Evaluation of 'quick and dirty' DNA extraction methods for marker-assisted selection in rice (Oryza sativa L.)

Six simple methods for extracting DNA from rice seedlings were evaluated for marker‐assisted selection (MAS). The assessment of each method was based on PCR amplification of SSR markers, DNA yield and purity, time and cost. Based on these criteria, two methods were selected as being superior to other methods. The best two methods included the standard method developed at the International Rice Research Institute (IRRI), which utilizes a sodium dodecyl sulfate extraction buffer followed by chloroform/isoamyl alcohol extraction and a previously published method using sodium hydroxide and Tris. These two methods produced nearly identical PCR amplification results. The sodium hydroxide method is considerably simpler, quicker and cheaper than the standard IRRI method, and may be particularly useful for many applications of MAS or high‐resolution mapping. This method was also adapted into an effective high throughput method utilizing 96‐well plates emphasizing its versatility.     

Molecular marker analysis of genetic variation characterizing a grasspea (Lathyrus sativus) collection from central Italy

Levels of genetic similarity characterizing 20 grasspea (Lathyrus sativus L.) populations collected in central Italy (17 populations in the Marche region and three populations in the Abruzzo region) were analysed with amplified fragment length polymorphism (AFLP) molecular markers. Two main clusters were found: one included large‐seeded populations from farms that were not market‐oriented (named Household populations) and the second, small‐seeded populations, cultivated in market‐oriented farms (named Commercial populations). Relationships among populations collected in different regions were found, although one population of the Abruzzo region was placed between the two main clusters, suggesting a possible further genetic differentiation within this grasspea germplasm collection. Principal component analysis based on AFLP marker frequency was effective in identifying polymorphic markers showing high discriminating ability between clusters H and C. In particular, seven markers showing high positive and three markers with low negative PC1 scores showed an almost cluster‐specific distribution. These results will be useful for enhancing Italian grasspea germplasm use in plant‐breeding programmes and for extending grasspea cultivation within the sustainable agricultural systems of central Italy.     

基于Glb 1基因序列的西南地区玉米地方品种的系统进化

为探讨我国西南地区玉米地方品种Glb 1基因序列多态性,对40份西南地区玉米地方品种的Glb 1基因序列测序,并与Gen Bank中来自普通玉米、小颖玉米、繁茂玉米和摩擦禾的19条同源序列比对。结果表明,来自西南地区四川、重庆、云南和贵州的玉米地方品种分别有35、25、33和25个多态性位点,其遗传多态性分别是小颖玉米的87%、86%、85%和71%,8个种群遗传多态性指数的大小排序为小颖玉米、四川地方品种、重庆地方品种、云南地方品种、贵州地方品种、普通玉米、磨擦禾和繁茂玉米,Tajima’D、FuLi’D和FuLi,F检验表明Glb 1基因是中性进化基因。比较种群间DNA序列的共有多态性和固定差异,玉米地方品种与小颖玉米、繁茂玉米、磨擦禾间存在较高的固定差异和较低的共有多态性。Glb 1基因的系统进化关系及网状关系分析为玉蜀黍属的分类系统提供了佐证,四川玉米地方品种有较高的遗传多态性支持我国玉米由印度经西藏传入四川的论点。     

水稻抗病基因同源序列多态性与品种鉴定

利用已知植物抗病基因不同保守序列设计的S1/AS3和XLRR for/XLRR rev两对引物对22个水稻品种DNA进行PCR扩增和聚丙烯酰胺凝胶电泳.分析结果表明水稻抗病基因同源序列类型丰富,2对引物共扩增出143条带,品种间有差异的谱带92条,根据谱带差异可将供试品种完全鉴别出来.证明抗病基因同源序列分析可以用于水稻品种鉴定.     

Ectomycorrhizal fungi identification in single and pooled root samples: terminal restriction fragment length polymorphism (TRFLP) and morphotyping compared

We describe here the results of a study conducted to evaluate a terminal restriction fragment length polymorphism (TRFLP) approach targeting rRNA genes for determination of ectomycorrhizal (ECM) communities colonizing the roots of loblolly pine (Pinus taeda L.). Root tips separated from soil cores were classified according to morphological characteristics and DNA was then extracted from each group of morphotyped tips. Labeled primers were used to generate terminal restriction fragments (TRF) for molecular fingerprinting of root colonizing fungi and to determine how well TRFLP could be used to discriminate between ectomycorrhizal types. Morphotypes generally correlated well with specific TRFs and sequence analysis confirmed that TRFs could be used to discriminate among fungal types. Sequence analysis indicated that important ECM fungi including Russulaceae, Thelephorales, and Tricholomataceae could be fingerprinted with TRFLP. In addition, a fixed proportion of the DNA extracted from each morphotype from the same core was used in a pooling experiment used to assess whether previously identified fungal species types could be distinguished from one another within reconstructed communities. Since some morphotypes share TRFs, dual analysis of ITS1 and ITS2 was necessary for accurate fingerprinting of fungal types. Approximately, 5.0±0.3 phylotypes were detected per core with TRFLP-corrected morphotyping as compared to 4.0±0.4 phylotypes using TRFLP on pooled community samples. TRFLP made on experimental sporocarp communities suggested that reduced ECM richness with TRFLP may be partly caused by differences in the amount of DNA available for PCR and primer bias. Nonetheless, TRFLP on pooled morphotypes accounted for more than 93% of colonized root tips. The method can be used to facilitate analysis of mycorrhizal communities using root tips collected from soil cores.     

