Cloning of AFLP markers linked to resistance to Peronosclerospora sorghi in maize

Genetic mapping of resistance genes for sorghum downy mildew (SDM) in maize revealed multiple-locus inheritance. A combination of AFLP (amplified fragment length polymorphism) technique with bulked segregant analysis (BSA) was applied to map the genes involved in the resistance to SDM (Peronosclerospora sorghi) in a recombinant inbred population. Three AFLP markers were identified and mapped to chromosomes 1 and 9, in regions previously associated with SDM resistance. One other AFLP marker was found to be associated with disease susceptibility but could not be linked to any chromosome. These four AFLP fragments were isolated, cloned and sequenced. A BLAST search of the GenBank database showed that none of these four sequences was closely related to resistance genes that have been reported previously. Sequence-characterized amplified regions (SCARs) were produced and used to assess the presence of SDM resistance genes and characterize specific genotypes. These markers may be useful in marker-assisted breeding programs.     

A biological and molecular characterization of some Egyptian barley genotypes which are resistant to net blotch disease

A survey for resistance against net blotch disease (caused by Pyrenophora teres) was performed on some Egyptian barley landraces and some selected resistance and susceptible standard German barley genotypes. The results indicated that most of the Egyptian barley landraces are extremely resistant to the disease. Molecular analysis using RAPD and AFLP showed unique banding profiles for the different genotypes, and specific AFLP markers for the Egyptian genotypes were identified. The effectiveness of RAPD and AFLP for identifying different barley genotypes of different origins and with different reactions against P. teres was discussed. The results of the biological evaluation and molecular characterization done in this study can be seen as the starting point needed to identify the valuable net blotch resistant Egyptian barley germplasm at both the phenotype and genotype levels and draw the attention of breeders and banks of natural plant genetic resources towards this valuable yet neglected germplasm.     

龙舌兰麻种质资源抗斑马纹病鉴定研究

通过分离培养斑马纹病病原菌,人工接种鉴定不同龙舌兰麻种质的抗斑马纹病的特性.结果表明,番麻、东368、墨引6、墨引12、墨引7、墨引5、假7、马盖麻、东109、金边孤叶龙舌和兰墨引4号11份种质为高抗种质,病斑扩展速度和病情严重度可作为龙舌兰麻抗病性快速鉴定技术手段.     

用SSR和AFLP技术分析花生抗青枯病种质遗传多样性的比较

由Ralstonia solanacearum E.F.Smith引起的青枯病是若干亚洲和非洲国家花生生产的重要限制因子,利用抗病品种是防治这一病害最好的措施。虽然一大批抗青枯病花生种质资源材料已被鉴定出来,但对其遗传多样性没有足够的研究,限制了在育种中的有效利用。本研究以31份对青枯病具有不同抗性的栽培种花生种质为材料,通过简单序列重复(SSR)和扩增片段长度多态性(AFLP)技术分析了它们的遗传多样性。通过78对SSR引物和126对AFLP引物的鉴定,筛选出能显示抗青枯病种质多态性的SSR引物29对和AFLP引物32对。所选用的29对多态性SSR引物共扩增91条多态性带,平均每对引物扩增3.14条多态性带;32对多态性AFLP引物共扩增72条多态性带,平均扩增2.25条多态性带。在所筛选引物中,4对SSR引物(14H06,7G02,3A8,16C6)和1对AFLP引物(P1M62)检测花生多态性的效果优于其他引物。SSR分析获得的31个花生种质的遗传距离为0.12-0.94,平均为0.53,而AFLP分析获得的遗传距离为0.06～0.57,平均为0.25,基于SSR分析的遗传距离大于基于AFLP分析的遗传距离,疏枝亚种组的遗传分化相对大于密枝亚种组。基于两种分析方法所获得的聚类结果基本一致,但SSR数据聚类结果与栽培种花生的形态分类系统更为吻合。根据分析结果,对构建青枯病抗性遗传图谱群体的核心亲本和抗性育种策略提出了建议。     

AFLP studies on downy-mildew-resistant and downy-mildew-susceptible genotypes of opium poppy

