The construction of a linkage map of Alstroemeria aurea by AFLP markers

An AFLP based linkage map has been generated for the ornamental crop species Alstroemeria aurea. In view of the large genome size of Alstroemeria (25,000 Mb) the number of selective nucleotides for AFLP amplification was increased to EcoRI+4/MseI+4 to generate fingerprints of moderate complexity. In addition, markers were generated with the enzyme combination Sse/Mse, where Sse8387I is an8-cutter, thereby reducing AFLP template complexity. Segregation of 374AFLP polymorphisms was recorded in the F₁ of an intraspecific A. aurea cross (A002 × A003). The map consisted of 8 A002 and 10A003 linkage groups with 122 and 214 markers covering 306.3 and605.6 cM, respectively. The two maps were integrated by using the21% of the AFLP markers that were heterozygous in both parents, and31% of the markers remained unlinked. Pollen color was assigned to linkage group A002-6. The enzyme combinations EcoRI+4/MseI+4 and Sse+2/MseI+3 generated 80 and 30 clear bands per lane with 16 and 9 polymorphic markers, respectively. Twenty percent of the EcoRI+4/MseI+4 primer combinations resulted in fingerprints that were disturbed by a few excessively thick bands (55 out of 288 primer combinations). We conclude that fingerprints and markers generated with the eight-cutter enzyme Sse8387I, in combination with+2/+3 selective nucleotides (Sse+2/MseI+3) are superior toEcoRI+4/MseI+4. This revised version was published online in August 2006 with corrections to the Cover Date.     

Genetic relationships between Southern African Solanum retroflexum Dun. and other related species measured by morphological and DNA markers

In this study, the genetic relationship between 14 genotypes of black nightshade, most which were part of the Solanum nigrum complex, was investigated. Fifteen morphological characters were measured and used to compile a dendrogram. Amplified fragment length polymorphism (AFLP) markers were also used to assess the level of polymorphism between the 14 Solanum genotypes. Three EcoR I/Mse I primer combinations with three selective nucleotides per primer were used for screening the respective genotypes. Multiple polymorphisms could be detected to the extent that all the genotypes studied could be distinguished, using any single primer combination, thus showing the usefulness of AFLP's for this purpose. Up to 43 polymorphic bands were detected with a single primer combination among the 14 different genotypes. The three primer combinations generated a total of 359 bands, of which 222 (62%) were clearly polymorphic. This data was used to compile a dendrogram. Both the morphological and AFLP marker analysis clearly separated the different genotypes into similar groups. This revised version was published online in August 2006 with corrections to the Cover Date.     

Genetic diversity and relationships among pea cultivars revealed by RAPDs and AFLPs

In order to obtain an overview of the genetic diversity present within the set of pea cultivars released in Germany, 21 cultivars were analysed at the DNA level by random amplified polymorphic DNAs (RAPDs) and amplified fragment length polymorphisms (AFLPs), as well as for agronomic traits. Yield of grain cultivars ranged from 2.95 to 3.87 t/ha. Based on the screening of 60 RAPD primers and 32 Eco RI + 3/Mse I+3 AFLP primer combinations, 20 RAPD primers and 11 Eco RI + 3/MseI+ 3 primer combinations generating polymorphic and distinct fragments were chosen for estimation of genetic diversity. Twenty RAPD primers amplified a total of 314 scorable bands ranging from about 262 bp to 1996 bp. Of these, 175 fragments (55.7%) were polymorphic. Based on these data, genetic similarity (GS) was estimated between 0.80 (‘Lisa’ vs.‘Grapis’) and 0.94 (‘Bohatyr’ vs. ‘Sponsor’; mean GS = 0.88). Eleven AFLP primer combinations led to the amplification of 949 scorable fragments ranging from 43 to 805 bp and of these, 462 (48.7%) were polymorphic. Genetic similarity based on AFLPs was calculated between 0.85 (‘Lisa’ vs.‘Laser’) and 0.94 (‘Bohatyr’ vs. ‘Sponsor’, mean GS = 0.90). Correlation of genetic similarity estimated on RAPDs and AFLPs was estimated at r = 0.79** using Spearman's rank correlation coefficient and at r = 0.84 by the Mantel test, respectively. UPGMA cluster analysis carried out on these data separately for RAPDs and AFLPs and on the combined data reflected, to some extent, pedigree relationships and cophenetic correlations (r = 0.89 for RAPDs, r = 0.88 for AFLPs, and r = 0.93 RAPDs + AFLPs) indicate a good fit of respective clusters to genetic similarity data. The correlation of cluster analyses to pedigree information and the impact on parental genotype selection is discussed.     

