (Antheraea mylitta) populations

Indian tasar silkmoth, Antheraea mylitta is an economically important wild silkmoth species distributed across India. A number of morphologically and ethologically well-defined ecotypes are known for this species that differ in their primary food plant specificity. Most of these ecotypes do not interbreed in nature, but are able to produce offspring under captive conditions. Microsatellite markers were developed for A. mylitta, and out of these, ten well-behaved microsatellite loci were used to analyze the population structure of different ecoraces. A total of 154 individual moths belonging to eight different ecoraces, were screened at each locus. Hierarchical analysis of population structure using Analysis of MOlecular VAriance (AMOVA) revealed significant structuring (F-ST = 0.154) and considerable inbreeding (F-IS = 0.505). A significant isolation by distance was also observed. The number of possible population clusters was investigated using distance method, Bayesian algorithm and self organization maps (SOM). The first two methods revealed two distinct clusters, whereas the SOM showed the different ecoraces not to be clearly differentiated. These results suggest that although there is a large degree of phenotypic variation among the different ecoraces of A. mylitta, genetically they are not very different, and the phenotypic differences may largely be a result of their respective ecology.

Limited genetic structure of Gulf Menhaden (Brevoortia patronus), as revealed by microsatellite markers developed for the genus Brevoortia (Clupeidae)

Long-term sustainable management of wild populations should be based on management actions that account for the genetic structure among populations. Knowledge of genetic structure and of the degree of demographic exchange between discreet [sic] populations allows managers to better define management units. However, adequate gene loci for population assessments are not always available. In this study, variable co-dominant DNA loci in the heavily exploited marine genus Brevoortia were developed with a microsatellite-enriched DNA library for the Gulf Menhaden (Brevoortia patronus). Microsatellite marker discovery was followed by genetic characterization of 4 endemic North American Brevoortia species, by using 14 novel loci as well as 5 previously described loci. Power analysis of these loci for use in species identification and genetic stock structure was used to assess their potential to improve the stock definition in the menhaden fishery of the Gulf of Mexico. These loci could be used to reliably identify menhaden species in the Gulf of Mexico with an estimated error rate of α=0.0001. Similarly, a power analysis completed on the basis of observed allele frequencies in Gulf Menhaden indicated that these markers can be used to detect very small levels of genetic divergence (Fst≈0.004) among simulated populations, with sample sizes as small as n=50 individuals. A cursory analysis of genetic structure among Gulf Menhaden sampled throughout the Gulf of Mexico indicated limited genetic structure among sampling locations, although the available sampling did not reach the target number (n=50) necessary to detect minimal values of significant structure.

Population structure of king mackerel (Scomberomorus cavalla) around peninsular Florida, as revealed by microsatellite DNA

A total of 1006 king mackerel (Scomberomorus cavalla) representing 20 discrete samples collected between 1996 and 1998 along the east (Atlantic) and west (Gulf) coasts of Florida and the Florida Keys were assayed for allelic variation at seven nuclear-encoded microsatellites. No significant deviations from Hardy-Weinberg equilibrium expectations were found for six of the microsatellites, and genotypes at all microsatellites were independent. Allele distributions at each microsatellite were independent of sex and age of individuals. Homogeneity tests of spatial distributions of alleles at the microsatellites revealed two weakly divergent “genetic” subpopulations or stocks of king mackerel in Florida waters—one along the Atlantic coast and one along the Gulf coast. Homogeneity tests of allele distributions when samples were pooled along seasonal (temporal) boundaries, consistent with the temporal boundaries used currently for stock assessment and allocation of the king mackerel resource, were nonsignificant. The degree of genetic divergence between the two “genetic” stocks was small: on average, only 0.19% of the total genetic variance across all samples assayed occurred between the two regions. Cluster analysis, assignment tests, and spatial autocorrelation analysis did not generate patterns that were consistent with either geographic or spatial-temporal boundaries. King mackerel sampled from the Florida Keys could not be assigned unequivocally to either “genetic” stock. The genetic data were not consistent with current spatial-temporal boundaries employed in stock assessment and allocation of the king mackerel resource. The genetic differences between king mackerel in the Atlantic versus those in the Gulf most likely stem from reduced gene flow (migration) between the Atlantic and Gulf in relation to gene flow (migration) along the Atlantic and Gulf coasts of peninsular Florida. This difference is consistent with findings for other marine fishes where data indicate that the southern Florida peninsula serves (or has served) as a biogeographic boundary.