Development of EST‐SSR markers for diversity and breeding studies in opium poppy

All publicly available opium poppy expressed sequence tag (EST) sequences, totalling 20 885, were assembled into unigenes and examined for simple sequence repeats (SSRs). Nearly 19% of the 14 957 unigenes contained SSRs with 4% harbouring more than one SSR. Average density of the SSRs was 1 SSR per 3.6 kb of non‐redundant EST sequence. Trinucleotide SSRs were most frequently identified (39%), and many of the most prevalent motifs were AT‐rich. Flanking primers were designed for 86% of the SSRs and 67 primer pairs were tested on 37 opium poppy accessions and seven related species. All markers were transferable to the related species. Polymorphism information content (PIC) values for the markers were intermediate for comparisons within opium poppy (average of 0.27) and slightly higher for comparisons across species (average of 0.29). The markers were found to be useful for diversity analysis as they successfully distinguished among Turkish opium poppy accessions and land races.     

东北地区东方蜜蜂遗传分化和多样性分析

调查我国东北地区东方蜜蜂Apis cerana,分析其遗传分化并探讨隔离机制,明确东北东方蜜蜂种群遗传特征和多样性水平,探究蜜蜂种群遗传分化和遗传多样性相关规律,建立并完善东方蜜蜂种群遗传学。利用33个形态标记和38个微卫星DNA标记,对东北7个样点共461群东方蜜蜂及北京平谷、浙江金华、四川平昌、海南儋州外群作遗传分化、遗传特征和遗传多样性分析。形态标记、微卫星DNA标记分析结果均显示,分布于长白山山脉抚松、安图、敦化、本溪、宽甸东方蜜蜂聚为同一种群。长白山种群肘脉指数、翅长、第五背板绒毛带长、第四腹板长等形态指标数值较高。微卫星各位点等位基因以1、2种为主,频率达80%以上的21个。遗传多样性结果显示,长白山种群的观察杂合度、有效等位基因数、多态信息含量等数值较低。东北地区东方蜜蜂主要分布在吉林、辽宁长白山山区,形成长白山东方蜜蜂种群。该种群与北京、四川、浙江、海南东方蜜蜂发生明显遗传分化,遗传多样性水平较低。分化主要原因为辽宁省长白山以西平原地区,东方蜜蜂逐渐消失,基因流阻断。长白山东方蜜蜂种群遗传多样性水平较低,环境和人为因素导致种群数量减少。     

Low level of polymorphism detected by SSR probes in bread wheat

In-gel hybridization patterns were studied in a set of nine diverse bread wheat ( Triticum aestivum L. em. Thell) genotypes using 23 simple sequence repeat (SSR) probes in combination with 14 different restriction enzymes. Multilocus fingerprints due to SSR probes, shown earlier to be characteristic of a majority of plant genomes, were not obtained and only a very low level of polymorphism was detected when using as many as 142 probe-enzyme combinations. The hybridization of a prominent solitary high molecular weight fragment (> 23 kb) with a number of SSR probes suggested the presence of these SSRs (microsatellites) within the long stretches of repeated DNA sequences. This indicates that the genome of bread wheat differs from that of other plants in the organization and distribution of SSRs and that SSR probes detect very little polymorphism.     

Genetic diversity and population structure of burbot Lota lota in Germany: Implications for conservation and management

The genetic structure of the gadiform fish species, burbot Lota lota L., was investigated across Germany to derive management options for facilitating the preservation of genetic diversity. Sequence analysis of the mitochondrial control region (n = 244) and microsatellite analysis (n = 861) of specimens from 20 sites revealed genetic structuring between major river basins, and particularly between lake and river habitats. The admixture zone between the Eurasian and West European phylogenetic clades in Lake Constance was confirmed and expanded to include the drainage basins of the rivers Rhine and Schlei/Trave. Haplotype distribution and private haplotypes in single river basins indicated population differentiation and imply that German burbot constituted an important part of the entire species' diversity. The derived genetic structuring has implications for future stocking programmes and the preservation of the adaptive potential of burbot, a guiding species for oligotrophic lakes in Europe.     

Genetic relationships and hybrid vigour in olive (Olea europaea L.) by microsatellites

The olive (Olea europaea) is one of the most important oleaginous crops of the Mediterranean basin. Increased demand for olive oil creates a need for new olive varieties to help meet the requirements of the global market. However, olive breeding has been handicapped by such varied challenges as a prolonged juvenile period, agrotechnical problems and insufficient genetic knowledge. The use of DNA markers has the potential to overcome these problems and increase the effectiveness of classical breeding programmes. In this study, co‐dominant polymorphic simple sequence repeats (SSRs) were used as markers to analyse the genetic relationships between several local and other ‘non‐native’ olive cultivars. Cluster analysis revealed four major groups among the 15 cultivars examined in this study. Table and oil cultivars were clustered in different groups. However, the clusters did not differentiate between cultivars of different geographical origins. In addition, we used the data gathered to analyse genetic relationships to evaluate the effects of heterosis in agricultural traits. Genetic distances between cultivars were determined based on the SSR genotype data and were used for evaluating the possible effects of heterosis in various F₁ populations. Interestingly, phenotypic data of F₁ progenies from crosses between different cultivars indicated the potential effects of heterosis as expressed in several traits. Genetic distance between parents was significantly correlated to F₁ performance for three traits: percentage of dry fruit weight, oil content and commercial oil production. Thus, crosses between olive cultivars exhibiting relatively extensive genetic distances one from the other are expected to result in better progeny performance in future Olea breeding programmes. Our study linked assessment of biodiversity of commercial olive cultivars with the application of this information in olive breeding programmes for selection of specific parents to generate superior new cultivars.     