Clustering of amplified fragment length polymorphism markers in a linkage map of rye

Amplified fragment length polymorphisms (AFLPs) are now widely used in DNA fingerprinting and genetic diversity studies, the construction of dense genetic maps and in fine mapping of agronomically important traits. The AFLP markers have been chosen as a source to extend and saturate a linkage map of rye, which has previously been generated by means of restriction fragment length polymorphism, random amplified polymorphic DNA, simple sequence repeat and isozyme markers. Gaps between linkage groups, which were known to be part of chromosome 2R, have been closed, thus allowing the determination of their correct order. Eighteen EcoRI‐MseI primer combinations were screened for polymorphism and yielded 148 polymorphic bands out of a total of 1180. The level of polymorphism among the different primer combinations varied from 5.7% to 33.3%. Eight primer combinations, which revealed most polymorphisms, were further analysed in all individuals of the F₂ mapping population. Seventy‐one out of 80 polymorphic loci could be integrated into the linkage map, thereby increasing the total number of markers to 182. However, 46% of the mapped AFLP markers constituted four major clusters located on chromosomes 2R, 5R and 7R, predominantly in proximity to the centromere. The integration of AFLP markers caused an increase of 215 cM, which resulted in a total map length of almost 1100 cM.     

AFLP markers distinguishing an early mutant of Flame Seedless grape

Molecular markers have been frequently used to differentiate grape species and cultivars. There are fewer cases where molecular markers have been used to differentiate grape clones within a cultivar, or for the demarcation of somatic mutants from parental clones. This study reports the first successful utility of AFLPs for the differentiation of somatic mutants from their parental grapevine line, and discusses the potential for similar AFLP applications. The somatic mutant analysed demonstrates earlier budburst characteristics than the Flame Seedless line from which is arose. Analysis of 64 AFLP primer combinations in silver stained polyacrylamide produced in excess of 3000 markers in Vitis vinifera, and provided two markers which differentiated the somatic mutant, from its parental line. One marker was 440 bpin length and was produced with primer combinationEcoR1-AT and Mse1-CTT. The second marker was 340 bp in length and generated with primer combination EcoR1-TC and Mse1-CAC. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic diversity among selected Argentinean garlic clones (Allium sativum L.) using AFLP (Amplified Fragment Length Polymorphism)

Genetic diversity of eight selected Argentinean garlic clones (Allium sativum L.) were investigated at the DNA level with the amplified fragment length polymorphism DNA (AFLP) procedure. A total of 405 unambiguous bands were identified by six primer combinations of EcoR I +3 and Mse I +3, of those, 398 showed a clear polymorphism, representing 98% of the total bands. A presence/absence matrix was constructed with the polymorphic bands, and a dendrogram was obtained from it with the UPGMA method. The accessions showed different levels of similarity ranging between 0.24 and 0.97, using the coefficient of Jaccard. The dendrogram showed six arbitrary groups. Accessions typically considered as different clones show similarities between 0.97 and 0.495. The garlic clones were clustered according to the physiological group and bulb color. We could detect an association between AFLP and the geographical origin of the clones. The potential use of AFLP could allow not only the differentiation among species, but also between botanical varieties and well-defined ecotype groups. This is the first report of the use of AFLP to characterize Argentinean garlic clones. This revised version was published online in August 2006 with corrections to the Cover Date.     

Genetic diversity in cornsalad (Valerianella locusta) and related species as determined by AFLP markers

Fifteen amplified fragment length polymorphism (AFLP) EcoRI/MseI‐based primer combinations with five selective bases (Eco RI‐ANN, MseI‐CN) were used to estimate genetic diversity among 45 line varieties of cultivated cornsalad and 19 genebank accessions classified into nine different species related to cornsalad. Polymorphic fragments were scored for calculation of Jaccard's coefficient of genetic similarity (GS). The average GS estimate in elite germplasm (GS = 0.90) was substantially higher than in exotic germplasm (GS = 0.47). UPGMA‐cluster analysis revealed genetic relationships among recently bred varieties, old varieties and genebank accessions. Analysis of molecular variance indicated almost threefold variability within sets compared with between sets due to a high level of polymorphism among wild species. Sources for increasing genetic diversity in elite germplasm of cornsalad were suggested and a duplicate among the genebank accessions was detected. AFLPs could be considered a powerful tool for genetic diversity estimation in cornsalad germplasm and are recommended for systematic fingerprinting of remaining cornsalad species.     