中国部分板栗品种的SSR标记

从板栗(Castanea mollissima)燕红中分离到25个SSR标记。为了鉴定这些SSR位点,在重复序列的两侧侧翼序列设计引物,并通过化学荧光检测法对6个板栗样品进行检测,共检测到20个多态性位点,每个位点的等位基因数为2~6个。挑选12个位点,通过半自动系统ABI PRISM377对中国北方24个板栗品种进行分析,这12个标记显示了高达75个等位基因的遗传多态性,每个位点的等位基因为4~10个,平均为6.3个,预期的平均杂合性为0.743(介于0.680~0.845),观察值为0.829(介于0.730~0.930)。无效等位基因估算频率显示为3个位点的正向价值,除了一个位点外,这些价值都很低,鉴定几率为7.01×10-11,亲缘关系鉴定几率非常高,为0.999,足够用于花粉流的研究。     

牛类胰岛素生长因子结合蛋白3基因PCR-SSCP分析

采用PCR-SSCP技术分析了牛类胰岛素生长因子结合蛋白3(IGFBP3)基因在南阳牛、鲁西牛和中国西门塔尔牛3个牛品种中的遗传多态性。结果表明:IGFBP3的第3外显子不存在遗传多态性;第2外显子在3个牛品种中检测到了AA、AB和BB基因型,而且A等位基因为3个牛群体的优势等位基因,分布较高。3个品种中,中国西门塔尔牛AA基因型频率最高,达到0.6615,而鲁西牛和南阳牛则相对较低,分别为0.4043和0.3095。对第2外显子的多态片段测序分析表明:位于IGFBP3基因第8069bp处发生单碱基突变T→C,并导致了苯丙氨酸变为亮氨酸。     

Search for Polymorphism in Exon 2 of the Equine Leptin Gene

The aim of the current research was to investigate the possible occurrence of a single nucleotide polymorphism (SNP) in exon 2 of the equine leptin gene in obese mares determined to be hyperleptinemic. Three experiments were conducted: one to determine the prevalence of hyperleptinemic horses in the resident herd; another to complete the sequencing of exon 2 and flanking introns of the equine leptin gene, which had been partially sequenced by others; and a third to compare the exon 2 sequences of obese, hyperleptinemic mares with those of obese mares not displaying hyperleptinemia. In experiment 1, jugular blood was collected from 31 mares, and they were categorized by age, body condition score (BCS), and average plasma leptin concentrations. Mean BCS was correlated (P < .001) with leptin concentrations; age was not. Five obese, hyperleptinemic and five obese, nonhyperleptinemic mares were selected to study the possibility of polymorphism in exon 2. First, in experiment 2, forward and reverse primers were designed from the Bos taurus leptin gene (GenBank Accession # U50365) to identify and subsequently clone the equine leptin gene to provide material for sequencing. The multiple copies of genomic DNA were then used for sequencing of exon 2 and the flanking introns. Comparative genomics was used to further identify and characterize regulatory elements in the 5' and 3' flanking regions of the leptin gene. In experiment 3, DNA from the five mares of each type (previously selected in experiment 1) was extracted, and exon 2 was sequenced and analyzed for possible SNP. The sequences of exon 2 for the 10 mares were identical; thus, no polymorphism was present. It was concluded that approximately one third of obese, pasture-maintained mares and geldings in the resident herd display hyperleptinemia relative to other horses of similar body condition, but this condition was not associated with the occurrence of SNP in exon 2 of the leptin gene.     

中国荷斯坦牛GlyCAM1基因遗传多态性的研究

以273头中国荷斯坦牛为研究对象,利用CRS—PCR、PCR—SSCP及DNA测序技术检测了GlyCAM1基因外显子3、内含子3的遗传多态性。结果表明：GlyCAM1基因分别在外显子3和内含子3的第2081（A／C）、2417（C／T）位存在突变,2个位点的等位基因频率A／B分别为0．7525,0．2475和0．9046,0．0954;经菇。适合性检验,中国荷斯坦牛内含子3的突变达到Hardy—Weinberg平衡状态（P〉0．05）,但其外显子3的突变未达到Hardy—Weinberg平衡状态（P〈0．05）。