Detection of genetic diversity among populations of sweet cherry (Prunus avium L.) by AFLPs

Summary

The high degree of polymorphism of AFLPs provides an efficient system for identification and genome analysis of sweet cherry (Prunus avium) cultivars and selections. The cultivars of sweet cherry have usually been characterized by assessment of phenotypic and pomological traits. AFLP markers were employed to identify 38 sweet cherry accessions and estimate the genetic diversity among this material. Ten of 18 tested primer combinations were informative with up to 80 bands per primer combination. Seven to 33% of the amplfied bands were polymorphic depending upon primer combination. Allcultivars and selections tested could be clearly identified. The objective of this work was to demonstrate the usefulness of molecular markers in revealing the genetic diversity among different sweet cherry genotypes.     

Use of ISSR,SRAP, and RAPD markers to assess genetic diversity in Turkish melons

The genetic relationships among 63 melon (Cucumis melo L.) genotypes collected from various regions of Turkey were determined by comparing their molecular ISSR, SRAP, and RAPD markers with those of 19 foreign melon genotypes to investigate the taxonomic relationships and genetic variation of Turkish melon germplasm. Total 162 polymorphic markers (69, 18, and 75 obtained from ISSR, SRAP, and RAPD primers, respectively) were used to define the genetic similarity among the melon genotypes by dendrogram or two and three dimensional scalings. The average similarity (SM coefficient) between any two pairs of accessions examined as estimated by molecular variation was 0.73 ± 0.48. Within-group genetic similarities ranged between 0.46 and 0.96. Related genotypes or genotypes collected from similar regions were partitioned to similar clusters. Southeastern Anatolian genotypes were distinctly apart from group inodorus and group cantalupensis (sweet) genotypes. This reinforced the position of Turkey in the secondary genetic diversity center of melon. The genetic diversity among Turkish genotypes (H = 0.28 and I = 0.42) was only a little less than that of the world accessions (H = 0.30 and I = 0.45). On the other hand, the percentage of polymorphic loci among Turkish melon genotypes (90.7%) was even higher than that of the world accessions (87.6%).     

樱桃SRAP-PCR体系优化及其遗传多样性分析

选取亲缘关系较远的3个不同基因型樱桃资源为试材,对影响SRAP标记PCR反应的模板、Mg2+、dNTPs、Taq酶及引物浓度进行了优化,建立了适合于樱桃SRAP标记的扩增体系。反应体系具体为:模板DNA75ng,dNTPs0.2mmol·L-1,Mg2+2.5mmol·L-1,引物0.3μmol·L-1,Taq酶1.0U,反应总体积20μL。采用优化的扩增体系,对45个樱桃种质材料进行了遗传多样性分析,筛选8对扩增清晰且多态性高的引物组合,检测位点共227个,其中多态性位点192个,占84.6%。应用NTSYS-pc软件进行聚类分析(UPGMA),结果表明45个樱桃品种可分为欧洲甜樱桃和中国樱桃2大类,品种间遗传相似系数在0.52～0.98;其中中国樱桃与甜樱桃种间的相似系数最小,表明2类种质具有不同的遗传背景;而组群内的不同品种资源表现了较高的遗传相似性。SRAP分子标记的聚类分析揭示了樱桃品种间亲缘关系与地理分布以及来源相关。     

Assessment of genetic diversity of Latvian and Swedish sweet cherry (Prunus avium L.) genetic resources collections by using SSR (microsatellite) markers

Three previously described highly polymorphic SSR (microsatellite) primer pairs were tested on 126 sweet cherry (Prunus avium L.) accessions to adapt a fast, reliable method for preliminary screening of sweet cherry germplasm collections and to compare two sweet cherry germplasm collections: at the Latvia State Institute of Fruit-Growing, Dobele (LIFG-Dobele) and at the Division of Horticultural Genetics and Plant Breeding at Balsgård, Department of Crop Sciences, Swedish University of Agricultural Sciences (SLU-Balsgård). The SSR loci were highly polymorphic with 4–10 different alleles and 5–18 genotypes. Heterozygosity values ranged from 0.431 to 0.809, gene diversity (PIC) values ranged from 0.400 to 0.753, and the discriminating power of each locus varied from 0.631 to 0.894. The combined discriminating power of all loci was highly effective (0.996). Sixteen identical accession groups with the same allele profile were discovered in both collections. This study demonstrated that SSR fingerprinting with the three primer pairs tested, can be used for preliminary characterization of sweet cherry germplasm collections.     