Hybrid performance and AFLP- based genetic similarity in faba bean

Successful prediction of heterosis and performance of F₁-hybrids from the genetic similarity of their parents based on molecular markers has been reported in several crops and can be very helpful in hybrid breeding. The relationship between genetic similarities based on amplified fragment length polymorphism (AFLP) of 18 European faba bean lines and their hybrid performance and heterosis was investigated. Parental lines, 62 F₁-hybrids and their F₂-progenies were evaluated in field trials in four environments in Germany for seed yield, 1,000-seed weight and plant height. Results clearly demonstrated a stable superiority of the hybrids over their inbred parents and elite check cultivars, and showed a marked and varying amount of heterosis. Parental seed yield and F₂-hybrid yield were promising as predictors for F₁-hybrids. AFLP analysis of the 18 inbred lines using 26 EcoRI/MseI primer combinations resulted in 1202 polymorphic fragments. Cluster analysis based on genetic similarity estimates unambiguously identified pedigree-related inbred lines. No clear separation of the 18 inbred lines into subgroups was detected. Correlation coefficients between genetic similarity estimates and either heterosis or F₁-hybrid performance were small and not useful. Also correlations between specific genetic similarity and specific combining ability were too small for all traits to be of predictive value. Results showed that AFLP-based genetic similarities are not useful to predict the performance of hybrids or heterosis within the elite European faba bean gene pool.     

花生DNA分子标记的研究Ⅲ不同限制酶组合AFLP分析效果比较

采用了28对EcoR I/Mse I AFLP选择性扩增引物、64对Pst I/Taq I AFLP选择性扩增引物,对 作图亲本24-3、B4及其杂种F1进行了分析,统计了两种限制酶组合的扩增总带数、多态性带数及多 态性率。秩和检验结果说明,两种限制酶组合AFLP每对引物扩增出的总带数无显著差异,而多态性条 带数目以及多态性率均有极显著的差异。证明尝试不同的限制酶组合可望揭示出花生品系材料间更 为丰富的遗传变异性。     

花生DNA分子标记的研究 III 不同限制酶组合AFLP分析效果比较

采用了28对EcoR I/Mse I AFLP选择性扩增引物、64对Pst I /Taq I AFLP选择性扩增引物，对作图亲本24-3、B4及其杂种F1进行了分析，统计了两种限制酶组合的扩增总带数、多态性带数及多态性率。秩和检验结果说明，两种限制酶组合AFLP每对引物扩增出的总带数无显著差异，而多态性条带数目以及多态性率均有极显著的差异。证明尝试不同的限制酶组合可望揭示出花生品系材料间更为丰富的遗传变异性。     

Identification of polymorphic DNA markers in cultivated peanut (Arachis hypogaea L.)

The detection of DNA polymorphism in cultivated peanut (Arachis hypogaea L.) is reported here for the first time. The DNA amplification fingerprinting (DAF) and amplified fragment length polymorphism (AFLP) approaches were tested for their potential to detect genetic variation in peanut. The AFLP approach was more efficient as 43% of the primer combinations detected polymorphic DNA markers in contrast to 3% with the DAF approach. However, the number of polymorphic bands identified using primers selected in both approaches was comparable. In the DAF study, when 559 primers of varying types were screened, 17 (mostly 10-mer types) detected polymorphism producing an average of 3.7 polymorphic bands per primer with a total of 63 polymorphic markers. In the AFLP study, when 64 primer combinations (three selective nucleotides) corresponding to restriction enzymes Eco RI and Mse I were screened, 28 detected polymorphism. On an average, 6.7% of bands obtained from these 28 primer pairs were polymorphic resulting in a total of 111 AFLP markers. Our results demonstrate that both AFLP and DAF approaches can be employed to generate DNA markers in peanut and thus have potential in the marker-assisted genetic improvement and germplasm evaluation of this economically important crop. This revised version was published online in July 2006 with corrections to the Cover Date.     