Downy mildew (DM) caused by Peronospora arborescens, is a serious disease in opium poppy (Papaver somniferum), which has a world-wide spread. The establishment of DM-resistant cultivars appears to be a sustainable way to control the disease. In this paper, we present the results of a study aimed at the identification of amplified fragment length polymorphism (AFLP) markers for DM-resistance in opium poppy. Three opium poppy genotypes (inbred over about 10 years): Pps-1 (DM-resistant), Jawahar-16 (DM-susceptible) and H-9 (DM-susceptible) were crossed in a diallel manner and the F1 progeny along with the parents were subjected to AFLP analysis of chloroplast (cp) and nuclear DNA with seven and nine EcoRI / MseI primer combinations, respectively. cpDNA AFLP analysis identified 24 Pps-1 (DM-resistant)-specific unique fragments that were found to be maternally inherited in both the crosses, Pps-1 × Jawahar-16 and Pps-1 × H-9. In the case of nuclear DNA AFLP analysis, it was found that 17 fragments inherited from Pps-1 were common to the reciprocal crosses of both (i) Pps-1 and Jawahar-16 as well as (ii) Pps-1 and H-9. This is the first molecular investigation on the identification of polymorphism between DM-resistant and DM-susceptible opium poppy genotypes and development of DM-resistant opium poppy genotype-specific AFLP markers. These AFLP markers could be used in future genetic studies for analysis of linkage to the downy mildew resistance trait.     

A high-resolution map of the H1 locus harbouring resistance to the potato cyst nematode Globodera rostochiensis

The resistance gene H1 confers resistance to the potato cyst nematode Globodera rostochiensis and is located at the distal end of the long arm of chromosome V of potato. For marker enrichment of the H1 locus, a bulked segregant analysis (BSA) was carried out using 704 AFLP primer combinations. A second source of markers tightly linked to H1 is the ultra-high-density (UHD) genetic map of the potato cross SH × RH. This map has been produced with 387 AFLP primer combinations and consists of 10,365 AFLP markers in 1,118 bins (). Comparing these two methods revealed that BSA resulted in one marker/cM and the UHD map in four markers/cM in the H1 interval. Subsequently, a high-resolution genetic map of the H1 locus has been developed using a segregating F₁ SH × RH population consisting of 1,209 genotypes. Two PCR-based markers were designed at either side of the H1 gene to screen the 1,209 genotypes for recombination events. In the high-resolution genetic map, two of the four co-segregating AFLP markers could be separated from the H1 gene. Marker EM1 is located at a distance of 0.2 cM, and marker EM14 is located at a distance of 0.8 cM. The other two co-segregating markers CM1 (in coupling) and EM15 (in repulsion) could not be separated from the H1 gene.Communicated by J.G. Wenzel     

AFLP analysis of genetic relationships among aromatic grapevines (<Emphasis Type="Italic">Vitis vinifera</Emphasis>)

Genotypic diversity has been detected among aromatic grapevines (Vitis vinifera) by molecular markers (AFLPs). The 22 primer-pairs generated a total of 1,331 bands of which 564 (40%) were polymorphic over all the genotypes. The bootstrap analysis pointed out that a large number of polymorphic bands (200–400) has to be used for a better estimation of the genetic distances among genotypes; 383 polymorphic AFLP bands were used for the cluster and the principal coordinate analyses because they did not present missing data across all the genotypes. The cluster analysis (UPGMA), based on polymorphic AFLP markers, revealed no relationship between the Moscato and Malvasia grapevines. The Malvasias, unlike the Moscatos distinguished by their distinct muscat aroma, have to be considered a more complex group because it includes muscat and non-muscat grapevines. The principal coordinate analysis (PCO) confirmed the pattern of the cluster analysis only for those varieties which presented a low coefficient of dissimilarity, while for the other varieties there was no correspondence between the two analyses. The pattern of aggregation among aromatic grapevines in the cluster and principal coordinate analyses does not support any classification that might include an aromatic grapevine group in V. vinifera. Even though some synonyms and homonyms are present among aromatic grapevines (V. vinifera), genetic diversity exists among genotypes in AFLP markers.Communicated by H.F. Linskens     

