A new DNA extraction method for high-throughput marker analysis in a large-genome species such as Triticum aestivum

Gene mapping and marker‐assisted selection in complex, polyploid genomes still relies strongly on restriction fragment length polymorphism (RFLP) analysis, as conversion of RFLP to polymerase chain reaction (PCR) markers can be very difficult. DNA extraction in amounts suitable for RFLP analysis represents the most time‐consuming and labour‐intensive step in molecular marker analysis of plant populations. In this paper, a new flexible method for plant DNA extraction is presented. It allows a high‐throughput of samples in a short time without the need for freezing or lyophilizing the plant material. The method allows the isolation of genomic DNA with a yield of 100 μg for a minimal amount of 200 mg of leaf material. This is sufficient for work with large‐genome plant species such as hexaploid wheat, where 20 μg of genomic DNA are required for a single RFLP analysis.     

Resistance to soybean cyst nematode and molecular polymorphism in various sources of Peking soybean

Summary Cultivar Peking has been extensively used as a source of resistance to Race 3 and Race 5 of soybean cyst nematode, Heterodera glycines I., and Peking genes for resistance are present in a wide range of resistant soybean cultivars. Peking is also used as a host differential in the soybean cyst nematode race classification system. Thirteen Peking lines maintained in the USDA Soybean Germplasm Collection and in several breeding programs were surveyed using RFLP and RAPD markers for genetic characterization. Based on the molecular diversity combined with reaction to soybean cyst nematode, Peking genotypes from a common original source were identified. Peking lines PI 297543 (introduction from Hungary), and PI 438496A, PI 438496B and PI 438496C (introductions from Russia) represented unrelated germplasms. Identified molecular polymorphism can be used to validate the genetic purity of Peking lines used as host differentials for soybean cyst nematode classification system as well as utilization of an individual germplasm line in genetic-breeding programs.     

Mitochondrial DNA variation in foxtail millet, Setaria italica (L.) P. Beauv.

Mitochondrial DNA (mtDNA) wascharacterized by RFLPs in 94 strains offoxtail millet, Setaria italica (L.)P. Beauv. Three RFLP patterns wereobserved by using rice atp6 as aprobe, and were designated as types I-III. Difference between types I and II seems tobe attributed to recombination between twoatp6 genes. In East and SoutheastAsia and Afghanistan, both of types I andII were found, while type I was predominantin India, Central Asia and Europe. InChina, type III was also found. Chinesestrains showed higher gene diversity thanthose from other regions. This resultcoincided the previous studies on isozymesand nuclear RFLPs.     

Mapping of QTL controlling NIR predicted hot water extract and grain nitrogen content in a spring barley cross using marker-regression

A restriction fragment length polymorphism (RFLP) map constructed from 99 doubled haploid lines of a cross between two spring barley varieties (‘;Blenheim’בKym’) was used to map QTL controlling hot water extract and grain nitrogen content (predicted by analysis with near-infrared reflectance spectroscopy). Eight QTL affecting predicted hot water extract were identified by a marker-regression approach. The largest effects were found on chromosomes 3HL, associated with the denso dwarfing gene which is present in‘Blenheim’and conferred poorer predicted hot water extract quality, and 4HL. Other QTL were detected on chromosomes IHS. IHL. 2HS, 2HL. 5HL and 6HS. Analysis of single markers by analysis of variance detected an additional effect on chromosome 1H. Eight QTL affecting predicted grain nitrogen content were identified by marker-regression, on chromosomes 1HS, 1HL. 2HL. 5HS, 6H, 7HS and 7HL. There was also evidence for an additional QTL on chromosome 5HL. The positions of the grain nitrogen content QTL on 5HS and 5HL are comparable to QTL on wheat chromosomes 5A and 5D that affect grain protein content. The denso gene had no detectable effect on grain nitrogen content.     

A comparative analysis of the genetic relationships between rye cultivars using RFLP and RAPD markers

Summary The genetic similarities of eight closely related rye cultivars were estimated using two molecular marking techniques: restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD). Cultivars were evaluated for variation by 11 random cDNA and genomic clones used in combination with four restriction enzymes and 40 decamer primers. A total of 53 polymorphic RFLP fragments and 94 polymorphic RAPD fragments were observed. Based on the presence/absence of fragments, two genetic similarity matrices were calculated which were then used in cluster analysis. Differences between pair of cultivars were observed in RFLP and RAPD dendrograms. RFLP analysis produced estimates of genetic relationships more in accordance with the partially known pedigree of the cultivars than did RAPD analysis. The use of bulk samples of DNA in these analyses affected the sensitivity of RAPD assays more strongly. Dendrograms which took into account all fragments produced, either by RFLP or RAPD, reflected better the relationships between cultivars than did dendrograms based on only one type of marker. This reflects the importance of the number of markers used in determining the genetic relationships between genotypes.     