A comparison of genetic relationship measures in strawberry (Fragaria × ananassa Duch.) based on AFLPs, RAPDs, and pedigree data

Nineteen of the major strawberry (Fragaria × ananassa Duch.) cultivars grown in the UnitedStates and Canada were examined for AFLP markerpolymorphisms. For the AFLP reactions, the EcoRI-ACC primer was used in combination with fourMseI primers (MseI-CAC, MseI-CAG,MseI-CAT, or MseI-CTT). Each set ofprimers produced 46–66 scorable fragments ranging insize between 50 and 500 bp. The polymorphic fragmentsproduced from each set of primers were more thansufficient to distinguish among all the cultivars,demonstrating the usefulness of AFLP markers forcultivar identification. Similarity coefficients werecalculated based on data from 228 AFLP markers anddata from 15 previously characterized RAPD markers. The RAPD markers had been specifically selected forfingerprinting purposes because they succesfullydistinguish 41 strawberry cultivars, including the 19cultivars analyzed in this study. Separatedendrograms were constructed based on analysis of theAFLP and RAPD marker data using a neighbor-joiningalgorithm. The dendrograms were compared and found tobe very different. Correlations between similaritycoefficients calculated from AFLP marker data,similarity coefficients calculated from RAPD markerdata, and coefficients of coancestry calculated frompedigree information were evaluated. Interestingly,a better correlation with the coefficients ofcoancestry was observed with the RAPD marker data thanwith the AFLP marker data.     

Genetic Variation for Regenerative Response in Immature Leaflet Cultures of the Cultivated Peanut,Arachis hypogaea*

Forty-seven genotypes of the cultivated peanut, Arachis hypogaea L., were screened for regenerative responses of immature leaflet cultures on two media varying in the auxin: cytokinin ratio. Statistical analysis revealed significant media and genotype differences for rhizogenesis plus media differences for callus proliferation but no differences for shoot formation. When genotypes were categorized according to botanical types, differences between subspecies were observed for rhizogenesis. Cultivars of the Virginia botanical type (subspecies hypogaea) were significantly different from either the Valencia or Spanish botanical types (both subspecies fastigiata). Overall, rhizogenesis varied from 0 to 70% among all genotypes. Shoot production was low (30%) while callus proliferation was extremely high (70%).     

Molecular characterization of <Emphasis Type="Italic">Fusarium</Emphasis> head blight resistance from wheat variety Wangshuibai

Fusarium head blight (FHB) is a destructive disease of wheat worldwide. FHB resistance genes from Sumai 3 and its derivatives such as Ning 7840 have been well characterized through molecular mapping. In this study, resistance genes in Wangshuibai, a Chinese landrace with high and stable FHB resistance, were analyzed through molecular mapping. A population of 104 F₂-derived F₇ recombinant inbred lines (RILs) was developed from the cross between resistant landrace Wangshuibai and susceptible variety Alondras. A total of 32 informative amplified fragment length polymorphism (AFLP) primer pairs (EcoRI/MseI) amplified 410 AFLP markers segregating among the RILs. Among them, 250 markers were mapped in 23 linkage groups covering a genetic distance of 2,430 cM. In addition, 90 simple sequence repeat (SSR) markers were integrated into the AFLP map. Fifteen markers associated with three quantitative trait loci (QTL) for FHB resistance (P < 0.01) were located on two chromosomes. One QTL was mapped on 1B and two others were mapped on 3B. One QTL on 3BS showed a major effect and explained up to 23.8% of the phenotypic variation for type II FHB resistance.     

Simple sequence repeat markers for botanical varieties of cultivated peanut (Arachis hypogaea L.)

Cultivated peanut (Arachis hypogaea L.) consists of six botanical varieties. Identification of DNA markers associated with botanical varieties would be useful in plant genotyping, germplasm management, and evolutionary studies. We have developed 130 simple sequence repeat (SSR) markers in peanut, 38 of which were used in this study because of their ability in detecting genetic polymorphism among 24 peanut accessions. Eight SSR markers were found useful to classify botanical varieties. Among them, six SSR markers were specific to botanical varieties fastigiata and vulgaris, one to botanical varieties hypogaea and hirsuta, and one to botanical varieties peruviana, and aequatoriana. Also, three of them derived from peanut expressed sequence tags (ESTs) were associated with putative genes. As botanical varieties have different morphological traits and belong to different subspecies in A. hypogaea, these markers might be associated with genes involved in the expression of morphological traits. The results also suggested that SSRs (also called microsatellites) might play a role in shaping evolution of cultivated peanut. Multiplex PCR of botanical variety-specific markers could be applied to facilitate efficient genotyping of the peanut lines.     

