AFLP, ISSR and RAPD markers reveal high levels of genetic diversity among Lupinus spp.

The Lupinus genus includes a number of important crop species. The use of defined nucleotide sequences for the analysis of genetic diversity among these species has revealed modest levels of diversity. The aim of this study was to access AFLP, ISSR and RAPD markers to evaluate the genetic diversity among L. albus, L. angustifolius, L. cosentinii, L. hispanicus, L. luteus, L. mutabilis, L. pilosus and L. polyphyllus. Unexpectedly, low levels of genetic similarity were found (ranging from 0.205 to 0.432), regardless of the type of molecular marker used. Nevertheless, these techniques consistently showed a greater genetic similarity between L. pilosus and L. cosentinii, L. mutabilis and L. polyphyllus and among L. luteus, L. hispanicus and L. angustifolius, clearly separating the Old World from the New World species. Such low genetic similarity among Lupinus spp. is most unlikely to be due to differences in coding sequences but could be the result of a long diverging process concerning non‐coding regions, which would represent a very important proportion of these genomes.     

AFLP in Triticum aestivum L.: patterns of genetic diversity and genome distribution

The amplified fragment length polymorphism (AFLP) procedure was applied to a diverse panel of wheat (Triticum aestivum L. em. Thell.) accessions and sixty-nine of the recombinant inbred lines (RILs) from the widely used genetic mapping population derived from the cross of Opata 85 and W7984. Most (76.8%) bands were monomorphic among T. aestivum accessions. The majority of bands monomorphic in T. aestivum also were present in the synthetic wheat parent (W7984). Ten primer pairs generated 153 polymorphic AFLP bands, 140 of which could be assigned to a chromosome location and were relatively evenly distributed on the genetic linkage map. AFLP loci in T. aestivum were distributed throughout the genome; they generally have only one detectable sequence variant; and they exhibit monogenic dominant mendelian inheritance. Frequencies of polymorphic bands in the germplasm sampled are in the range that enables informative cluster analyses as well as map-based diversity and association analysis studies. AFLP bands mapped to individual loci in the Opata 85/W7984 RIL population will frequently be polymorphic in other crosses or germplasm, irrespective of whether the band arises from the T. aestivum parent or the synthetic wheat parent. This revised version was published online in July 2006 with corrections to the Cover Date.     

Assessment of genetic variability of olive varieties by microsatellite and AFLP markers

Fourteen developed microsatellite markers were characterized for their use in genotyping and diversity studies of olive varieties. After optimisation of microsatellite assay and allele sizing, ninety-six alleles were found in nineteen varieties, with an average of 6.8 alleles per locus. The characteristics of the microsatellite markers were used to identify markers that can be reliably applied for variety genotyping. Such features were the generation of complex banding patterns supported by underlying allele sequences, `short allele dominance', an unstable repeat structure and a low number of alleles. AFLP analysis was performed on the same set of olive varieties using eight primer pair combinations. The genetic relationships among nineteen olive varieties were compared on the basis of microsatellite and AFLP polymorphisms. Genetic distances between all pairwise combinations of the varieties were calculated using Jaccard's coefficient of similarity and dendrograms were constructed by the UPGMA method. The results of clustering analysis with both molecular systems showed the common genetic background of Tuscan varieties, and genetic divergence within Slovene olive germplasm. Slovenian varieties ‘Buga’, ‘Štorta’ and ‘Samo’ might represent regionally selected olives, while ‘Zelenjak’ and ‘Črnica’ are probably derived from the Central Italian region. The predominant local ‘Istrska belica’ was introduced to Slovenia independently from the other regional varieties and showed the lowest genetic similarity with the other regional varieties. This revised version was published online in August 2006 with corrections to the Cover Date.     

Genetic similarity among European winter triticale elite germplasms assessed with AFLP and comparisons with SSR and pedigree data

Genetic similarities (GS) based on molecular markers are well suited for direct exploration of relationships within a germplasm pool. The objectives of this study were to: (i) assess the genetic diversity in the European winter triticale germplasm by using AFLP markers, and (ii) compare the GS estimates of AFLP markers, simple sequence repeat (SSR) markers and MALÉCOT's coancestry coefficient (f). A representative set of 127 European winter triticale varieties and breeding lines, previously investigated with SSR, was assessed with 10 PstI/TaqI primer combinations (PC). AFLP analysis identified 344 polymorphic fragments with an average polymorphic information content per PC of 0.25 and a marker index of 8.56. GS‐values between genotypes (calculated after DICE) averaged 0.61 for AFLP and 0.43 for SSR. The mean f‐value was 0.06. Dendrograms based on ‘unweighted pair‐group method and arithmetic average’ showed no clear groupings within the triticale germplasm pool, but smaller clusters were consistently found. Both molecular marker systems were superior to the coancestry coefficient for genetic diversity assessment within the elite triticale germplasm.     

