Cyclic genotyping strategies. III: A comparison of predictive methods for group genotyping

This paper provides an investigation into some of the key practical issues for minimizing the cost of DNA testing. Previous studies focused on maximizing the utility of genotyping by prioritizing individuals for genotyping. For logistical reasons, individuals may have to be genotyped in groups rather than individually, and the best group to genotype is expected to differ from the same-sized group chosen when individuals are genotyped sequentially. In a calibration step, simulated populations and full knowledge of genotypes were used to discover the best group(s) to genotype. The characteristics of these groups were then targeted in an optimization step, using normally available information for group formation in targeted populations. Contrasts were made among predictive indices for: (i) individuals, with genotyping between each individual; (ii) individuals, with genotyping occurring group-at-a-time; and (iii) groups, using group variables as criteria. The results of this investigation allow the determination of the value of moving from individual to group genotyping, reveal the favourable attributes of individuals for group formation, and lead to methods to form groups for genotyping. The approach used has applications in reducing genotyping costs in both experimental and commercial populations for both quantitative trait loci (QTL) detection and monitoring.     

Comparison of genotyping by sequencing and microsatellite markers for unravelling population structure in the clonal fungus Verticillium dahliae

Microsatellite genotyping of a large sample of isolates of Verticillium dahliae from diverse locations recently identified seven distinct genotypic clusters. However, these clusters were not put in the context of phenotypes known to be correlated with clonal lineages in V. dahliae. The objective of this study was to compare clusters defined by microsatellite markers with clonal lineages defined by single‐nucleotide polymorphisms (SNPs) and vegetative compatibility groups (VCGs). Genotyping isolates known to belong to specific clonal lineages (based on SNPs) with microsatellite markers determined the correspondence of clusters and lineages. All but one cluster corresponded to a known clonal lineage, allowing analysis of correlations of phenotypes with microsatellite genotypes from other studies. As shown previously, most race 1 isolates are in lineage 2A, and most isolates with the defoliating pathotype are in lineage 1A. Phylogenetic incompatibility was used to test for recombination or homoplasy caused by hypervariable microsatellite loci; incompatibility was highly correlated with the number of alleles per locus, suggesting that homoplasy caused by parallel evolution of microsatellite alleles is the cause of incompatibility. Microsatellite genotyping of lineage 1A isolates from cotton and olive in Spain over a 29‐year period revealed remarkably little variation; these markers did not mutate enough to provide insight on the spatial and temporal expansion of this clone. Overall, this study showed that microsatellite genotyping can be used to identify clonal lineages in V. dahliae, which has predictive power for inferring phenotypes of phytopathological relevance such as race and pathotype.     

基于PFGE技术的酸乳中乳酸菌的基因分型

采用选择性培养基分离酸乳中的保加利亚乳杆菌和嗜热链球菌,并利用生理生化和糖发酵实验结合16S rDNA同源性分析对分离所得菌株进行鉴定,获得保加利亚乳杆菌和嗜热链球菌分离菌株。利用脉冲场凝胶电泳（pulsed field gel electrophoresis,PFGE）分别对保加利亚乳杆菌和嗜热链球菌分离株进行基因分型。结果表明：所检测的酸乳样品中均含有保加利亚乳杆菌和嗜热链球菌;仅有2 种酸乳的嗜热链球菌为同一菌株,其余酸乳中的保加利亚乳杆菌及嗜热链球菌均为不同菌株。PFGE可以对保加利亚乳杆菌和嗜热     

A high‐resolution einkorn (Triticum monococcum L.) linkage map involving wild,domesticated and feral einkorn genotypes

A high‐resolution consensus linkage map of Triticum monococcum was assembled from two separate maps involving domesticated, feral and wild einkorn wheat accessions. The genotyping‐by‐sequencing (GBS) approach based on DArTseq markers yielded overstretched maps. Deleting all markers with missing data and then converting dubious singletons to missing data produced two maps of about 1,380 cM, close to the published genome size. The consensus map spanned 1,562 cM, had one bin mapped every 0.92 cM and showed only one gap > 10 cM. Chromosome length varied between 151 cM (chromosome 4) and 270 cM (chromosome 7). The consensus map was compared to other A‐genome maps, and the sequences of genetically mapped DArTseq were used to anchor contigs of the T. monococcum, T. urartu and T. aestivum draft genomes based on sequence homology to assess colinearity and to assign mapped markers to the seven chromosomes of the bread wheat A‐genome. Finally, an in silico functional characterization of the sequences was performed. This high‐resolution map will facilitate QTL and association analysis and assist the genome assembly of the einkorn genome.     

