Genetic diversity of Aquilegia (Ranunculaceae) species and cultivars assessed by AFLPs

Species of the genus Aquilegia are exceptionally diverse in their floral morphology and color, commonly known as columbine. They are widely planted ornamentals and are highly attractive for hummingbirds. However, little is known about their genetic diversity. We examined the genetic diversity of the species and cultivars using amplified fragment length polymorphism (AFLP) markers. Sixteen EcoRI/MseI AFLP primer combinations produced 327 informative polymorphic bands, with a mean of 20.4 bands scored per primer. Jaccard's coefficient of similarity varied from 0.61 to 0.93, indicative of high levels of genetic variation. Cluster analysis using the unweighted pair group method with arithmetic mean algorithm placed the 64 accessions into two main clusters, each divided into two sub-clusters. The AFLP variability was significantly associated with the geographic origins, as the Asian species and the North American species grouped into two distinct clusters. The genetic diversity found among Aquilegia demonstrated the potential value of Chinese germplasm for cultivar improvement and for widening the genetic basis of breeding programs and breeding material selection. We concluded that AFLPs are informative and can provide significant insights for genetic diversity research in columbine species.     

Application of AFLPs to the characterization of grapevine Vitis vinifera L. genetic resources. A case study with accessions from Rioja (Spain)

 AFLPs were used to characterize 67 different grapevine accessions from a collection of D.O.Ca. Rioja in Spain. A correct selection of primers and selective nucleotides allowed us to maximize the number of amplified fragments analyzed per reaction yielding an average of 100 per reaction, 49% of which were polymorphic. Based on the presence or absence of amplified fragments for each genotype resulting from a reaction with two primer combinations, we have established the genetic similarity between the different accessions in the collection. These results allowed us to resolve different genotypes maintained under the same name (homonyms) and to identify the same genotype under different names (synonyms) thus permitting the elimination of redundant germplasm. Furthermore, by providing information on more than 50 polymorphic loci per reaction, a few reactions were sufficient to identify distinct AFLP patterns characteristic of specific clones, with different agronomic and organoleptic features, belonging to the same cultivar. The possibility for clonal identification, shown here for grapevines, can have important implications in the protection and management of clonal selections. Received: 1 February 1998/Accepted: 25 February 1998     

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.     

Genetic diversity in cultivated carioca common beans based on molecular marker analysis

A wide array of molecular markers has been used to investigate the genetic diversity among common bean species. However, the best combination of markers for studying such diversity among common bean cultivars has yet to be determined. Few reports have examined the genetic diversity of the carioca bean, commercially one of the most important common beans in Brazil. In this study, we examined the usefulness of two molecular marker systems (simple sequence repeats - SSRs and amplified fragment length polymorphisms - AFLPs) for assessing the genetic diversity of carioca beans. The amount of information provided by Roger's modified genetic distance was used to analyze SSR data and Jaccards similarity coefficient was used for AFLP data. Seventy SSRs were polymorphic and 20 AFLP primer combinations produced 635 polymorphic bands. Molecular analysis showed that carioca genotypes were quite diverse. AFLPs revealed greater genetic differentiation and variation within the carioca genotypes (Gst = 98% and Fst = 0.83, respectively) than SSRs and provided better resolution for clustering the carioca genotypes. SSRs and AFLPs were both suitable for assessing the genetic diversity of Brazilian carioca genotypes since the number of markers used in each system provided a low coefficient of variation. However, fingerprint profiles were generated faster with AFLPs, making them a better choice for assessing genetic diversity in the carioca germplasm.     

An analysis of genetic diversity in coconut (Cocos nucifera) populations from across the geographic range using sequence-tagged microsatellites (SSRs) and AFLPs

