AFLP-Based detection of DNA methylation

By using the isoschizomersHpa II andMsp I which display differential sensitivity to cytosine methylation, a modified amplified fragment length polymorphism (AFLP) technique has been developed to investigate DNA methylation profiles in eukaryotic organims. Genomic DNA was digested with a mixture ofEcoR I and one of the isoschizomers, and ligated to oligonucleotide adapters. After two rounds of selective PCR amplification, followed by DNA separation on a Long Ranger gel electrophoresis, a subset of restriction fragments can be displayed on an X-ray film. Comparison of AFLP banding patterns betweenHpa II andMsp I revealed the extent of DNA methylation. The technique has been successfully applied in this study to investigate DNA methylation profiles of apple (Malus domestica cv. Gala) genomic DNA extracted from leaves of field-grown adult trees andin vitro-grown shoot cultures. The results showed that up to 25 percent of AFLP bands were derived from methylated sequences, and among those, a few bands unique to either adult trees orin vitro shoots were observed. These results demonstrated that this protocol is effective in identifying methylated DNA profiles. Both first authors have contributed equally to this work.     

华北地区小丛红景天种群的AFLP遗传多样性

利用扩增片段长度多态性(AFLP)标记, 对分布于华北地区5个山脉的25个小丛红景天(Rhodiola dumulosa)自然种群的776个样品进行了遗传多样性和遗传结构的研究。结果表明: 华北地区小丛红景天种群具有较高的遗传多样性, 4对选择扩增引物共扩增出398条清晰的条带, 其中多态带312条, 种群的平均多态位点百分率为78.46%, 种群总的Nei’s基因多样性为0.364 9, 总Shannon多态性信息指数为0.542 2。华北地区小丛红景天种群间的遗传分化系数G_st = 0.150 7, 基因流Nm = 2.817 9, 表明种群间遗传分化较低, 有一定的基因交流。AMOVA分析结果也表明: 华北地区小丛红景天的遗传变异主要存在于种群内, 地理单元间有一定的遗传分化, 而种群间的遗传分化较低。STRUCTURE的分析和UPGMA聚类分析结果一致, 结果显示地理分布距离相近的种群优先聚在一起。Mantel检验也进一步证实, 华北地区小丛红景天种群的遗传距离与地理距离间呈显著的正相关关系(r = 0.512 9, p < 0.001)。种群的遗传多样性与海拔呈显著的负相关关系(p < 0.05), 而与坡向没有显著相关性。用Dfdist软件分析海拔对遗传多样性的影响, 结果表明没有显著的受选择位点。     

基于果蝇DNA甲基化的AFLP体系的建立及优化

以黑腹果蝇(Drosophila melanogaster)Canton-S品系为材料,采用改良SDS法提取高质量DNA,对连接、预扩增及选择性扩增进行分析,建立适用于果蝇基因组DNA甲基化多态性研究的AFLP优化体系:1)10μL连接体系加T4连接酶1 U,AluⅠ接头50 pmol,EcoR Ⅰ接头5 pmol,4℃反应12 h;2)25μL预扩增体系含Mg2+0.2 mmol/L,dNTPs 0.15 mmol/L,模板1.0μL,Taq DNA聚合酶2 U,E-00 50 ng,A-00 50 ng;3)25μL选择性扩增体系含Mg2+0.1 mmol/L,dNTPs 0.15 mmol/L,模板2.0μL,Taq DNA酶1.5 U、E+340 ng,A+3 40 ng.该体系稳定性高、重复性好,适用于果蝇基因组DNA甲基化多态性研究.     

AFLPMax: a user-friendly application for computing the optimal number of amplified fragment length polymorphism markers needed in phylogenetic reconstruction

Amplified fragment length polymorphisms (AFLPs) are widely used for phylogenetic inference especially in non-model species. Frequently, trees obtained with other nuclear or mitochondrial markers or with morphological information need additional resolution, increased branch support, or independent data sources (i.e. unlinked loci). In such cases, the use of AFLPs is a quick and cheap option. Computer simulation has shown that dominant AFLP markers lead to less accurate tree topologies than bi-allelic codominant markers such as SNPs, but this difference becomes negligible for shallow trees when using AFLP data sets that include a sufficiently large number of characters. Thus, determining how many AFLP characters are required to recover a given phylogeny is a key issue regarding the appropriateness of AFLPs for phylogenetic reconstruction. Here, we present a user-friendly, java-based graphical interface, AFLPMax, which executes an automatic pipeline of different programs providing the user with the optimal number of AFLP characters needed to recover a given phylogeny with high accuracy and support. Executables for Windows, linux and MacOS X operating systems, source code and user manual are available from: http://webs.uvigo.es/acraaj/AFLPMax.htm.     

pms3 is the locus causing the original photoperiod-sensitive male sterility mutation of‘Nongken 58S'

Photoperiod-sensitive genic male sterile (PSGMS) rice is a very useful germplasm for hybrid rice development. It was first found as a spontaneous mutant in a japonica cultivar 'Nongken 58'. pms3 on chromosome 12 was determined to be the locus where the original PSGMS mutation occurred, changing the normal cultivar Nongken 58 to PSGMS Nongken 58S. Large amounts of RAPD and AFLP analyses were also conducted for the fine mapping of the pms3 genomic region, which resulted in 4 molecular markers linked to pms3. Although these markers somewhat increased the marker density of this region, the pms3 locus is still located in a marker-sparse region.     