Genotyping by sequencing of 270 Indica rice varieties revealed genetic markers probably related to heavy metal accumulation

Genotyping by sequencing (GBS) has been applied to identify genetic markers in crops for trait association and breeding purposes. Here, we applied GBS technology to study a natural population of 270 Indica rice strains, which resulted in identification of 79,545 genomewide single nucleotide polymorphisms. Using these SNPs, we found the close relationship between the 270 Indica rice strains. Furthermore, we tested the feasibility of using these 270 Indica strains in studying important rice traits by analysing the cadmium and other metal accumulation of these strains and correlating the traits with genetic markers. We identified 32 SNPs to be associated with cadmium (Cd) accumulation, explaining 61.25% of the Cd concentration variances in grains. The genetic markers provided here are valuable resources for future rice studies, and further characterization of the candidate loci identified in this study can also aid the development of low Cd‐accumulating rice varieties.     

SNP检测方法在动物研究中的应用

单核苷酸多态性(SNP)作为第三代分子标记技术,以其数量丰富、遗传稳定性高、易于快速自动化检测等优点备受关注。该文综述了基于不同原理的SNP分型检测方法,包括几种常见的测序技术、基于酶学及杂交原理的分析检测技术和基于色谱及质谱的相关技术。阐释了方法的原理、分析了方法的优缺点及适用范围。总结了目前在动物遗传育种、亲缘鉴定、动物品种及产品溯源等方面的应用研究现状。为下一步开发更灵敏准确、简便易行、高通量及低成本的SNP检测方法及拓展SNP的应用领域提供参考。     

Genetic variability of Fusarium fujikuroi populations associated with bakanae of rice in Italy

The heterothallic ascomycete Fusarium fujikuroi (teleomorph: Gibberella fujikuroi) is the causal agent of bakanae of rice, a disease of increasing economic importance in the major rice‐producing areas in the world and a serious threat for Italian rice cultivation. A few studies have characterized F. fujikuroi isolates in America and the Philippines but no data are available on the genetic structure of Italian pathogen populations. Microsatellite SSRs are useful tools to study the intraspecific diversity at population level. In this study, 19 polymorphic SSRs have been identified and applied to characterize the genetic variation of 334 isolates of F. fujikuroi coming from eight Italian rice‐growing areas. A high degree of diversity at haplotype level has emerged: in the eight populations, 107 unique haplotypes were scored. Analysis of molecular variance (amova ) showed that 98% of genetic variability occurred within F. fujikuroi Italian populations, as confirmed by the allelic Shannon index ranging from 0.56 to 1.06. The presence of a 1:1 ratio of mating type alleles in six out of eight of the Italian fungal populations suggests a potential for sexual reproduction in the field. However, the high fraction of clonality (43%), confirmed by neighbour‐joining clustering analysis, and the high level of linkage disequilibrium observed, indicates that reproduction of F. fujikuroi is mostly clonal in Italy. All data suggest that the observed genetic variability was probably mediated by human activity and transmission by rice seeds.     

Founder representation and effective population size in old versus young breeds—genetic diversity of Finnish and Nordic Spitz