Molecular mapping of a new gene for resistance to frogeye leaf spot of soya bean in 'Peking'

Amplified fragment length polymorphism (AFLP) and microsatellite (simple sequence repeat, SSR) techniques were used to map the _RGSp_eking gene, which is resistant to most isolates of Cercospora sojina in the soya bean cultivar ‘Peking’. The mapping was conducted using a defined F₂ population derived from the cross of ‘Peking’(resistant) בLee’(susceptible). Of 64 EcoRI and MseI primer combinations, 30 produced polymorphisms between the two parents. The F₂ population, consisting of 116 individuals, was screened with the 30 AFLP primer pairs and three mapped SSR markers to detect markers possibly linked to Rcs_Peking. One AFLP marker amplified by primer pair E‐AAC/M‐CTA and one SSR marker Satt244 were identified to be linked to Res_Peking. The gene was located within a 2.1‐cM interval between markers AACCTA178 and Satt244, 1.1 cM from Satt244 and 1.0 cM from AACCTA178. Since the SSR markers Satt244 and Satt431 have been mapped to molecular linkage group (LG) J of soya bean, the Res_Peking resistance gene was putatively located on the LG J. This will provide soya bean breeders an opportunity to use these markers for marker‐assisted selection for frogeye leaf spot resistance in soya bean.     

Pod Yield Comparison of Pure-Line Peanut Selections Simultaneously Developed from Georgia and Zimbabwe Breeding Programs

Crosses were made between two widely grown U.S. peanut (Arachis hypogaea L.) cultivars, ‘Florunner’ and Florigiant’, and two genotypes adapted to growing conditions on the plateau of tropical Africa, ‘Makulu Red’ and 486 GKP. F₂ seed populations were equally divided between Georgia and Zimbabwe. Subsequently, pedigree selection was practised simultaneously at both locations in the early segregating generations. The highest yielding pureline selections were then interchanged, and combined yield evaluations were determined over three growing seasons at each location. ‘Florunner’, ‘Florigiant’, and the Georgia pureline selections tested at the Georgia location had significantly higher pod yields than ‘Makulu Red’, 486 GKP, and the Zimbabwe selections. Conversely, the mean yield of the Zimbabwe selections tested m Zimbabwe was significantly higher than that of the Georgia selections. Thus, the breeding environment under which selection is conducted among cross populations strongly influences the yield adaptability of selected peanut genotypes.     

A rapid and reliable PCR-restriction fragment length polymorphism (RFLP) marker for the identification of Amaranthus cruentus species

A rapid and reliable PCR-restriction fragment length polymorphism (RFLP) marker was developed to identify the Amaranthus cruentus species by comparing sequences of the starch branching enzyme (SBE) locus among the three cultivated grain amaranths. We determined the partial SBE genomic sequence in 72 accessions collected from diverse locations around the world by direct sequence analysis. Then, we aligned the gene sequences and searched for restriction enzyme cleavage sites specific to each species for use in the PCR-RFLP analysis. The result indicated that MseI would recognize the sequence 5′-T/TAA-3′ in intron 11 from A. cruentus SBE. A restriction analysis of the amplified 278-bp portion of the SBE gene using the MseI restriction enzyme resulted in species-specific RFLP patterns among A. cruentus, Amaranthus caudatus and Amaranthus hypochondriacus. Two different bands, 174-bp and 104-bp, were generated in A. cruentus, while A. caudatus and A. hypochondriacus remained undigested (278-bp). Thus, we propose that the PCR-RFLP analysis of the amaranth SBE gene provides a sensitive, rapid, simple and useful technique for identifying the A. cruentus species among the cultivated grain amaranths.     

利用SSR标记分析栽培种花生多态性及亲缘关系

利用11对SSR引物对24个花生栽培品种（包括四大类型）进行PCR扩增分析，其中4对检测到明显的多态性，共检测到33个等位基因变异，每一个位点上检测到的等位变异数为5～13个，平均为8.25个。根据扩增结果可以将24个品种中的21个相互区分。供试品种间的遗传相似系数值在0.2～1.0之间，平均为0.4788。根据UPGMA聚类分析结果,供试     

DNA分子标记技术在花生品种鉴定上的应用研究

利用10个中国花生品种,评估了AFL,SRAP,SSR和STS技术在区分相近来源花生品种上的应用效果。结果表明,AFLP和SRAP标记虽然可以揭示这些花生品种的遗传多样性,但在本研究采用的引物对条件下,单独使用任何一对引物不足以区分全部品种,提示我们需要考虑多对引物的配合,或者需要尝试更多的限制酶或引物对;从103对SSR或STS引物中筛选出9对引物,即Lec_1,Ah4_26,Ah4_4,SsS14,SHPAL_1,PG_71,PG_43,PM_36,PG_22,对所采用的10个花生品种的区分率均为100%。鉴于花生栽培种形态学、细胞学和生化标记贫乏,这9对高辨别力的SSR和STS引物对花生遗传研究和品种鉴定工作具有重要价值。