Untangling individual variation in natural populations: ecological, genetic and epigenetic correlates of long-term inequality in herbivory

Individual variation in ecologically important features of organisms is a crucial element in ecology and evolution, yet disentangling its underlying causes is difficult in natural populations. We applied a genomic scan approach using amplified fragment length polymorphism (AFLP) markers to quantify the genetic basis of long‐term individual differences in herbivory by mammals at a wild population of the violet Viola cazorlensis monitored for two decades. In addition, methylation‐sensitive amplified polymorphism (MSAP) analyses were used to investigate the association between browsing damage and epigenetic characteristics of individuals, an aspect that has been not previously explored for any wild plant. Structural equation modelling was used to identify likely causal structures linking genotypes, epigenotypes and herbivory. Individuals of V. cazorlensis differed widely in the incidence of browsing mammals over the 20‐year study period. Six AFLP markers (1.6% of total) were significantly related to herbivory, accounting altogether for 44% of population‐wide variance in herbivory levels. MSAP analyses revealed considerable epigenetic variation among individuals, and differential browsing damage was significantly related to variation in multilocus epigenotypes. In addition, variation across plants in epigenetic characteristics was related to variation in several herbivory‐related AFLP markers. Statistical comparison of alternative causal models suggested that individual differences in herbivory are the outcome of a complex causal structure where genotypes and epigenotypes are interconnected and have direct and indirect effects on herbivory. Insofar as methylation states of MSAP markers influential on herbivory are transgenerationally heritable, herbivore‐driven evolutionary changes at the study population will involve correlated changes in genotypic and epigenotypic distributions.     

A modified AFLP with fluorescence-labelled primers and automated DNA sequencer detection for efficient fingerprinting analysis in plants

A modified AFLP (amplified fragment length polymorphism) technique is described. Fluorescence-labelled primers were used in the selective amplifications. The amplified fragments were detected on denaturing polyacrylamide gels using an automated ALF DNA sequencer with the fragment option. The modified AFLP technique avoids the use of isotopes or silver staining, but gives a much higher resolution than other AFLP detection systems.     

番茄抗青枯病基因的AFLP分子标记

用番茄高抗青枯病品种“T51A”与高感青枯病品种“T9230”配制杂交组合,接种鉴定其正反交Ｆ₁代及Ｆ₂代分离群体的青枯病发生情况。结果表明,T51A对青枯病的抗性属于细胞质遗传,受1对杂合基因加性控制。用64个EcoRＩ/ＭseＩ引物组合对“T51A”、“T9230”两个亲本及其Ｆ₂代抗病和感病基因池进行AFLP分析,共扩增出约4200条可分辨的带,其中2条为稳定的差异。用“T51A”和“T9230”杂交产生的Ｆ₂代分离群体对2个特异条带与目的基因的遗传连锁性进行分析,发现特异条带AAG/CAT与暂定名为RRS-342的抗青枯病基因紧密连锁,二者之间的遗传距离为6.7 cM。将AAG/CAT片段回收、克隆和测序,成功地将其转化为SCAR标记,可以更加方便地用于对番茄青枯病基因的标记辅助选择。     

北京油鸡MSAP毛细管电泳荧光检测技术的建立

采用毛细管电泳荧光检测技术, 对北京油鸡肌肉组织基因组进行甲基化敏感扩增片段多态性(Methylation sensitive amplified polymorphism, MSAP)检测。通过对基因组DNA用量、预扩产物稀释倍数、选择性引物浓度、Mg2+浓度、dNTPs浓度和电泳内标量等6个主要参数进行分析和优化, 建立了适合北京油鸡基因组DNA甲基化分析的MSAP毛细管电泳荧光检测技术。重复实验表明, 建立的毛细管电泳荧光标记的MSAP检测技术能够自动地、高通量地分析北京油鸡基因组的DNA甲基化状态, 也适用于其他动植物基因组的DNA甲基化研究。     

AFLP标记在小香羊遗传多态性检测中的应用

研究了AFLP标记在研究小香羊遗传多态性方面应用的可行性和该山羊个体基因组DNA的AFLP扩增结果。实验应用10条AFLP引物,用PstI酶切,对15只小香羊基因组DNA进行AFLP反应,共获得113个AFLP标记,单引物获得的标记数在2～19之间,小香羊群体相似系数AFLP研究结果为0．913(0．814～0．980)。该研究为评价小香羊的遗传稳定性提供了相关的参数,准确评价尚待和其它品种对比研究后确定。     

荧光标记DNA扩增片段长度多态性方法的改进

采用常规PCR试剂和合成的接头和引物,其中MseⅠ引物为荧光标记物FAM标记,并对扩增片段长度多态性(AFLP)程序进行了改进,优化了PCR反应和电泳条件,建立了一个新的、有效的反应体系,降低了实验费用,经比较实验结果与AFLP荧光标记试剂盒的实验效果一致.     

