An improved semiautomated rapid method of extracting genomic DNA for molecular marker analysis in cocoa,<Emphasis Type="Italic">Theobroma cacao</Emphasis> L

DNA extraction is a time-consuming and expensive component of molecular marker analysis, constituting about 30–60% of the total time required for sample processing. Furthermore, the procedure for extracting high-quality DNA from tree species such as cocoa differs from extraction protocols suitable for other crop plants. This is accompanied by problems in collecting leaf tissues from field-grown cocoa trees, where storage facilities are not available and where transporting samples to laboratory for immediate refrigeration is usually impossible. We preserved cocoa leaf tissues in the field in an NaCl-CTAB-azide solution (as described in Rogstad, 1992), which did not require immediate refrigeration. This method also allowed preservation of leaf tissues for a few days during transportation and protected leaf tissues from bacterial and fungal attacks. Once transported to the laboratory, the samples were stored at 4°C for almost 1 y. To isolate good-quality DNA from stored leaf tissues, a rapid semiautomated and relatively high-throughput protocol was established. The procedure followed a modified CTAB/β-mercaptoethanol method of DNA extraction in a 96-well plate, and an automated system (i.e., GenoGrinder 2000) was used to grind the leaf tissues. The quality of DNA was not affected by long storage, and the quantity obtained per sample was adequate for about 1000 PCR reactions. Thus, this method allowed isolation of about 200 samples per day at a cost of $0.60 per sample and is a relatively high-throughput, low-cost extraction compared with conventional methods that use manual grinding and/or expensive kits.     

长江春大豆核心种质构建及分析

利用长江春大豆初选核心种质SSR(simple sequence repeat）标记和农艺性状表型等基础数据,对用不同个体取样方法以及不同数据类型建立的核心种质进行评价,目的是确定中国大豆（Glycine max）核心种质的最佳取样策略提供依据,结果表明,根据SSR分子数据聚类,采用类内随机取样,类内以遗传相似性系数取样以及仅依据遗传相似性系数取样都可用于大豆核心种质构建,但是综合不同评价参数发现,以类内随机取样最佳,类内按遗传相似性系数取样次之,单独以遗传相似性系数取样较差。分析不同SSR等位变异保留比例的遗传多样性指数发现,当保留90%和80%的SSR等位变异时,核心种质具有更高的遗传多样性,由于与SSR分子数据种质遗传关系评价的不一致性,农艺性状等基础数据虽然可用来构建核心种质,但其SSR分子水平代表性相对较低,本研究结果还表明,用不同方法或同一方法不同重复次数取样建立的核心种质具有异质性,且这种异质性随核心种质取样比例的降低而增大,因此,虽然可依据不同数据类型确定相应的方法建立核心种质,但综合表型和分子数据建立的核心种质更具有代表性。     

Assessing wheat (Triticum aestivum L.) genetic diversity using quality traits, amplified fragment length polymorphisms, simple sequence repeats and proteome analysis

The genetic diversity among 10 Iranian bread wheat (Triticum aestivum) genotypes was analysed using 12 quality traits, 320 amplified fragment length polymorphisms (AFLP) polymorphic fragments, 491 simple sequence repeats (SSR) alleles and 294 proteome markers. The results revealed that the genotypes differed for quality traits, AFLP, SSR and proteome markers. The average genetic diversity based on quality traits (0.684 with a range of 0.266–0.997) was higher than AFLP (0.502 with a range of 0.328–0.717), SSR (0.503 with a range of 0.409–0.595) and proteome (0.464 with a range of 0.264–0.870) markers. Although there were apparent similarities between the groupings of particular genotypes, the overall correspondence between the distance matrices appeared to be rather low. In this study, the cluster analysis based on AFLP data showed the closest agreement with genotypes’ regions of origin or pedigree information. In addition to the genetic diversity assessment, specific proteins with known function were detected uniquely for the studied genotypes. Our results suggest that the classification based on quality traits and genotypic markers of these wheat genotypes will be useful for wheat breeders to plan crosses for positive traits.     

Development of simple sequence repeat (SSR) markers for the assessment of gene flow and genetic diversity in pigeonpea (Cajanus cajan)

Pigeonpea (Cajanus cajan) is an important subsistence crop in India where traditional landraces and improved hybrids are grown alongside each other. Gene flow may result in genetic erosion of these landraces and their wild relatives, whilst transgene escape from future genetically engineered varieties is another potential hazard. To assess the impact of these factors gene flow needs to be measured. A set of 10 simple sequence repeat markers have been developed, which exhibit polymorphism across a range of pigeonpea varieties. Use of these markers also offers an efficient system for the assessment of genetic diversity within populations of pigeonpea.     