Stability of fingerprints of Solanum tuberosum plants derived from conventional tubers and vitrotubers

The restriction and amplification patterns of potato genomic DNA of eight different cultivars and breeding clones has been studied. The analysis was carried out by polyacrylamide high resolution gels, and subsequent blotting and hybridization to potato DNA probes, and by agarose gel electrophoresis of the amplification products obtained with a variety of random primers. Fingerprinting of the genotypes was possible using two enzyme-probe combinations (Rsal-GP35 and Rsal-CP6) and three random primers (OPA4, OPA19 and OPG12). Based on the same techniques, a number of plants from the cvs. Monalisa and Spunta originated from the sprouting of in vitro-induced tubers (vitrotubers) were analysed to test the reproducibility of RFLP and PCR patterns. No variations were found with RFLP analysis. Some different RAPD patterns were seen, showing putatively vitrotuber-specific variations.     

水稻抗白叶枯病基因Xa23的RFLP标记定位及其STS标记的转化

用水稻抗白叶枯病基因Xa23的近等基因系CBB23与其感病轮回亲本金刚30（JG30）杂交，构建了包含2562个单株的F₂作图群体。用水稻白叶枯病广致病菌系P6进行抗性鉴定表明，F₂植株抗感分离比严格符合3：1。根据日本水稻基因组计划RGP水稻高密度图谱上的RFLP探针对F₂群体中的145个感病单株进行RFLP检测和连锁分析，获得了6个与Xa23紧密连锁的RFLP分子标记。其中RFLP标记C1003A靠着丝粒一侧，与Xa23的遗传图距为0.4cM，为Xa23的图位克隆奠定了重要基础。并将标记C1003A成功地转化为STS标记，为分子标记辅助选择育种（MAS）提供了方便有效的分子标记。     

4个绵羊品种褪黑激素受体1a基因第二外显子PCR-RFLP分析

采用2种限制性内切核酸酶MnlI和只Rsu I，对常年发情的小尾寒羊和湖羊以及季节性发情的多赛特羊和萨福克羊共146只绵羊褪黑激素受体1a(MTNRlA)基因外显子2的824bp扩增产物进行PCR-RFLP分析。结果表明：1)MTNRlA基因外显子2的605位碱基处表现出MnlI酶切多态性。小尾寒羊、萨福克羊和湖羊只出现2种基因型：AB(303bp，236bp／67bp)、1313(236bp／67bp，236bp／67bp)：多赛特羊出现3种基因型：AA(303bp，303bp)、AB(303bp，236bp／67bp)、BB(236bp／67bp，236bp／67bp)。2)MTNR1A基因外显子2的604位碱基处表现出只Rsa I酶切多态性。4个绵羊品种都出现3种基因型：AA(290bp，290bp)、AB(290bp，267bp／23bp)、BB(267bp／23bp，267bp／23bp)。X^2适合性检验结果表明，小尾寒羊和多赛特羊MTNRlA基因第二外显子在M22ZI酶切位点没有达到Hardy-Weinberg平衡状态(0．01＜P＜0．05)，萨福克羊和湖羊MTNRlA基因第二外显子在RsaI酶切位点已经达到Hardy—Weinberg平衡状态(P＞0．05)；4个绵羊品种MTNRlA基因第二外显子在只Rsa I酶切位点都已经达到Hardy-Weinberg平衡状态(P＞0．05)。     

玉米C型胞质不育恢复基因Rf4的RFLP定位

采用田间育性鉴定和RFLP分子标记的方法对玉米C型胞质不育的恢复基因进行了研究 .田间育性结果表明 ,玉米C型胞质不育有两对重叠恢复基因 ,通过对A192近等基因系和 (CMS C2 37×A6 19)BC1分离群体的RFLP分子标记 ,将玉米的C型胞质不育恢复基因Rf4定位在第 8染色体短臂上 ,与RFLP探针npi2 2 0和csu2 9连锁     

玉米分子标记技术的研究进展

主要介绍了AFLP、RAPD、RFLP和SSR分子标记技术在玉米育种中的应用，以及近年来分子标记技术在玉米遗传多样性，QTL定位等研究方面的应用。应用分子标记技术，不仅对玉米种质资源的发现、鉴定、分类整理和开发利用有一定作用，而且能进一步挖掘与玉米高产、优质高抗有关的基因，从而进行基因的定位和标记，提高玉米育种的效率。同时运用该技术对玉米抗逆性基因进行精确定位，为这些抗逆基因的克隆、转化及利用打下良好的基础，为分子标记辅助选择育种提供了条件，加快育种进程。