河北省番茄上致病疫霉Phytophthora infestans群体结构的分析

番茄晚疫病是河北省番茄生产上最具毁灭性的病害之一,对引起该病害的致病疫霉群体结构进行分析有利于病害的防治。利用对峙培养法和菌落直径法对2007-2008年采自河北省保定、沧州和唐山分离自番茄的49个致病疫霉菌株进行了交配型和甲霜灵抗性的表型测定,结果表明该群体所有菌株均为A1交配型,以甲霜灵敏感菌株为主,抗性菌株仅7株。利用聚合酶链式反应-限制性片段长度多态性(PCR-RFLP)、简单序列重复(SSR)和扩增片段长度多态性(AFLP)等分子技术对该群体的基因型进行了分析,结果表明供试菌株线粒体基因型均为Ia型,共鉴定出了Ⅰ、Ⅱ和Ⅲ3种SSR基因型,AFLP聚类分析在相似系数0.87时可以形成α、β和γ等3个不同的分支。河北省所有番茄上致病疫霉菌株均分布在α分支上,该分支又可进一步分为7个亚分支。AFLP亚分支与甲霜灵抗性和地理来源均无明显相关性,但Ⅱ型SSR与甲霜灵抗性和地理来源有明显的相关性。综合表型和基因型数据说明河北省番茄上致病疫霉群体结构比较单一,遗传多样性程度较低。     

Phytophthora infestans isolates from Northern China show high virulence diversity but low genotypic diversity

Phenotypic and genotypic characteristics of 48 Phytophthora infestans isolates , collected in five provinces in Northern China between 1997 and 2003, were determined and compared with reference isolates. Characterisation included mating type, virulence, mitochondrial DNA (mtDNA) haplotype and DNA fingerprinting patterns based on simple sequence repeats (SSR) and amplified fragment length polymorphisms (AFLP). All isolates had the A1 mating type, mtDNA haplotype IIa and an identical SSR genotype (designated as SG-01-01) that differed from SSR genotypes found in the reference isolates, including those representing the 'old' US-1 lineage that dominated the P. infestans population worldwide prior to 1980. In contrast, the virulence spectra were highly variable and virulence to all resistance genes present in the standard differential set ( R1 to R11 ) was found. AFLP analysis revealed some diversity; eight different AFLP genotypes were found that could be grouped into two major clusters. This study shows that there is very little genotypic diversity in the P. infestans population in Northern China. The occurrence of many different races within this rather uniform population is discussed in the framework of recent insights into the molecular determinants of avirulence in potato– P. infestans 'gene-for-gene' interactions.     

Establishment of a local map of AFLP markers around the powdery mildew resistance gene Run1 in grapevine and assessment of their usefulness for marker assisted selection

The monogenic dominant genetic determinism of total resistance to powdery mildew, introduced from Muscadinia rotundifolia into Vitis vinifera, was further assessed in BC₄ and BC₅ full-sib families. A BC₅ population of 157 individuals was used to select AFLP markers linked to the resistance gene, Run1. Thirteen AFLP markers were selected and a local map was constructed around the Run1 gene. Ten markers among the 13 were found to co-segregate with the resistance gene. The usefulness of these 13 AFLP markers for the selection of Run1-carrying genotypes was further assessed through their analysis in a set of 22 Run1-carrying resistant genotypes and 16 susceptible genotypes. Three markers out of the 13 analysed were found to be absent in all susceptible genotypes and present in all resistant individuals, and may thus represent good tools for the marker-assisted selection of grapevine varieties resistant to powdery mildew. A recombination event among the markers that were originally found to co-segregate was observed in one of the resistant individuals, showing that recombination is possible in this region and may therefore be observed in larger populations. Received: 18 October 2000 / Accepted: 4 April 2001     

Investigations on chemical control of zebra disease in Agave hybrid no. 11648

In laboratory, greenhouse and field experiments, mancozeb and copper compounds controlled the leaf-spot phase of zebra disease caused by Phytophthora arecae, one of the pathogens responsible for this serious disease in Tanzania's high-yielding Agave hybrid no. 11648. As the copper compounds were phytotoxic, Dithane M-45 (Mancozeb) is recommended as a protectant during the rainy season.     

Characterisation of powdery mildew resistance in a segregating diploid rose population

Powdery mildew (Podosphaera pannoso) is one of the most serious fungal diseases on both greenhouse and field grown roses. Improvement of disease resistance is a major selection aim for garden rose breeders. For rose cultivars, being mostly tetraptoid, it is complicated to develop molecular markers for resistance. Hence, a segregating diploid population was established from a cross between 'Yesterday', a commercial available rose variety susceptible to powdery mildew, and R. wichurana, a rose species with resistance to certain isolates of powdery mildew. A progeny of 94 seedlings was planted in the field. The segregation of powdery mildew resistance was studied in this population by means of a bioassay with two different monoconidial isolates of powdery mildew. Based on the response to these inoculations different groups were selected: a first group of genotypes was susceptible to both isolates, other groups were susceptible to one of both isolates and a last group was resistant to both tested isolates. The disease resistance inherits for both isolates in a quantitative way. A genetic map based on AFLP and SSR markers was established and will be used for QTL analysis of powdery mildew resistance.     