大豆SSR技术研究进展

微卫星DNA是重复单位少于 6个核苷酸的简单重复序列。在大部分真核细胞的基因组中有着广泛分布 ,呈孟德尔式遗传。以此为基础发展起来的SSR标记是一种共显性分子标记 ,遗传多态性丰富。本文着重介绍近年来SSR技术应用在大豆遗传图谱构建、基因研究、品种鉴定和分子标记辅助育种等方面取得的进展 ,并初步预测该方法在大豆研究中的发展方向。     

Development and characterization of microsatellite markers in Cucumis

This study provides a set of useful SSR markers and describes their development, characterization and application for diversity studies.Sixty one Cucumis SSR markers were developed, most of them (46) from melon (Cucumis melo L.) genomic libraries. Forty of the markers (30 melon and 10 cucumber SSRs) were evaluated for length polymorphism in a sample of 13 melon genotypes and 11 cucumber (Cucumis sativus L.) genotypes. PCR-amplification revealed up to six size alleles among the melon genotypes and up to five alleles among the cucumber genotypes, with mean gene-diversity values of 0.52 and 0.28 for melon and cucumber, respectively. These differences are in accordance with the known narrower genetic background of the cucumber. SSR data were applied to phylogenetic analysis among the melon and cucumber genotypes. A clear distinction between the ’exotic’ groups and the sweet cultivated groups was demonstrated in melon. In cucumber, separation between the two sub-species, C.sativus var. sativus and C.sativus var. hardwickii,was obtained. Conservation of SSR loci between melon and cucumber was proven by sequence comparisons. Received: 17 April 2000 / Accepted: 16 May 2000     

Use of microsatellites to evaluate genetic diversity and species relationships in the genus Lycopersicon

In order to determine how informative a set of microsatellites from tomato is across the genus Lycopersicon, 17 microsatellite loci, derived from regions in and around genes, were tested on 31 accessions comprising the nine species of the genus. The microsatellite polymorphisms were used to estimate the distribution of diversity throughout the genus and to evaluate the efficacy of microsatellites for establishing species relationships in comparison with existing phylogeny reconstructions. Gene diversity and genetic distances were calculated. A high level of polymorphism was found, as well as a large number of alleles unique for species. The level of polymorphism detected with the microsatellite loci within and among species was highly correlated with the respective mating systems, cross-pollinating species having a significantly higher gene diversity compared to self-pollinating species. In general, microsatellite-based trees were consistent with a published RFLP-based dendrogram as well as with a published classification based on morphology and the mating system. A tree constructed with low-polymorphic loci (gene diversity <0.245) was shown to represent a more-reliable topology than a tree constructed with more-highly polymorphic loci. Received: 19 February 2001 / Accepted: 26 March 2001     

Seed quality QTLs identified in a molecular map of early maturing soybean

This study identified QTLs influencing seed quality characters in a cross of two early maturing soybean (Glycine max [L.] Merr.) cultivars (Ma.Belle and Proto) adapted to the short growing seasons of Central Europe. A molecular linkage map was constructed by using 113 SSR, 6 RAPD and 1 RFLP markers segregating in 82 individuals of an F₂ population. The map consists of 23 linkage groups and corresponds wellto previously published soybean maps. Using phenotypic data of the F₂-derived lines grown in five environments, four markers for protein content, three for oil content and eight for seed weight were identified. Four from fifteen seed quality QTL-regions identified in the present study were also found by other authors. Markers associated with seed weight QTLs were consistent across all environments and proved to have effects large enough to be useful in a marker-assisted breeding program, whereas protein and oil QTLs showed environmental interactions. Received: 9 October 2000 / Accepted: 26 February 2001     

应用红外荧光和加尾引物法进行向日葵SSR遗传分析中的多聚PCR和多聚凝胶电泳的优化

在向日葵(Helianthus annuus L.)自交系SSR遗传分析中,为了提高SSR荧光分析效率、简化分析程序和降低研究费用,我们进行了多聚PCR和多聚凝胶电泳两项技术的优化研究。结果表明,优化多聚PCR和多聚凝胶电泳的影响因子(如逐步降低的退火温度的循环数、各个多聚位点引物浓度的平衡、PCR缓冲液浓度以及Taq DNA多聚酶的浓度等)可以收到更好的实验效果。基于以上的优化研究,系统地提出了一套优化的加尾引物法的策略。同时,提出了在多聚PCR和多聚凝胶电泳中常常遇到的技术难题的有效防止和解决的方法。     

A high-throughput plant DNA extraction method for marker analysis

The use of molecular markers to improve crops depends on the availability of rapid and efficient DNA extraction methods. Here we describe a simple and inexpensive method to isolate plant DNA suitable for RFLP, AFLP, and simple sequence repeat (SSR) analysis. This procedure uses stainless steel ball bearings to grind 16 samples simultaneously using a high-speed flask shaker. The method used in routine laboratory exercises yields 120–144 DNA extractions in a day by a single person at a cost of $0.60 (AUD) per sample, doubling the throughput of conventional methods.