Genetic diversity estimates in Cicer using AFLP analysis

Amplified fragment length polymorphism (AFLP) analysis was used to evaluate the genetic variation among cultivated chickpea and wild Cicer relatives. In total, 214 marker loci were assessed, of which 211 were polymorphic (98.6%) across the 95 accessions that represented 17 species of Cicer. The genetic variation within a species was highest in C. pinnatifidum followed by C. reticulatum and lowest in C. macracanthum. Three main species groups were identified by UPGMA clustering using Nei's pair‐wise distance calculations. Group I included the cultivated species C. arietinum, C. reticulatum and C. echinospermum. Within this group, C. reticulatum accessions were clustered closest to the C. arietinum cultivars ‘Lasseter’, ‘Kaniva’ and ‘Bumper’, supporting the hypothesis that C. reticulatum is the most probable progenitor of the cultivated species. Cicer bijugum, C. judaicum and C. pinnatifidum were clustered together creating group II. Group III contained all nine perennial species assessed and two annual species C. yamashitae and C. cuneatum. The genetic distance detected between group I and group III (0.13) was equivalent to the genetic distance detected between group I and group II (the primary and annual tertiary species, respectively; 0.14). This indicated that the perennial tertiary species may be as valuable for increasing variation to incorporate novel germplasm in the cultigen as the annual tertiary species.     

Utility of AFLP markers for the assessment of genetic diversity within Brassica nigra germplasm

Genetic diversity of 18 Brassica nigra accessions was estimated using amplified fragment length polymorphism (AFLP) marker technology. Two B. rapa and two B. juncea accessions were selected as outliers in the study. Eight AFLP primer combinations generated a total of 426 bands, of which 79% were polymorphic. The UPGMA method was employed to construct a dendrogram based on the Jaccard's similarity coefficient. The accessions of B. rapa separated from those of B. nigra at a genetic similarity coefficient of 0.27 while those of B. juncea did so at 0.5. The genetic similarity coefficients within the B. nigra accessions ranged from 0.58 to 0.86. Based on these coefficients it was concluded that the B. nigra accessions show high levels of genetic variation. These results have significant implications in the crop improvement programmes for the agronomically important crop B. juncea, an amphidiploid of B. nigra and B. rapa. Two incorrectly labelled B. nigra accessions were also identified. These accessions were found to cluster with those of B. juncea accessions. This result demonstrates the great value of AFLP markers in the management of genebanks.     

Simplified AFLP procedure as a tool for identification of strawberry cultivars and advanced breeding lines

DNA polymorphisms among 6 cultivars of Fragaria × ananassa (Duch.) and 13salinity tolerant clones were evaluated using simplified – PstI based Amplified Fragment Length Polymorphism procedure(^PstIAFLP). Out of 129 amplification products obtained with 10 selective primers, 116 markers were polymorphic and could be used to distinguish all analyzed materials. Coordinate and cluster analyses revealed 2 main groups of clones and divided strawberry cultivars (CUL) and tested F₁ hybrids of ‘Sweet Heart’(HYB). Mean genetic similarities in groups of cultivars and selected breeding lines (SEL) were significantly higher (0.722 and0.706, respectively, p < 0.05) than in group of SH hybrids (0.485). Results suggest that ^PstIAFLP method is sufficient for effective identification and useful for assessing the level of genetic diversity in strawberry cultivars and breeding lines. The presented method can bean alternative multilocus marker system to widespread RAPD method. This revised version was published online in July 2006 with corrections to the Cover Date.     