Targeted mapping of a sugarcane rust resistance gene (Bru1) using bulked segregant analysis and AFLP markers

The presence of a major resistance gene (Bru1) for brown rust in the sugarcane cultivar R570 (2n about 115) was confirmed by analyzing segregation of rust resistance in a large population of 658 individuals, derived from selfing of clone R570. A subset of this population was analyzed with AFLP and bulked segregant analysis (BSA) to develop a detailed genetic map around the resistance gene. Four hundred and forty three primer pairs were used resulting in the identification of eight AFLP markers surrounding the resistance gene in an interval of 10 cM, with the closest markers located at 1.9 and 2.2 cM on each side of the gene. Efficiency of the AFLP/BSA applied to the complex polyploid genome of sugarcane is discussed, as well as the potential of the newly identified AFLP markers for developing a map-based cloning approach exploiting, synteny conservation with sorghum.Communicated by H. F. Linskens     

SSR and AFLP based genetic diversity of soybean germplasm differing in photoperiod sensitivity

Forty-four soybean genotypes with different photoperiod response were selected after screening of 1000 soybean accessions under artificial condition and were profiled using 40 SSR and 5 AFLP primer pairs. The average polymorphism information content (PIC) for SSR and AFLP marker systems was 0.507 and 0.120, respectively. Clustering of genotypes was done using UPGMA method for SSR and AFLP and correlation was 0.337 and 0.504, respectively. Mantel's correlation coefficients between Jaccard's similarity coefficient and the cophenetic values were fairly high in both the marker systems (SSR = 0.924; AFLP = 0.958) indicating very good fit for the clustering pattern. UPGMA based cluster analysis classified soybean genotypes into four major groups with fairly moderate bootstrap support. These major clusters corresponded with the photoperiod response and place of origin. The results indicate that the photoperiod insensitive genotypes, 11/2/1939 (EC 325097) and MACS 330 would be better choice for broadening the genetic base of soybean for this trait.     

Assessment of genetic diversity among faba bean genotypes using agro-morphological and molecular markers

Forty faba bean (Vicia faba L.) genotypes were evaluated for their agro-morphological performance and molecular diversity under Central Region of Saudi Arabia conditions during 2010–11 and 2011–12 seasons. Field performance results showed that faba genotypes exhibited a significant amount of variation for their agro-morphological studied parameters. Giza40 recorded the tallest genotype (139.5 cm), highest number of seeds per plants (100.8), and the highest seed yield per plant (70.8 g). The best performing genotypes were Giza40, FLIP03-014FB, Gazira1 and Goff1. Genetic variability among genotypes was determined using Sequence Related Amplified Polymorphism (SRAP) and Amplified Fragment Length Polymorphism (AFLP) markers. A total of 183 amplified fragments (alleles) and 1758 polymorphic fragments (bands) in SRAP and 202 alleles and 716 bands in AFLP were obtained using six SRAP and four AFLP primer combinations respectively. Polymorphism information content (PIC) values for AFLP and SRAP markers were higher than 0.8, indicating the existence of a considerable amount of genetic diversity among faba tested genotypes. The UPGMA based clustering of faba genotypes was largely based on origin and/or genetic background. Result of cluster analysis based on SRAP showed weak and not significant correlation while, it was highly significant based on AFLP analysis with agro-morphological characters (r = 0.01, p > 0.54 and r = 0.26, p < 0.004 respectively). Combined SRAP and AFLP markers proved to be significantly useful for genetic diversity assessment at molecular level. They exhibited high discrimination power, and were able to distinguish the faba bean genotypes with high efficiency and accuracy levels.     

Identification and chromosomal location of major genes for resistance to Pyrenophora teres in a doubled-haploid barley population.

Net blotch, caused by Pyrenophora teres, is one of the most economically important diseases of barley worldwide. Here, we used a barley doubled-haploid population derived from the lines SM89010 and Q21861 to identify major quantitative trait loci (QTLs) associated with seedling resistance to P. teres f. teres (net-type net blotch (NTNB)) and P. teres f. maculata (spot-type net blotch (STNB)). A map consisting of simple sequence repeat (SSR) and amplified fragment length polymorphism (AFLP) markers was used to identify chromosome locations of resistance loci. Major QTLs for NTNB and STNB resistance were located on chromosomes 6H and 4H, respectively. The 6H locus (NTNB) accounted for as much as 89% of the disease variation, whereas the 4H locus (STNB resistance) accounted for 64%. The markers closely linked to the resistance gene loci will be useful for marker-assisted selection.     