Genetic diversity in 31 individuals from 14 coconut populations across the entire geographic range (2–3 individuals per population) was assessed using sequence-tagged microsatellites (or simple sequence repeats, SSRs) and amplified fragment length polymorphism (AFLP). From the 39 SSR primer sets tested, only two gave patterns that could not be scored and used in the data analysis. The remainder included five SSRs that gave double-locus profiles in which one locus could still be scored separately. The 37 SSRs revealed between 2 and 16 alleles per locus and a total of 339 alleles in the 14 populations. Gene diversity (D = 1-Σp_i ²) ranged from 0.47 to 0.90. Two of the four Dwarf populations were homozygous at all 37 loci, which is consistent with their autogamous (self-fertilising) reproduction. One Dwarf population was heterozygous at one locus but the other (Niu Leka Dwarf), which is known to be cross-pollinating, showed high levels of heterozygosity. Generally, diversity was higher in populations from the South Pacific and South East Asia. Three SSR loci (CNZ46, CN2A5, CN11E6) gave distinct genotypes for all but two populations. The East African populations had higher heterozygosities than those from West Africa, and the populations from Tonga and Fiji generally had distinct alleles from those of the South Pacific. AFLP analysis with 12 primer combinations gave a total of 1106 bands, of which 303 were polymorphic (27%). Similarity matrices were constructed from the two data sets using the proportion of shared alleles for SSRs and a Jaccard coefficient for AFLPs. In each case cluster and principal co-ordinates analyses were performed, with the resultant dendrograms and plots revealing similar relationships among the populations for both approaches. There was generally a good separation of populations, and phenetic relationships were in agreement with those previously shown by RFLPs. The use of SSRs and AFLPs in genetic-diversity analysis for the establishment of germplasm collections is discussed. Received: 27 October 1998 / Accepted: 31 March 1999     

中国食用向日葵种质资源遗传变异的RAPD及AFLP分析

本研究采用RAPD和AFLP方法对23个中国不同地区的食用向日葵（Helianthus annuus L.)骨干品种进行了遗传变异分析,同时对两种标记系统进行了比较。26个RAPD引物产生了总计192条DNA条带,大小分布 于0.26kb-1.98kb之间,其中165条（86．12％）具有多态性,每条引物产生DNA条带的平均数为7．38。8对AFLP引物组合共产生了576条带,分布于100bp-500bp之间,其中的341条具有多态性,多态百分率为76．00％,每对引物组合产生DNA条带的平均数为72。RAPD方法检测的每位点有效等位基因数（1．76）大于AFLP（1．65）,AFLP标记位点的平均多态性信息量（PIC）（0．38）低于RAPD标记位点PIC（0．41）,但AFLP标记具有很高的多态性检测效率（Ai=38．52）。用RAPD标记分析23个食用向日葵材料的亲缘关系,Nei氏相似性系数分布在47．84％－82．06％,平均相似性系数为0．6495,而采用AFLP的Nei氏相似性系数分布在54．15％－83．52％,平均相似性系数为0．6884。RAPD数据的标准差为0．13,而AFLP数据的标准差为0．08。因此,采用RAPD和AFLP方法分析食用向日葵遗传变异,RAPD标记具有较低相似性系数和较高方差而AFLP则相反。源于两种不同标记的遗传相似矩阵的相关系数为0．51,说明采用RAPD和AFLP系统分析食用向日葵遗传变异得到的结果有一定的相关性,无论采用RAPD还是AFLP标记进行聚类分析,都将23个不同基因型的食用向日葵材料分成了三个类群。     

Genetic relationships of Aglaonema species and cultivars inferred from AFLP markers

BACKGROUND AND AIMS: Aglaonema is an important ornamental foliage plant genus, but genetic relationships among its species and cultivars have not been reported. This study analysed genetic relatedness of 54 cultivars derived from nine species using amplified fragment length polymorphism (AFLP) markers. METHODS: Initially, 48 EcoRI + 2/MseI + 3 primer set combinations were screened, from which six primer sets that showed clear scoreable and highly polymorphic fragments were selected and used for AFLP reactions. AFLP fragments were scored and entered into a binary data matrix as discrete variables. Jaccard's coefficient of similarity was calculated for all pair-wise comparisons among the 54 cultivars, and a dendrogram was constructed by the unweighted pair-group method using the arithmetic average (UPGMA). KEY RESULTS: The number of AFLP fragments generated per primer set ranged from 59 to 112 with fragment sizes varying from 50 to 565 bp. A total of 449 AFLP fragments was detected, of which 314 were polymorphic (70 %). All cultivars were clearly differentiated by their AFLP fingerprints. The 54 cultivars were divided into seven clusters; cultivars within each cluster generally share similar morphological characteristics. Cluster I contains 35 cultivars, most of them are interspecific hybrids developed mainly from A. commutatum, A. crispum or A. nitidum. However, Jaccard's similarity coefficients among these hybrids are 0.84 or higher, suggesting that these popular hybrid cultivars are genetically much closer than previously thought. This genetic similarity may imply that A. nitidum and A. crispum are likely progenitors of A. commutatum. CONCLUSIONS: Results of this study demonstrate the efficiency and ease of using AFLP markers for investigating genetic relationships of ornamental foliage plants, a group usually propagated vegetatively. The AFLP markers developed will help future Aglaonema cultivar identification, germplasm conservation and new cultivar development.     

