利用RFLP、SSR、AFLP和RAPD标记分析玉米自交系遗传多样性的比较研究

利用 ＲＦＬＰ、ＳＳＲ．ＡＦＬＰ和ＲＡＰＤ ４种分子标记方法研究了 １５个玉米（Ｚｅａ ｍａｙｓ Ｌ．）自交系的遗传多样性,同时对４种标记系统进行比较。在供试材料中筛选到具多态性的ＲＦＬＰ探针酶组合５６个,６６对ＳＳＲ引物,２０个ＲＡＰＤ引物和９个ＡＦＬＰ引物组合,分别检测到多态性带１６７、２０１、８７和１０８条。ＳＳＲ标记位点的平均多态性信息量（ＰＩＣ）最大（０．５４）,ＡＦＬＰ标记位点最小（０．３６）,但ＡＦＬＰ标记具有最高的多态性检测效率（Ａｉ,３２．２）。４种分子标记所得遗传相似系数相关性显著,比较相关系数表明 ＲＡＰＤ可靠性较低。依据 ４种分子标记结果将 １５个供试自交系划分为塘四平头、旅大红骨、兰卡斯特、瑞德和ＰＮ共５个类群,与系谱分析基本一致。认为ＳＳＲ和ＲＦＬＰ两种分子标记方法适合进行玉米种质遗传多样性的研究。     

Comparative study of the discriminating capacity of RAPD,AFLP and SSR markers and of their effectiveness in establishing genetic relationships in olive

RAPDs, AFLPs and SSRs were compared in terms of their informativeness and efficiency in a study of genetic diversity and relationships among 32 olive cultivars cultivated in Italy and Spain. SSRs presented a higher level of polymorphism and a greater information content, as assessed by the expected heterozygosity, than AFLPs and RAPDs. The lowest values of expected heterozygosity were obtained for AFLPs, which, nevertheless were the most efficient marker system due to their capacity to reveal the highest number of bands per reaction and because of the high values achieved for a considerable number of indexes. All three techniques discriminated the genotypes very effectively, but only SSRs were able to discriminate the cultivars Frantoio and Cellina. The correlation coefficients of similarity were statistically significant for all three marker systems used but were lower for the SSR data than for RAPDs and AFLPs. For all markers a high similarity in dendrogram topologies was obtained although some differences were observed. All the dendrograms, including that obtained by the combined use of all the marker data, reflect some relationships for most of the cultivars according to their geographic diffusion. AMOVA analysis detected greater genetic differentiation among cultivars within each country than it did between the two countries.Communicated by H.F. Linskens     

Comparative analysis on the genetic relatedness of Sorghum bicolor accessions from Southern Africa by RAPDs,AFLPs and SSRs

In order to get an overview on the genetic relatedness of sorghum (Sorghum bicolor) landraces and cultivars grown in low-input conditions of small-scale farming systems, 46 sorghum accessions derived from Southern Africa were evaluated on the basis of amplified fragment length polymorphism (AFLPs), random amplified polymorphic DNAs (RAPDs) and simple sequence repeats (SSRs). By this approach all sorghum accessions were uniquely fingerprinted by all marker systems. Mean genetic similarity was estimated at 0.88 based on RAPDs, 0.85 using AFLPs and 0.31 based on SSRs. In addition to this, genetic distance based on SSR data was estimated at 57 according to a stepwise mutation model (Deltamu-SSR). All UPGMA-clusters showed a good fit to the similarity estimates (AFLPs: r = 0.92; RAPDs: r = 0.88; SSRs: r = 0.87; Deltamu-SSRs: r = 0.85). By UPGMA-clustering two main clusters were built on all marker systems comprising landraces on the one hand and newly developed varieties on the other hand. Further sub-groupings were not unequivocal. Genetic diversity (H, DI) was estimated on a similar level within landraces and breeding varieties. Comparing the three approaches to each other, RAPD and AFLP similarity indices were highly correlated (r = 0.81), while the Spearman's rank correlation coefficient between SSRs and AFLPs was r = 0.57 and r = 0.51 between RAPDs and SSRs. Applying a stepwise mutation model on the SSR data resulted in an intermediate correlation coefficient between Deltamu-SSRs and AFLPs (r = 0.66) and RAPDs ( r = 0.67), respectively, while SSRs and Deltamu-SSRs showed a lower correlation coefficient (r = 0.52). The highest bootstrap probabilities were found using AFLPs (56% on average) while SSR, Deltamu-SSR and RAPD-based similarity estimates had low mean bootstrap probabilities (24%, 27%, 30%, respectively). The coefficient of variation (CV) of the estimated genetic similarity decreased with an increasing number of bands and was lowest using AFLPs.     