Integration of AFLP markers into an RFLP-based map of durum wheat

Amplified fragment length polymorphism (AFLP) is a powerful technique which can readily be applied to a wide range of species for mapping purposes. AFLPs were added to a linkage map of durum wheat constructed using restriction fragment length polymorphisms (RFLPs). The mapping population included 65 recombinant inbred lines derived from a cross between the durum wheat cultivar ‘Messapia’ and accession ‘MG4343’ of the wild Triticum turgidum ssp. dicoccoides (Körn.). Genomic DNA was digested with MseI (4‐cutter) and Sse83871 (8‐cutter). Using a silver‐staining protocol, 14 primer combinations revealed 421 clearly scorable amplicons including 100 polymorphisms. The presence of nine pairs of bands linked in repulsion phase with each pair generated by one primer combination suggested the presence of codominant alleles; sequence analysis of four band pairs confirmed their codominant nature. The integration of 80 AFLP loci extended the map in several telomeric regions, reduced the size of four large gaps present in the previous map, and eliminated one gap. The new map obtained after the inclusion of the 80 AFLP loci and eight additional RFLP loci spans 2063cM which represent a 52.6% increment compared with the previous map. Compared with the distribution of RFLPs, no significant clustering of AFLP markers was observed.     

Assessment of genetic diversity in clover species from Sardinia, Italy, using AFLP analysis

Two species, Trifolium glomeratum and T. nigrescens, from Sardinia, Italy, were analysed for genetic diversity using amplified fragment length polymorphism (AFLP). Variation between and within populations was compared between the inbreeder, T. glomeratum, and the outbreeder, T. nigrescens. Four AFLP primer combinations resulted in a total of 292 loci, of which 75% were polymorphic in T. glomeratum and 85% in Trifolium nigrescens. Variation was highest between populations in both species, but the difference between populations was greater in T. glomeratum (Fst = 0.17), compared with T. nigrescens (Fst = 0.02). Cluster analysis and principal coordinates analysis were used to verify the relationships found. The high level of genetic variation within populations in both species is attributed to the movement of sheep between paddocks, the existence of both species in Sardinia for thousands of years and the persistence of a long‐lived seedbank due to the production of large numbers of small seeds with high levels of hard seededness.     

Genetic characterization and identification of new accessions from Syria in an olive germplasm bank by means of RAPD markers

Thirty-two olive cultivar accessions from Syria, most of them obtained from collecting expeditions, were characterized by means of RAPD markers before being introduced in the World Germplasm Bank of Cordoba. A total of 79 polymorphic bands(6.1 polymorphisms per primer) out of 93(7.1 bands per primer) were scored for the13 primers used, corresponding to 84.9% of the amplification products. Thirty-one different genotypes were clearly discriminated. Differences were not found among the amplification profiles from different individuals of the same cultivar. Only two cases of mislabeling or errors of planting were found. Fourteen accessions corresponding to 6 homonyms were discriminated by RAPDs as different genotypes. The dendrogram obtained by RAPD analysis included three major groups. Some evidence of relationships of the Syrian accessions studied according to their geographic origin and/or diffusion was found. For instance, cultivars from the Central Syria (Tadmur/Palmyra)such as Toffahi', ‘Abbadi Abo Gabra’-1033,‘Abo Kanani’, ‘Shami’-1041, ‘Abbadi Shalal’ ‘Adgam’-844 and ‘Majhol’-1013 clustered in Group 1 and 2. Six cultivars from Northern Syria clustered in Group 2. But it was not found a geographic structure for the cultivars from South and West of Syria. These results agree with the hypothesis of autochthonous origin of most of the olive cultivars. Some associations between cultivars from Central Syria and their fruit size were observed. This suggests that fruit size was a criterion of local selection in olive cultivars of this area. This revised version was published online in July 2006 with corrections to the Cover Date.     

RAPD genetic diversity of Albanian olive germplasm and its relationships with other Mediterranean countries

RAPD markers were used for the study of 19Albanian olive cultivars and two wild olives (oleasters). A total of 76polymorphic bands (4.8 polymorphic markers per primer) out of 107 reproducible were obtained using 16 primers. The number of bands per primer ranged from 4 to 10,whereas the number of polymorphic bands ranged from 1 to 9, corresponding to 71%of the total amplification products. All the accessions could be identified by the combination of four primers: OPA-19;OPA-02; OPK-16 and OPP-19. The dendrogram,based on Jaccard's index, included three major groups according to their origin: 1)most of the cultivars from the area of Berat (South of Albania) 2) cultivars from the Centre and Centre-North of Albania and3) cultivars from the Centre and North-West of Albania along with the oleaster from Elbasan. In order to evaluate the origin of Albanian cultivars they were compared to those diffused in other countries like Greece, Italy and Turkey, due to geographical and historical affinity among these countries, by using a one way AMOVA. Although most of the genetic diversity was attributable to differences among cultivars within each country (91.47%) significantφ-values among countries(φ_st = 0.085; p < 0.001)suggested the existence of RAPD phenotypic differentiation. Significant φ-values in all pairs formed by Albania with the other countries were observed. These results are consistent with the autochthonous origin of Albanian cultivars. This revised version was published online in August 2006 with corrections to the Cover Date.     