Finnish Spitz is 130‐year‐old breed and has been highly popular in Finland throughout its history. Nordic Spitz is very similar to Finnish Spitz by origin and use, but is a relatively recent breed with much smaller population size. To see how breed age and breeding history have influenced the current population, we performed comprehensive population genetic analysis using pedigree data of 28,119 Finnish and 9,009 Nordic Spitzes combined with genomewide single nucleotide polymorphism (SNP) data from 135 Finnish and 110 Nordic Spitzes. We found that the Finnish Spitz has undergone repeated male bottlenecks resulting in dramatic loss of genetic diversity, reflected by 20 effective founders (f_a) and mean heterozygosity (Hz) of 0.313. The realized effective population size in the breed based on pedigree analysis () is 168, whereas the genetic effective population size (N_eg) computed the decay of linkage disequilibrium (r²) is only 57 individuals. Nordic Spitz, although once been near extinction, has not been exposed to similar repeated bottlenecks than Finnish Spitz and had f_a of 27 individuals. However, due to the smaller total population size, the breed has also smaller effective population size than Finnish Spitz ( = 98 and N_eg = 49). Interestingly, the r² data show that the effective population size has contracted dramatically since the establishment of the breed, emphasizing the role of breed standards as constrains for the breeding population. Despite the small population size, Nordic Spitz still maintains SNP heterozygosity levels similar to mixed breed dogs (mean Hz = 0.409). Our study demonstrates that although pedigree analyses cannot provide estimates of the present diversity within a breed, the effective population sizes inferred from them correlate with the genotyping results. The genetic relationships of the northern Spitz breeds and the benefits of the open breed registry are discussed.     

Genetic populations of Bacillus anthracis isolates from Korea

Bacillus (B.) anthracis is the pathogen that causes fatal anthrax. Strain-specific detection of this bacterium using molecular approaches has enhanced our knowledge of microbial population genetics. In the present study, we employed molecular approaches including multiple-locus variable-number tandem repeat analysis (MLVA) and canonical single-nucleotide polymorphism (canSNP) analysis to perform molecular typing of B. anthracis strains isolated in Korea. According to the MLVA, 17 B. anthracis isolates were classified into A3a, A3b, and B1 clusters. The canSNP analyses subdivided the B. anthracis isolates into two of the three previously recognized major lineages (A and B). B. anthracis isolates from Korea were found to belong to four canSNP sub-groups (B.Br.001/2, A.Br.005/006, A.Br.001/002, and A.Br.Ames). The A.Br.001/002 and A.Br.Ames sub-lineages are closely related genotypes frequently found in central Asia and most isolates were. On the other hand, B. anthracis CH isolates were analyzed that belonged to the B.Br.001/002 sub-group which found in southern Africa, Europe and California (USA). B.Br.001/002 genotype is new lineage of B. anthracis in Korea that was not found before. This discovery will be helpful for the creation of marker systems and might be the result of human activity through the development of agriculture and increased international trade in Korea.     

Characterization of Methicillin‐Resistant Staphylococcus aureus Isolates from Pig Carcasses in Hong Kong

This study describes the isolation and characterization of methicillin‐resistant Staphylococcus aureus (MRSA) from slaughtered pigs sampled from local markets in Hong Kong. The nares of 400 slaughtered pigs were cultured and MRSA isolates characterized for the presence of antibiotic‐resistance determinants, toxins and SCCmec and spa types using PCR. Clonality was investigated using PFGE and MLST. The prevalence of MRSA colonization of slaughter pigs was 39.3%, the majority (92%) harbouring SCCmec type IVb. Of the 157 samples yielding MRSA, 13 had two distinct MRSA strains present. Spa type t899 was predominant, with only 5/170 isolates displaying closely related types (t4474, t1939, t2922 and t5390). PFGE with sma1 and MLST confirmed the strains as ST9. Most isolates were multidrug resistant. Tetracycline resistance (97%) was mainly attributable to tet(K) with only 3% of isolates additionally harbouring tet(M). Resistance to erythromycin (89%) and chloramphenicol (71%) was associated with the presence of erm(C), and fex( A), respectively. No strains carried cfr and there was no resistance to linezolid, although minimum inhibitory concentration (MICs) were close to the resistance break point. Resistance to clindamycin (99%), ciprofloxacin(78%), quinopristin–dalfopristin (44%) and cotrimoxazole (32%) was common, but remained low for fusidic acid (4%) and rifampicin (2%). All strains were negative for PVL, exfoliative, and enterotoxins. This survey confirmed the uniformity of MRSA isolates in pigs from several regions of China, in contrast to more diversified characteristics reported in European studies. Colonization rates were higher than previously reported. Isolates were resistant to a wide range of antibiotics, but resistance was not detected to linezolid, nitrofurantoin, vancomycin or tigecycline. Although the clinical importance of ST9 in humans is uncertain, continued surveillance, in particular of those occupationally‐exposed, is recommended.