AFLP and SSR marker analysis of grape rootstocks in Indian grape germplasm

Twenty-one rootstock accessions were analyzed with seven grape microsatellite (SSR) primers and seven AFLP primer combinations. SSR primers detected 56 alleles across 21 genotypes and primer heterozygosity varied from 0.617 to 0.856. Similarly 252 AFLP bands were obtained with seven primer pairs. The average similarity index for different rootstocks was relatively low and ranged from 0.068 to 0.36 for AFLP data and from 0.13 to 0.36 for SSR data. A combination of three SSR primers was sufficient to distinguish 21 rootstocks. In cluster analysis majority of rootstocks belonging to same species grouped together. Although the dendrograms obtained with two marker systems were not identical, rootstocks belonging to same species or having common parents were grouped together in both the dendrograms.     

Genetic Identification and Conservation of Local Turkish Grapevine (<Emphasis Type="Italic">Vitis vinifera</Emphasis> L.) Genotypes on the Edge of Extinction

In the second half of the nineteenth century, intensive renovation of vineyards took place due to the losses caused by phylloxera and local varieties were mostly replaced by several worldwide cultivars. Shift in genotypic structure in favor of modern cultivars resulted in the decrease or even disappearance of regionally typical local varieties. A total of sixty five Turkish grape genotypes, including 5 references (four cultivars and one rootstock), were genotyped with 16 SSR and 15 SRAP markers. Sixteen SSR primers generated a total of 60 SSR amplicons in which 43 were polymorphic with 73.4% average polymorphism percentage. A total of 111 well-resolved clear DNA bands were obtained from 15 SRAP primers. Of these bands, 53 were highly polymorphic with an average of 47.74%. Cluster analysis based on pooled marker data generated a well resolved grouping pattern. The analyzed genotypes grouped basing on their geographical belongings. There were many cultivar pairs on the dendrogram most of which occurred between 0.75 and 0.90 levels. SSR and SRAP data revealed a wide genetic variability as well as certain synonyms among the historical grape varieties cultivated for decades in local vineyards lengthwise the mountainous regions of Konya, Karaman and Mersin provinces. All the genotypes have been maintained in a grapevine germplasm glasshouse. Preservation and use of these endangered genotypes will be helpful to avoid genetic erosion and diversity loss in this part of Turkey. Also, the molecular data generated in this study could be of great use in determining the optimal breeding strategies to allow continued progress in grapevine breeding.     

Genetic relatedness of sweet cherry (Prunus avium L.) cultivars from Ukraine determined by microsatellite markers

Microsatellite (SSR) markers were used to characterise 23 sweet cherry cultivars of Ukrainian, and four cultivars of non-Ukrainian, origin. To assess their genetic diversity and relatedness, 11 pairs of primers were applied to microsatellite loci, resulting in amplification of 66 SSR alleles. The mean value of the number of different alleles, and the polymorphic index content, amount to 7.333 and 0.700, respectively, demonstrating a significant genetic diversity of the investigated sweet cherry cultivars. Four highly polymorphic SSR loci (EMPAS02, EMPAS06, PceGA34, UDP98-412), which belong to the list recommended by the European Cooperative Program for Plant Genetic Resources, can be used as a minimum genetic marker set for identification of the majority of the studied cultivars; however, for successful discrimination of the most similar cultivars, more markers, located on all chromosomes of sweet cherry, appear to be necessary. Application of unweighted variable-group method using averages clustering allowed elucidation of the relatedness among the sweet cherry varieties, and showed that the Ukrainian cultivars combine genetic material of local, western European, and probably Caucasian origin; however, the origin of several cultivars still remains unclear, and should be studied additionally.     

Assessment of genetic diversity in common bean (Phaseolus vulgaris L.) germplasm using amplified fragment length polymorphism (AFLP)

AFLP technique was applied to assess genetic diversity among 44 common bean accessions that included 6 exotic accessions, 15 Indian land races and 23 released varieties. Eight AFLP primer pairs were used that produced 820 products of which 698 were polymorphic (85.12%). Wide variations were observed among all the accessions for the number of amplification products, percent polymorphism and average polymorphism information content (PIC). The Jaccard's similarity indices (J) based on the AFLP profiles were subjected to UPGMA cluster analysis. The dendrogram generated revealed seven major groups. Seventeen out of 23 released varieties were restricted to clusters VI and VII. The value of r = 0.934 in Mantel's test for cophenetic corrlelation applied to the cluster analysis indicated the high fitness of the accessions to a group. The germplasm used in the present study had narrow genetic base, although moderate to high genetic diversity was observed. The details of diversity analysis and the potential use of Indian common bean accessions in common bean breeding programme are provided in the present study.     