118份甘蔗种质资源遗传多样性的AFLP分析

采用AFLP标记技术,对来自中国、澳大利亚、美国、菲律宾、巴西和法国的118份甘蔗种质进行遗传多样性分析。采用10对引物组合共扩增出1310条谱带,其中多态性条带为1195条,多态性条带比率为91.2%。118份种质间的相似性系数在0.583~0.929之间,平均为0.750,多态信息量为0.2332,每个位点的有效等位基因数为1.3789。结果显示在相似性系数0.69处切割时,可划分为2个类群,第I类群包括粤糖00-236、云蔗04-622和SP80-1816;第II类群有115份种质,在相似性系数0.74处切割时,可将第II类群划分为5个亚群。各国甘蔗种质亲缘关系较近,其中美国种质遗传多样性相对较丰富。     

Genetic diversity and phylogenetic relationships among accessions of hop, Humulus lupulus, as determined by amplified fragment length polymorphism fingerprinting compared with pedigree data

DNA fingerprinting using amplified fragment length polymorphisms (AFLPs) was successfully employed to detect genetic relationships and variability among 90 hop cultivars and breeding lines comprising a collection of the world's hop germplasm. Seven AFLP primer combinations produced a total of 347 fragments of which 151 (43.5%)) were polymorphic. One‐hundred and thirty informative, highly reproducible DNA polymorphisms were used to estimate the genetic similarity (GS) which varied between 1.0 (e.g. ‘Saazer’ vs. ‘Tettnanger’) and 1.17 (‘Columbus’ vs. ‘Tettnanger’, ‘Spalter’ and ‘Saazer’). UPGMA (unweighted pair‐group method with arithmetic averages) clustering revealed two main clusters, reflecting the two main sources of origin and the two main breeding objectives: one cluster of mainly European origin representing the aroma pool and a second cluster associating accessions with European germplasm infiltrated by wild American genes with less aroma quality, but a higher bittering potential. Each main branch was composed of four or three subclusters with subgroups, respectively. Assignment of almost all genotypes in the dendrogram was consistent with the pedigree data as far as they are known. Consequently, AFLPs are shown to be suitable for assessing the genetic variability in hop germplasm and are useful for describing the genetic relationships among cultivars and accessions, which allows phylogenetic questions to be addressed.     

Assessment of genetic diversity in selected breeding lines and cultivars of canola quality Brassica juncea and their implications for canola breeding

AFLP markers were used to assess the genetic diversity of 77 breeding lines from three of the world's major canola qualityBrassica juncea breeding programs from Canada (Agriculture and Agri-Food Canada and Saskatchewan Wheat Pool) and Australia (Agriculture Victoria). The objectives of the paper were to assess the genetic diversity within and between these three breeding programs and to assess genetic diversity of the canola quality germplasm as compared to mustard quality B. juncea. Fifteen lines of mustard quality B. juncea from India, China, Russia and Australia were also included in the investigation. Ten EcoR1/Mse1 based primer pairs generated 751 scorable fragments with an average of 26 polymorphic bands per primer pair (35%). Similarity coefficients were calculated using the Simple Matching coefficient and adendrogram was developed using the UPGMA procedure, resulting in germplasm being partitioned into five main groups. Line specific markers were discovered that have potential in enhancing the efficiency of individual breeding programs using breeding techniques like accelerated backcrossing. Further understanding the genetic diversity within and between programs has implications for future breeding and collaboration within and between the three programs. This revised version was published online in July 2006 with corrections to the Cover Date.     

AFLP analysis of genetic diversity within and between populations of perennial ryegrass (Lolium perenne L.)

Amplified fragment length polymorphism (AFLP) analysis has been used to measure genetic diversity in perennial ryegrass (Lolium perenne L.) and to relate intra- and interpopulation variation to breeding history. Cluster analysis of AFLP data from contrasting populations showed features consistent with the origins of these varieties. Significant differences in intrapopulation diversity were detected and partial separation of different cultivars was observed. Restricted base cultivars, derived from small numbers of foundation clones, were suitable for this type of study, allowing near complete discrimination of closely related cultivars. Analysis of bulked samples was based on the pooling of genomic DNA from 20 individuals from 6 selected populations. Cluster analysis of AFLP data from bulked samples produced a phenogram showing relationships consistent with the results of individual analysis. AFLP profiling provides an important tool for the detection and quantification of genetic variation in perennial ryegrass. This revised version was published online in August 2006 with corrections to the Cover Date.     