Nuclear and chloroplast DNA variability and phylogeny of Iranian apples (Malus domestica)

This study was conducted to reveal genetic diversity among 23 local apple genotypes using nuclear (RAPD) and chloroplast DNA (PCR-cpRFLP) markers. Eleven RAPD primers and four cpDNA primer combinations were used in this study. RAPD primers produced a total of 77 polymorphic fragments with an average of seven bands per primer. The percentage of polymorphic bands (68.14 %) showed the efficiency of used RAPD primers in distinguishing all the genotypes considered. Genetic similarity between studied genotypes varied from 0.38 to 0.72 and cluster analysis showed the abundant diversity, indicating high intraspecific genetic variation between Iranian apple genotypes. From the four universal chloroplast primer pairs, three primer pairs amplified the fragments and their combinations showed polymorphic patterns and revealed intraspecific chloroplast variation. The information will facilitate germplasm identification, conservation and new cultivar development.     

Widespread amplification of amplified fragment length polymorphisms (AFLPs) in marine Antarctic animals

Although recent years have witnessed a rapid growth in the number of genetic studies of Antarctic organisms, relatively few studies have so far used nuclear markers, possibly due to the perceived cost and difficulty of isolating markers such as microsatellites. However, an often overlooked alternative is to use amplified fragment length polymorphisms (AFLPs), a versatile and low-cost method capable of generating large numbers of predominantly nuclear loci in virtually any organism. We conducted a literature review of population genetic studies of Antarctic organisms, finding that fewer than 10% used AFLPs. Moreover, a strong taxonomic bias was found, with studies employing mitochondrial DNA or microsatellites focussing predominantly on animals, while those using AFLPs were mostly of plants or lower organisms. Consequently, we explored the extent to which AFLPs amplify across a range of Antarctic marine animal taxa by genotyping eight individuals each of twelve different species, ranging from echinoderms through soft corals to pelagic fish, at four selective primer combinations. AFLPs readily amplified across all of the taxa, generating between 32 and 84 loci per species, with on average 56.5% of these being polymorphic. In general, levels of polymorphism bore little relationship with expectations based on larger populations of broadcast-spawning species being more variable, though we did find a tentative positive correlation between the number of AFLP bands amplified and a measure of effective population size. Our study lends further support for the utility and ease of use of AFLPs and their suitability for studies of Antarctic species across a wide range of taxa.     

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。     

Genetic diversity in colonial bentgrass (Agrostis capillaris L.) revealed by EcoRI-MseI and PstI-MseI AFLP markers.

Colonial bentgrass (Agrostis capillaris L.) is a potential source for genetic improvement of resistance to environmental stress and disease for other bentgrass species (Agrostis spp.). To conserve and study the existing genetic resources of colonial bentgrass for use in breeding, genetic diversity was investigated using amplified fragment length polymorphism (AFLP) markers. Included in this study were 22 accessions from US Department of Agriculture germplasm collected from 11 countries, in conjunction with 14 accessions from northern Spain and 3 commercial cultivars. Ten EcoRI-MseI and 6 PstI-MseI AFLP primer combinations produced 181 and 128 informative polymorphic bands, respectively. Cluster analysis of genetic similarity estimates revealed a high level of diversity in colonial bentgrass species with averages of 0.51 (EcoRI-MseI) and 0.63 (PstI-MseI). Greater genetic diversity was detected by the EcoRI-MseI AFLP primer combinations. A low but significant positive correlation (r = 0.44, p = 0.0099) between the 2 Jaccard similarity matrices was obtained by the Mantel test. Commercial cultivars of bentgrass showed a narrow genetic background. The assessment of genetic diversity among colonial bentgrass accessions suggested the potential value of the colonial bentgrass germplasm in turfgrass cultivar improvement.     