利用相关性分析鉴定与水稻根部性状表达相关的分子标记

84个水稻品种在营养液中生长,10天后测定每一品种的最大根长（Maximum Root Length,MRL)和根干重（Root Dry Weight,RDW)。选取其中有代表性的27个水稻品种,用扩增片段长度多态性（Amplified Fragment Length Polymor-phism,AFLP)技术进行基因组差异分析,通过计算差异带与性状表现间的相关系数,筛选与苗期水稻最大根长和根干重显著相关的分子标记,经过15对AFLP引物的筛选,有4对引物的7个片段的基因型表现与最大根长或（和）根干重显著相关,对其中的片段之一“T3P3f”进行克隆,测序后,设计特异PCR扩增引物“Z336”,进一步对84个水稻品种进行鉴定,统计分析后发现,Z2336与最大根长的相关系数为-0.193,相关性几近显著水平;与根干重的相关系数为-0.391,相关性达极显著水平,计算对根干重的差异解释率,可达15.3%,显示该标记与控制根干重性状表达的某个数量基因紧密连锁,它的存在对性状值的降低有显著的关系,进一步利用源于ZYQ8和JX17的加倍单倍体（double haploid,DH)分离群体进行基因定位,发现Z336位于水稻第11号染色体上,距离相邻的分子标记9.4cM。     

Loss of genetic diversity in the North American mayfly Ephemerella invaria associated with deforestation of headwater streams

1. Terrestrial dispersal by aquatic insects increases population connectivity in some stream species by allowing individuals to move outside the structure of the stream network. In addition, individual survival and reproductive success (as well as dispersal) are tightly linked to the quality of the terrestrial habitat. 2. In historically forested catchments, deforestation and altered land use have the potential to interfere with mayfly dispersal or mating behaviours by degrading the quality of the terrestrial matrix among headwater streams. We hypothesised that loss of tree cover in first‐order catchments would be associated with an increase in population substructure and a decrease in genetic diversity of mayfly populations. 3. To test this hypothesis, we investigated spatial patterns of genetic variation in the common mayfly Ephemerella invaria across a gradient of deforestation in the central piedmont region of eastern United States. Intraspecific genetic diversity and population substructure were estimated from data obtained using fluorescent amplified fragment length polymorphism (AFLP) markers. 4. We found that mayfly populations had low population substructure within headwater stream networks and that genetic diversity was strongly negatively correlated with mean deforestation of the first‐order catchments. The large‐scale pattern of population substructure followed a pattern of isolation by distance (IBD) in which genetic differentiation increases with geographical distance, but assignment tests placed a few individuals into populations 300 km away from the collection site. 5. Our results show that loss of genetic diversity in this widespread aquatic insect species is co‐occurring with deforestation of headwater streams. 6. Most arguments supporting protection of headwater streams in the United States have centred on the role of these streams as hydrological and biogeochemical conduits to downstream waters. Our work suggests that headwater stream land use, and specifically tree cover, may have a role in the maintenance of regional genetic diversity in some common aquatic insect species.     

Localization of the muscular dystrophy AM locus using a chicken linkage map constructed with the Kobe University resource family

A chicken linkage map, constructed with the Kobe University (KU) resource family, was used to locate the genetic locus for muscular dystrophy of abnormal muscle type (AM). The KU resource family is a backcross pedigree with 55 offspring produced from the mating of a White Leghorn F-line (WL-F) male and a hybrid female produced from a cross between the WL-F male and a female of the Fayoumi OPN line who was homozygous for the AM gene. In total, 872 loci were genotyped on the pedigree; 749 (86%) were informative and mapped to 38 linkage groups. These informative loci included 649 AFLPs, 93 MS, three functional genes, the AM locus, sex phenotype, and two red blood cell loci. The remaining 123 markers were unlinked. Nineteen of the 38 KU linkage groups were assigned to macrochromosomes 1-8 and 11 microchromosomes including chromosome W, while 19 linkage groups were unassigned. The total map was 3569 cM in length, with an average marker interval of 4.8 cM. The AM locus was mapped 130 cM from the distal end of chromosome 2q.     

基于AFLP标记的野生梅种质的鉴定

梅Prunus mume是我国原产的名花佳果。为有效保护和利用野生种质资源,文中采用了AFLP标记技术,结合形态表型特征分析,对65份野梅种质进行试验分析。首先从64对引物中筛选出MseⅠ-EcoRⅠ8对引物组合,扩增出1 002条多态性条带。按照Nei’72距离系数进行聚类,在Nei’72=0.26处,可区分西山野梅原变种、西山野梅原种、西山毛梅、厚叶梅、南大坪桃梅、嵩明小梅、曲梗常绿梅、蜡叶梅以及匍匐梅,这与形态学上变种或变型的划分一致。群体内变种单株样本遗传多样性丰富。基于梅花种质资源的遗传变异,建议今后需要对所有变种与变型进行有效的保护。