Identification of AFLP Makers Linked to Early-Bearing Gene in Walnut

Amplified fragment length polymorphism （AFLP）, a novel DNA fmgerprinting technique based on polymerase chain reaction （PCR） with high discriminatory power, has the advantages of high efficiency, rapidness, reliability, and rich polymorphism et. It has been widely used for screening molecular markers, constructing genetic linkage map, analyzing relationship and locating genes （Vos et al., 1995）. However, due to high price of reagents involved in AFLP and requirement of high technology, its potential use has been limited. For walnuts, there are two groups characterized with early-bearing and late-bearing trait, respectively. Early-bearing walnuts ramify early with short intemodes, and begin to blossom and bear fruits in the first or second year or so. Studies show that the gene associated with the early-bearing character is a kind of precious genetic resource in nature and has very high hereditable ability （Zheng et al., 2003; Yamamo et al., 1998; Fang et al., 1999）. Research and employment of this has important meaning not only for the varieties improvement of walnuts, controlling and realizing of earlybearing quality, but also for the studies of flower bud differentiation in fruit trees. This research, combined AFLP technology with bulked segregant analysis （BSA） to screen AFLP markers linked to early-bearing character in walnut, can be applied to early screening of walnut hybrids, constructing genetic linkage map, locating genes and improving varieties.     

黄瓜种质资源遗传多样性的AFLP分析

对23份不同来源的黄瓜材料进行了AFLP分析。18对引物组合共扩增出543条谱带,其中,多态性带为106条,多态率为19.5%。UPGA分类结果将23份黄瓜材料划分为三大类群:第1类群为野生型黄瓜的聚类;第2类群为美国类型黄瓜、荷兰类型黄瓜以及具有荷兰血统的黄瓜的聚类;第3类群中包含所有中国类型黄瓜和2个前苏联类型黄瓜。遗传相似性分析和聚类结果表明:野生黄瓜和栽培黄瓜之间的亲缘关系较远,AFLP标记的分类结果与材料的主要性状特点基本一致。     

Bulked AFLP analysis for the assessment of genetic diversity in white clover (Trifolium repens L.)

The use of bulked leaf samples from individual plants for amplified fragment length polymorphism (AFLP) analysis was evaluated as a tool for assessment of genetic diversity in white clover (Trifolium repens L.). Bulking of leaf samples produced slightly simpler AFLP profiles compared to the combined profiles of individual plants from the same cultivar. Approximately 90% of bands which were present in individual plants were present in bulked samples of the same cultivar. The majority of those absent were rare bands, shared by less than 25% of individual plants. Replicate bulk samples gave almost identical banding patterns, demonstrating the robustness of the bulked AFLP technique. Cluster analysis of AFLP data derived from individual plants resulted in a phenogram similar to that produced from data derived from bulked samples of the same plants. AFLP analysis of bulked samples detected significant amounts of genetic variability among 52 cultivars and accessions with genetic similarity values ranging from 0.42 to 0.92. However, cluster analysis of AFLP data only partially reflected the geographic origin of cultivars and accessions and was not congruent with cluster analysis based on variation for morphophysiological characters. Bulked AFLP analysis provides a powerful tool for rapid assessment of genetic variability in white clover and may also be used for cultivar identification. This revised version was published online in August 2006 with corrections to the Cover Date.     

AFLP分子标记鉴别大白菜品种

本试验采用AFLP技术,研究了90份来自7个不同栽培地区的大白菜品种材料。共筛选了20对引物,不同引物组合检测多态性谱带的能力有很大的差异,多态性谱带的数量从9条到32条不等。其中E—ACA／M—CTG是大白菜品种十分高效的引物组合,共产生7l条清晰的扩增带,其中有32条多态性谱带,多态性谱带的百分率为45．7％。通过该引物组合,能将90个品种全部区分开来。同时应用该引物组合检测2个大白菜杂交品种(北京新2号,京夏王)各10株,其中有1株北京新2号的谱带异常,其余同一品种不同单株的带型完全一致。表明AFLP标记用于研究品种指纹图谱,并鉴别品种是完全可行的。