Genetic relationship of cowpea (Vigna unguiculata) varieties from Senegal based on SSR markers

Genetic diversity and phylogenetic relationships among 22 local cowpea (Vigna unguiculata) varieties and inbred lines collected throughout Senegal were evaluated using simple sequence repeat molecular markers. A set of 49 primer combinations were developed from cowpea genomic/expressed sequence tags and evaluated for their ability to detect polymorphisms among the various cowpea genotypes. Forty-four primer combinations detected polymorphisms, with the remaining five primer sets failing to yield PCR amplification products. From one to 16 alleles were found among the informative primer combinations; their frequencies ranged from 0.60 to 0.95 (mean = 0.79). The genetic diversity of the sample varied from 0.08 to 0.42 (mean = 0.28). The polymorphic information content ranged from 0.08 to 0.33 (mean = 0.23). The local varieties clustered in the same group, except 53-3, 58-53, and 58-57; while Ndoute yellow pods, Ndoute violet pods and Baye Ngagne were in the second group. The photosensitive varieties (Ndoute yellow pods and Ndoute violet pods) were closely clustered in the second group and so were inbred line Mouride and local cultivar 58-57, which is also one of the parents for inbred line Mouride. These molecular markers could be used for selection and identification of elite varieties for cowpea improvement and germplasm management in Senegal.     

鉴定烟草种质资源SSR核心引物筛选和验证

核心引物对遗传多样性分析、种质资源鉴定、品种纯度和真实性检测、指纹图谱构建等研究具有重要价值。本研究利用均匀分布于烟草24条染色体的278对SSR引物,对20份亲缘关系相对较远的烟草材料进行初步筛选,筛选出32对引物。随后再加上10份亲缘关系较近的材料（共30份）对引物进行复筛,最终确定14对为核心引物。将14对引物对39份烟草材料进行进行系谱分析、品种遗传多样性分析和农艺性状分析聚类,结果表明该套SSR核心引物适用于烟草种质资源鉴定和遗传多样性分析。     

Genetic diversity of loquat germplasm (Eriobotrya japonica (Thunb) Lindl) assessed by SSR markers.

Genetic relationships among 40 loquat (Eriobotrya japonica (Thunb) Lindl) accessions that originated from different countries and that are part of the germplasm collection of the Instituto Valenciano de Investigaciones Agrarias (IVIA) (Valencia, Spain) were evaluated using microsatellites. Thirty primer pairs flanking microsatellites previously identified in Malus x domestica (Borkh.) were assayed. Thirteen of them amplified polymorphic products and unambiguously distinguished 34 genotypes from the 40 accessions analyzed. Six accessions showing identical marker patterns were Spanish local varieties thought to have been derived from 'Algerie' by a mutational process very common in loquat species. A total of 39 alleles were detected in the population studied, with a mean value of 2.4 alleles per locus. The expected and observed heterozygosities were 0.46 and 51% on average, respectively, leading to a negative value of the Wright's fixation index (-0.20). The values of these parameters indicate a smaller degree of genetic diversity in the set of loquat accessions analyzed than in other members of the Rosaceae family. Unweighted pair-group method (UPGMA) cluster analysis, based on Nei's genetic distance, generally grouped genotypes according to their geographic origins and pedigrees. The high number of alleles and the high expected heterozygosity detected with SSR markers developed in Malus x domestica (Borkh.) make them a suitable tool for loquat cultivar identification, confirming microsatellite marker transportability among genera in the Rosaceae family.     

AFLP assessment of genetic variability in cassava accessions (Manihot esculenta) resistant and susceptible to the cassava bacterial blight (CBB).