不同生态类型西瓜种质资源遗传多样性的SSR分析

采用SSR分子标记技术对96份不同生态型西瓜种质资源的遗传多样性进行研究。从398对引物中筛选出38对扩增条带清晰、多态性高的引物分析供试材料,共得到7043条清晰可辨条带,其中多态性条带826条,占11.7%。平均每对引物对96份材料扩增出185.34条带,其中21.74条具有多态性。96份材料间的相似系数为0.42～0.99。聚类分析结果表明,野生西瓜与栽培西瓜的亲缘关系较远。在栽培品种内华北生态型、华南生态型和西北生态型西瓜的亲缘关系较近,说明我国西瓜种质资源同源性较高,遗传基础狭窄,有必要引入更多种质资源以拓宽西瓜育种背景。     

甜樱桃品种SSR指纹检索系统的开发及遗传多样性分析

用SSR技术开发了甜樱桃( Prunus avium ) 指纹检索系统并进行遗传多样性分析。18对樱桃、桃和杏的引物在19份甜樱桃及2份草原樱桃( P. fruticosa) 品种中共扩增出83个等位位点, 每个 SSR位点的等位位点数2 ～8 个, 平均416 个, 多态性信息量( PIC) 变化范围为0.38 ～0.80, 平均为 0.64。7个甜樱桃品种具有特殊位点或特殊带型。利用UDP98-414、UDP98-406、UDP96-001及PMS40等4 对引物开发的指纹检索系统, 可以区分18个甜樱桃品种。根据遗传距离进行聚类分析, 19个甜樱桃品种 分成2组, 甜樱桃品种间的聚类结果基本反映了供试材料之间的亲缘关系。     

Variety identification and comparative analysis of genetic diversity in yardlong bean (Vigna unguiculata spp. sesquipedalis) using morphological characters,SSR and ISSR analysis

Genetic diversity and relatedness of 23 yardlong bean (Vigna unguiculata spp. sesquipedalis) accessions and 7 accessions of a hybrid between cowpea (V. unguiculata spp. unguiculata) and yardlong bean (dwarf yardlong bean) in Thailand were estimated using morphological characters, simple sequence repeat (SSR) and inter-simple sequence repeat (ISSR) markers. In addition, two mungbean (Vignaradiata (L.) Wilczek) and two blackgram (Vigna mungo (L.) Hepper) accessions were also used as outgroup species for molecular analysis. Five morphological characters were diverse among most accessions. However, five groups of 2–3 accessions could not be distinguished from one another based on these morphological characters alone. Unweighted pair-group arithmetic average (UPGMA) analysis of these characters separated these 30 accessions into 2 major groups; the yardlong bean group and the dwarf yardlong bean group. Eleven of the sixteen SSR primers yielded clear SSRs, ten of which were polymorphic (90.91% polymorphism), detecting a total of 54 alleles with an average of 4.91 alleles per locus. These 10 polymorphic SSR markers successfully distinguished 28 yardlong bean and dwarf yardlong bean accessions. The polymorphic information content (PIC) among genotypes varied from 0.251 to 0.752 with an average of 0.597. Among the 16 ISSR primers used, a total of 312 ISSR fragments were amplified for these three Vigna species, revealing the polymorphism percentage of 91.03%. The average ISSR PIC value (0.197) with the range of 0.137–0.276 was lower than that of SSR. Nevertheless, the average marker index of this multilocus marker was 3.495, which was higher than that of SSR (0.669), owing to the differences in the effective multiplex ratio. In addition, Mantel test cophenetic correlation coefficient was higher for ISSR (0.566) than that of SSR (0.198). These results indicated higher efficiency of ISSR for estimating the levels of genetic diversity and relationships among yardlong beans and dwarf yardlong beans in this study. Pair-wise coefficients of SSR- and ISSR-based genetic similarity among all yardlong bean and dwarf yardlong bean accessions averaged 0.87 and 0.91, respectively, suggesting a narrow genetic base that emphasizes the need to broaden genetic diversity to ensure continued breeding success. Clustering of genotypes within groups was not similar when SSR and ISSR derived dendrograms from UPGMA analysis were compared. It appeared that ISSR was the most effective marker system in determining the genetic variability and relationships among yardlong bean and dwarf yardlong bean accessions and differentiating three Vigna species. In addition, ISSR was also most useful for variety identification since all 30 yardlong beans and dwarf yardlong bean accessions can be effectively distinguished by only four ISSR primers with the highest PIC values.     