Direct comparison of levels of genetic variation among barley accessions detected by RFLPs, AFLPs, SSRs and RAPDs

 RFLPs, AFLPs, RAPDs and SSRs were used to determine the genetic relationships among 18 cultivated barley accessions and the results compared to pedigree relationships where these were available. All of the approaches were able to uniquely fingerprint each of the accessions. The four assays differed in the amount of polymorphism detected. For example, all 13 SSR primers were polymorphic, with an average of 5.7 alleles per primer set, while nearly 54% of the fragments generated using AFLPs were monomorphic. The highest diversity index was observed for AFLPs (0.937) and the lowest for RFLP (0.322). Principal co-ordinate analysis (PCoA) clearly separated the spring types from the winter types using RFLP and AFLP data with the two-row winter types forming an intermediate group. Only a small group of spring types clustered together using SSR data with the two-row and six-row winter varieties more widely dispersed. Direct comparisons between genetic similarity (GS) estimates revealed by each of the assays were measured by a number of approaches. Spearman rank correlation ranked over 70% of the pairwise comparisons between AFLPs and RFLPs in the same order. SSRs had the lowest values when compared to the other three assays. These results are discussed in terms of the choice of appropriate technology for different aspects of germplasm evaluation.     

Molecular marker-based genetic diversity assessment of Striga-resistant maize inbred lines

Striga-resistant maize inbred lines are of interest to maize breeding programs in the savannas of Africa where the parasitic weed is endemic and causes severe yield losses in tropical maize. Assessment of the genetic diversity of such inbred lines is useful for their systematic and efficient use in a breeding program. Diversity analysis of 41 Striga-resistant maize inbred lines was conducted using amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers to examine the genetic relationships among these lines and to determine the level of genetic diversity that exists within and between their source populations. The two marker systems generated 262 and 101 polymorphic fragments, respectively. Genetic similarity (GS) values among all possible pairs of inbred lines varied from 0.45 to 0.95, with a mean of 0.61±0.002 for AFLPs, and from 0.21 to 0.92, with a mean of 0.48±0.003, for SSRs. The inbred lines from each source population exhibited a broad range of GS values with the two types of markers. Both AFLPs and SSRs revealed similar levels of within population genetic variation for all source populations. Cluster and principal component analysis of GS estimates with the two markers revealed clear differentiation of the Striga-resistant inbred lines into groups according to their source populations. There was clear separation between early- and late-maturing Striga-resistant inbred lines. Considering the paucity of germplasm with good levels of resistance to Striga in maize, the broad genetic diversity detected within and among source populations demonstrates the genetic potential that exists to improve maize for resistance to Striga.     

Comparison of PCR-based marker systems for the analysis of genetic relationships in cultivated potato

The application of AFLPs, RAPDs and SSRs to examine genetic relationships in the primary northwestern European cultivated potato gene pool was investigated. Sixteen potato cultivars were genotyped using five AFLP primer combinations, 14 RAPD primers, and 17 database-derived SSR primer pairs. All three approaches successfully discriminated between the 16 cultivars using a minimum of one assay. Similarity matrices produced for each marker type on the basis of Nei and Li coefficients showed low correlations when compared with different statistical tests. Dendrograms were produced from these data for each marker system. The usefulness of each system was examined in terms of number of loci revealed (effective multiplex ratio, or EMR) and the amount of polymorphism detected (diversity index, or DI). AFLPs had the highest EMR, and SSRs the highest DI. A single parameter, marker index (MI), which is the product of DI and EMR, was used to evaluate the overall utility of each marker system. The use of these PCR-based marker systems in potato improvement and statutory applications is discussed.Abbreviations: PCR, polymerase chain reaction; AFLP, amplified fragment length polymorphism; RAPD, randomly amplified polymorphic DNA; DNA, deoxyribonucleic acid; EMR, effective multiplex ratio; DI, diversity index; MI, marker index; RFLP, restriction fragment length polymorphism.     

The comparison of RFLP,RAPD, AFLP and SSR (microsatellite) markers for germplasm analysis

The utility of RFLP (restriction fragment length polymorphism), RAPD (random-amplified polymorphic DNA), AFLP (amplified fragment length polymorphism) and SSR (simple sequence repeat, microsatellite) markers in soybean germplasm analysis was determined by evaluating information content (expected heterozygosity), number of loci simultaneously analyzed per experiment (multiplex ratio) and effectiveness in assessing relationships between accessions. SSR markers have the highest expected heterozygosity (0.60), while AFLP markers have the highest effective multiplex ratio (19). A single parameter, defined as the marker index, which is the product of expected heterozygosity and multiplex ratio, may be used to evaluate overall utility of a marker system. A comparison of genetic similarity matrices revealed that, if the comparison involved both cultivated (Glycine max) and wild soybean (Glycine soja) accessions, estimates based on RFLPs, AFLPs and SSRs are highly correlated, indicating congruence between these assays. However, correlations of RAPD marker data with those obtained using other marker systems were lower. This is because RAPDs produce higher estimates of interspecific similarities. If the comparisons involvedG. max only, then overall correlations between marker systems are significantly lower. WithinG. max, RAPD and AFLP similarity estimates are more closely correlated than those involving other marker systems.Abbreviations RFLP restriction fragment length plymorphism - RAPD random-amplified polymorphic DNA - AFLP amplified fragment length polymorphism - SSR simple sequence repeat - PCR polymerase chain reaction - TBE Tris-borate-EDTA buffer - MI marker index - SENA sum of effective numbers of alleles     