Cassava bacterial blight (CBB) is caused by Xanthomonas axonopodis pv. manihotis (Xam). Resistance is found in Manihot esculenta and, in addition, has been introgressed from a wild relative, M. glaziovii. The resistance is thought to be polygenic and additively inherited. Ninety-three varieties of M. esculenta (Crantz) were assessed by AFLPs for genetic diversity and for resistance to CBB. AFLP analysis was performed using two primer combinations and a 79.2% level of polymorphism was found. The phenogram obtained showed between 74% and 96% genetic similarity among all cassava accessions analysed. The analysis permitted the unique identification of each individual. Two Xam strains were used for resistance screening. Variation in the reaction of cassava varieties to Xam strains was observed for all plant accessions. The correlation of resistance to both strains, had a coefficient of 0.53, suggesting the independence of resistance to each strain. Multiple correspondence analysis showed a random distribution of the resistance/susceptibility response with respect to overall genetic diversity as measured by AFLP analysis. A total heterozygosity index was calculated to determine the diversity within clusters as well as among them. Our results demonstrate that resistance to CBB is broadly distributed in cassava germplasm and that AFLP analysis is an effective and efficient means of providing quantitative estimates of genetic similarities among cassava accessions.     

Comparative assessment of DNA fingerprinting techniques (RAPD, ISSR and AFLP) for genetic analysis of cashew (Anacardium occidentale L.) accessions of India.

Nineteen cashew accessions were analysed with 50 random primers, 12 ISSR primers and 6 AFLP primer pairs to compare the efficiency and utility of these techniques for detecting variation in cashew germplasm. Each marker system could discriminate between all of the accessions, albeit with varied efficiency of polymorphism detection. AFLP exhibited maximum discrimination efficiency with a genotype index of 1. The utility of each molecular marker technique, expressed as marker index, was estimated as a function of average band informativeness and effective multiplex ratio. Marker index was calculated to be more than 10 times higher in AFLP than in RAPD and ISSR. Similarity matrices were determined based on the data generated by molecular and morphometric analyses, and compared for congruency. AFLP displayed no correspondence with RAPD and ISSR. Correlation between ISSR and RAPD similarity matrices was low but significant (r = 0.63; p < 0.005). The similarity matrix based on morphometric markers exhibited no correlation with any of the molecular markers. AFLP, with its superior marker utility, was concluded to be the marker of choice for cashew genetic analysis.     

茄子种质遗传多样性及群体结构的SRAP分析

采用SRAP标记法对183份茄子(Solanum melongena)种质资源的遗传关系和群体结构进行分析。结果表明, 33对多态性SRAP引物组合共扩增出215条清晰稳定的多态性条带, 平均每对引物组合产生7条多态性条带。183份茄子种质的遗传相似系数介于0.276-0.813之间, 平均值为0.623, 表明茄子种质资源间遗传背景存在一定的差异。在遗传距离为0.345 6处可将183份茄子种质分为4组。通过群体结构分析可将种质划分为4个群体, 不同群体间的界限十分明显, 且群体间的基因渗透较高。     

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     

西瓜核心种质的AFLP指纹图谱和SCAR标记

西瓜(Citrullus lanatus (Thunb.) Mansf.)种质资源的鉴定与评价是对其有效利用的基础.以往的研究表明, 西瓜是一种遗传资源特别狭窄的作物,在用同工酶、RAPD及SSR技术对西瓜种质资源进行鉴定时,发现很难将品种完全区分开来.本研究利用高效可靠的AFLP技术,对30个西瓜核心种质材料进行了遗传分析,最终建立了这30个材料的DNA指纹图谱.在该图谱中,每个材料均有其独特的"指纹",材料之间可以相互区分开来.为了进一步利用AFLP分子标记,将重要抗病种质材料"PI296341"的AFLP特异带转化成了生产上可以直接利用的SCAR标记.     

基于SRAP和TRAP标记的河北野生狗牙根种质遗传多样性分析

以SRAP和TRAP 2种标记技术对36份狗牙根材料的遗传多样性及亲缘关系进行了分析,其中包含34份河北省野生狗牙根种质资源。分别由238对SRAP和85对TRAP引物组合中筛选获得具有多态性的SRAP和TRAP引物组合各10对,PCR扩增总条带分别为186和161条,多态性条带156和132条,平均每对引物扩增出多态性条带各15.6和13.2条,多态性位点比率分别为83.4%和81.0%。2种标记合并进行聚类分析,所有供试的36份狗牙根材料遗传相似系数GS=0.519~0.983,平均为0.7。当GS=0.68时,可将36份供试材料分为4个类群。本研究结果表明河北野生狗牙根种质资源存在较丰富的遗传多样性,可为种质资源保护和选育优良狗牙根新品种提供科学依据。