Use of PCR-based methodologies for the determination of DNA diversity between Saccharum varieties

Summary In this study, two PCR-based methodologies were evaluated for potential use in the determination of DNA diversity between 20 commercial sugarcane hybrids and 6 outgroup varieties of S. spontaneum, S. officinarum and hybrids from early in the genealogy. The first method involved PCR amplification of sugarcane DNA in the presence of random, decamer primers (RAPDs), while the second protocol utilized specific microsatellite and telomere sequences as primers. A total of 41 RAPD primers (356 loci) were screened across the varieties of which 15 (160 loci) were used in the calculation of DNA diversity (expressed% similarity). This varied from 61 to 95%, with most of the commercial varieties showing more than 80% similarity in their DNA. The RAPD data indicated that there had been a gradual decline in DNA diversity (84% reduction) from the early inter-specific crosses to the commercial hybrids, probably as a result of backcrossing and in-breeding strategies used in the previous 5 to 6 generations of sugarcane breeding. The microsatellite and telomere data produced a much greater range in DNA similarity values (25–91%), probably due to the fact that these primers detect highly variable regions of the genome. It is suggested that these specific primers would not be suitable for determination of DNA diversity, but could be used more effectively in the development of a methodology for routine, rapid identification of sugarcane varieties.     

Population structure,genetic diversity,and sexual state of the rice brown spot pathogen Bipolaris oryzae from three Asian countries

Bipolaris oryzae causes brown spot in rice (Oryza sativa) inflicting substantial grain yield losses worldwide. Knowledge of the population structure, genetic diversity and sexual recombination of the fungal pathogen can help to implement effective disease management strategies. In this study, B. oryzae isolates sampled from Iran, the Philippines and Japan were analysed with 12 simple‐sequence repeat (SSR) markers, newly developed from the genome sequence of the fungus. Among the 288 B. oryzae isolates genotyped, 278 unique haplotypes were identified. High genotype numbers (richness) with even distribution (evenness) were found within the collection sites. Both mating types, MAT1‐1 and MAT1‐2, were present in each collection area, and the sexual state was induced under controlled conditions with production of viable ascospores. However, the tests of linkage disequilibrium rejected of the hypothesis of random mating. Discriminant analysis of principal components (DAPC) revealed that the B. oryzae collection formed three clusters, each consisting of isolates from different collection sites. Analysis of molecular variance (amova ) showed that genetic variation among clusters was 18.7%, with the rest of the variation distributed within clusters (R_ST = 0.187, P < 0.001). Statistically significant pairwise genetic differentiation was found between the clusters. These results show that Asian B. oryzae isolates are genetically diverse, and, overall, distributed in three groups. These findings will be helpful in managing the disease and guide the use of representative isolates needed for selection of resistant rice varieties.     

The potential of microsatellites for high throughput genetic diversity assessment in wheat and barley

Microsatellite (SSR) profiles from 65 wheats and 135 barleys have been generated, involving 14 and 22 loci, respectively. The wheat and barley varieties were chosen to represent the bulk of the area sown to these crops in the UK over the past 70 years. The profiling identified genotypic mixtures in some seed samples. Null alleles were common in wheat, but rare in barley. We describe attempts to increase the efficiency of data acquisition. High resolution agarose gel electrophoresis was unable to satisfactorily resolve 1–2 repeat unit differences in the common size range for SSR loci, and was therefore unsuitable for mass screening of allelic variants. Multiplex PCR was very dependent on the choice of primer combinations and seldom produced amplifications as consistently as when primer pairs were used individually. Background (non-specific) amplification was common to many primer pairs, and this hindered the use of both multiplex PCR and multiple sample loading. Sequential sample loading was the most effective strategy, although this was the least time-efficient of the measures used.     

Application of microsatellites from maize to teosinte and other relatives of maize

Teosinte comprises different Zea species (Zea mays, Zea diploperennis, Zea perennis, Zea luxurians) that can be crossed with cultivated maize (Z. mays ssp. mays). Nine microsatellites from maize were applied to different teosinte species in order to evaluate their usefulness in markerbased exploitation of these genetic resources. The same microsatellites were tested with rye, barley, and sorghum as potential molecular markers for these species. Almost all microsatellite × teosinte combinations yielded polymerase chain reaction (PCR) fragments in the range of cultivated maize. Using an F₂ population of a cross between maize inbred A188 and an individual of Zea mays ssp. mexicana, amplification products for maize and teosinte originated from the same genomic location for each of nine microsatellites investigated. PCR fragments of reduced intensity were generally obtained by applying maize microsatellites to rye, barley and sorghum. Polymorphisms among accessions within teosinte (sub)species occurred frequently. In contrast, no polymorphisms were obtained within rye, barley, and sorghum. Hence, application of maize microsatellites to teosinte for fingerprinting or marker-assisted introgression of genomic regions from teosinte into cultivated maize appears promising.