Construction of a combined sorghum linkage map from two recombinant inbred populations using AFLP,SSR, RFLP,and RAPD markers,and comparison with other sorghum maps

Sorghum [Sorghum bicolor (L.) Moench] is an important crop in the semi-arid tropics that also receives growing attention in genetic research. A comprehensive reference map of the sorghum genome would be an essential research tool. Here, a combined sorghum linkage map from two recombinant inbred populations was constructed using AFLP, SSR, RFLP and RAPD markers. The map was aligned with other published sorghum maps which are briefly reviewed. The two recombinant inbred populations (RIPs) analyzed in this study consisted of 225 (RIP 1) and 226 (RIP 2) F_3:5 lines, developed from the crosses IS 9830 2 E 36-1 (RIP 1) and N 13 2 E 36-1 (RIP 2), respectively. The genetic map of RIP 1 had a total length of 1,265 cM (Haldane), with 187 markers (125 AFLPs, 45 SSRs, 14 RFLPs, 3 RAPDs) distributed over ten linkage groups. The map of RIP 2 spanned 1,410 cM and contained 228 markers (158 AFLPs, 54 SSRs, 16 RFLPs) in 12 linkage groups. The combined map of the two RIPs contained 339 markers (249 AFLPs, 63 SSRs, 24 RFLPs, 3 RAPDs) on 11 linkage groups and had a length of 1,424 cM. It was in good agreement with other sorghum linkage maps, from which it deviated by a few apparent inversions, deletions, and additional distal regions.     

Genetic diversity analysis in sorghum germplasm as estimated by AFLP, SSR and morpho-agronomical markers

A comparison of the different methods of the estimation of genetic diversity is important to evaluate their utility as a tool in germplasm conservation and plant breeding. Amplified fragment length polymorphism (AFLP), microsatellites or SSR and morphological traits markers were used to evaluate 45 sorghum germplasm for genetic diversity assessment and discrimination power. The mean polymorphism information content (PIC) values were 0.65 (AFLPs) and 0.46 (SSRs). The average pairwise genetic distance estimates were 0.57 (morphological traits), 0.62 (AFLPs) and 0.60 (SSRs) markers data sets. The Shannon diversity index was higher for morphological traits (0.678) than AFLP (0.487) and SSR (0.539). The correlation coefficients obtained by the Mantel matrix correspondence test, which was used to compare the cophenetic matrices for the different markers, showed that estimated values of genetic relationship given for AFLP and SSR markers, as well as for morphological and SSR markers were significantly related (p <0.001). However, morphological and AFLP data showed non-significant correlation (p >0.05). Both data sets from AFLP and SSR allowed all accessions to be uniquely identified; two accessions could not be distinguished by the morphological data. In summary, AFLP and SSR markers proved to be efficient tools in assessing the genetic variability among sorghum genotypes. The patterns of variation appeared to be consistent for the three marker systems, and they can be used for designing breeding programmes, conservation of germplasm and management of sorghum genetic resources.     

Genetic diversity analysis of elite European maize (Zea mays L.) inbred lines using AFLP, SSR, and SNP markers reveals ascertainment bias for a subset of SNPs

Recent advances in high-throughput sequencing technologies have triggered a shift toward single-nucleotide polymorphism (SNP) markers. A systematic bias can be introduced if SNPs are ascertained in a small panel of genotypes and then used for characterizing a larger population (ascertainment bias). With the objective of evaluating a potential ascertainment bias of the Illumina MaizeSNP50 array with respect to elite European maize dent and flint inbred lines, we compared the genetic diversity among these materials based on 731 amplified fragment length polymorphisms (AFLPs), 186 simple sequence repeats (SSRs), 41,434 SNPs of the MaizeSNP50 array (SNP-A), and two subsets of it, i.e., 30,068 Panzea (SNP-P) and 11,366 Syngenta markers (SNP-S). We evaluated the bias effects on major allele frequency, allele number, gene diversity, modified Roger’s distance (MRD), and on molecular variance (AMOVA). We revealed ascertainment bias in SNP-A, compared to AFLPs and SSRs. It affected especially European flint lines analyzed with markers (SNP-S) specifically developed to maximize differences among North American dent germplasm. The bias affected all genetic parameters, but did not substantially alter the relative distances between inbred lines within groups. For these reasons, we conclude that the SNP markers of the MaizeSNP50 array can be employed for breeding purposes in the investigated material. However, attention should be paid in case of comparisons between genotypes belonging to different heterotic groups. In this case, it is advisable to prefer a marker subset with potentially low ascertainment bias, like in our case the SNP-P marker set.