Significance tests and weighted values for AFLP similarities, based on Arabidopsis in silico AFLP fragment length distributions

Many AFLP studies include relatively unrelated genotypes that contribute noise to data sets instead of signal. We developed: (1) estimates of expected AFLP similarities between unrelated genotypes, (2) significance tests for AFLP similarities, enabling the detection of unrelated genotypes, and (3) weighted similarity coefficients, including band position information. Detection of unrelated genotypes and use of weighted similarity coefficients will make the analysis of AFLP data sets more informative and more reliable. Test statistics and weighted coefficients were developed for total numbers of shared bands and for Dice, Jaccard, Nei and Li, and simple matching (dis)similarity coefficients. Theoretical and in silico AFLP fragment length distributions (FLDs) were examined as a basis for the tests. The in silico AFLP FLD based on the Arabidopsis thaliana genome sequence was the most appropriate for angiosperms. The G + C content of the selective nucleotides in the in silico AFLP procedure significantly influenced the FLD. Therefore, separate test statistics were calculated for AFLP procedures with high, average, and low G + C contents in the selective nucleotides. The test statistics are generally applicable for angiosperms with a G + C content of approximately 35-40%, but represent conservative estimates for genotypes with higher G + C contents. For the latter, test statistics based on a rice genome sequence are more appropriate.     

AFLP-Based Genetic Diversity and Its Comparison with Diversity Based on SSR, SAMPL, and Phenotypic Traits in Bread Wheat

Data on AFLP (eight primer pairs) and 14 phenotypic traits, collected on 55 elite and exotic bread wheat genotypes, were utilized for estimations of genetic diversity. We earlier used these 55 genotypes for a similar study using SSRs and SAMPL. As many as 615 scorable AFLP bands visualized included 287 (46.6%) polymorphic bands. The phenotypic traits included yield and its component traits, as well as physiomorphological traits like flag leaf area. Dendrograms were prepared using cluster analysis based on Jaccard's similarity coefficients in case of AFLP and on squared Euclidean distances in case of phenotypic traits. PCA was conducted using AFLP data and a PCA plot was prepared, which was compared with clustering patterns in two dendrograms, one each for AFLP and phenotypic traits. The results were also compared with published results that included studies conducted elsewhere using entirely different wheat germplasm and our own SSR and SAMPL studies based on the same 55 genotypes used in the present study. It was shown that molecular markers are superior to phenotypic traits and that AFLP and SAMPL are superior to other molecular markers for estimation of genetic diversity. On the basis of AFLP analysis and keeping in view the yield performance and stability, a pair of genotypes (E3876 and E677) was recommended for hybridization in order to develop superior cultivars.     

羊草种质基因组DNA的AFLP多态性研究

羊草是禾本科牧草之王 ,在当前我国西部生态建设和草原畜牧业发展中发挥着重要作用。用AFLP方法对2 7份我国不同地区分布的羊草 (Leymuschinensis (Trin .)Tzvel)材料进行了基因组DNA多态性分析 ,8对AFLP引物组合在 2 7个不同羊草基因型中共扩增出 5 37条带 ,产生出的DNA片段大小分布在 75bp - 5 30bp之间。其中单态性带 89条 ,占 16 .6 % ,多态性带 32 9条 ,占 6 1.3%。平均每对引物组合扩增的DNA带数为 6 6 .13,总的多态性比率为 78.84%。AFLP多态信息含量PIC值分布于 0 .0 - 0 .5之间 ,平均PIC值为 0 .2 16 ,出现的PIC最大值 (0 .5 )约占AFLP标记的 8.5 % ,说明羊草基因组DNA的多态性比较丰富。以 5 37个AFLP标记为原始数据 ,根据Nei和Li的方法对 2 7份羊草材料进行遗传变异和聚类分析的结果表明 :羊草种内有高频率的遗传变异发生 ,且与地理分布和生态环境密切相关 ;2 7份羊草不同基因型被划分为四大类群 ,不同类群相互间的遗传距离相对较大 ,在树状图中表现为较远的亲缘关系。对羊草种内遗传变异发生的原因和品种的形成进行了初步讨论。