萱草部分野生种和栽培品种亲缘关系的AFLP分析

 借助AFLP标记对35份萱草野生种和栽培品种进行亲缘关系研究, 结果表明, 7对引物组合对萱草共扩增出条带380条, 其中多态性条带357条, 平均多态性达到93.39% , 单对引物扩增条带19～85条, 平均每对引物组合扩增多态性条带51条。7对引物扩增出的多态性条带均超过90.0%。种质资源相似系数为0.3822～0.9656, 平均相似系数为0.7039。UPGMA聚类结果将供试材料分为3类, 即早花、中花和晚花类, 同一产地的品种基本能聚在一起。AFLP标记技术能较好地从分子水平揭示萱草种质资源的亲缘关系。     

AFLP analysis of genetic diversity in low chill requiring walnut (Juglans regia L.) genotypes from Hatay,Turkey

In this study, the genetic relatedness of 22 low chill requiring walnut genotypes adapted to the south east Mediterranean region of Turkey was analysed by amplified fragment length polymorphism (AFLP) markers. Relatively low level of genetic variation was detected among the genotypes examined by five AFLP primer combinations, suggesting that these walnut genotypes selected predominantly for their low chill requirement have relatively narrow genetic base. In addition, the geographical proximity of the genotypes analysed was not correlated with their level of genetic relatedness. These results have implications for walnut breeding and conservation.     

Genetic diversity and population genetic structure of pomegranate (Punica granatum L.) in Iran using AFLP markers

Pomegranate (Punica granatum L.) is one of the oldest known edible fruits. It is native to Iran and spread from Iran to other areas. In this study amplified fragment length polymorphism (AFLP) was used to detect intra- and inter-population genetic diversity of pomegranate. A group of 67 accessions belonged to 4 populations from Iran was studied using eight primer combinations. A total of 221 scorable bands were amplified, of which, 118 (54.13%) were polymorphic. Resolving power (Rp) ranged from 5.70 to 9.21, and the average of polymorphism information content (PIC) per primer pair was 0.40. According to Nei's gene diversity and allelic statistics, Isfahan population had a highest genetic diversity (H = 0.3646, I = 0.5327, N_e = 1.6467). Coefficient of gene differentiation between populations (GST) was 0.124, indicated that mainly proportion of genetic variation (87.6%), was within populations and the remaining (12.4%) of the variation was among populations that, also supported by analysis of molecular variance (AMOVA). The gene flow (Nm) varied from 0.969 to 10.404 between pair-wise populations and was 3.504 among all of the populations. The Jaccard similarity coefficient between individuals ranged from 0.26 to 0.88. The UPGMA dendrogram clustered all 67 accessions into 6 groups. In some cases accessions from same region were grouped together but in most cases, there was gene exchange. To study the genetic relationships among populations, a principal coordinate analysis (PCoA) based on Nei's genetic distances was performed. Results of this study showed that AFLP marker can be a useful tool for investigating the genetic diversity of pomegranate genotypes.     

Comparative analysis of genetic diversity in Citrus germplasm collection using AFLP,SSAP, SAMPL and SSR markers

In this study we evaluate the informativeness and efficiency of Amplified Fragment Length Polymorphism (AFLP), Sequence-Specific Amplified Polymorphism (S-SAP), Selectively Amplified Microsatellite Polymorphic Loci (SAMPL) and Simple Sequence Repeat (SSR) markers for genetic diversity, phylogenetic relationship among the Citrus species and mapping ability of the marker system. The SSR exhibited relatively higher level of polymorphism information content in terms of the expected heterozygosity, than that of the AFLPs, SSAPs and SAMPLs. For each marker system, average level of the discriminating potential was very close to the actual discriminating potential. Similarity matrices showed weak, yet significant correlations when Mantel's test was applied. The highest positive (0.72) correlation was found between the AFLP and SSAP markers. The SSR and SAMPL markers were poorly correlated. The dendrogram topology among the four marker systems had high similarity. Taken together, the SSAP and SAMPL were highly efficient in detecting genetic similarity in Citrus, while the SSR may be more useful for segregation studies and genome mapping in Citrus. The SSAP and SAMPL markers could be useful for Citrus genome mapping in combination with AFLP and SSR markers. To our knowledge, this was the first detail report of a comparison of performances among AFLP, SSR and retrotrasposon based molecular marker technique on a set of samples of Citrus. Our result provides guidance for future efficient use of these molecular methods in genetic analysis of Citrus sp. and its relatives.     

Comparative analysis of genetic diversity in Tunisian apricot germplasm using AFLP and SSR markers

Eighty-one accessions representing apricot germplasm in Tunisia were collected from different areas of cultivation and fingerprinted using amplified fragment length polymorphism (AFLP) and microsatellites (SSR) markers. A total of 339 polymorphic markers were revealed using 5 AFLP primers combinations and 24 SSR loci. AFLP and SSR markers expressed a high level of polymorphism allowing the distinction of the accessions with an efficiency coefficient of discrimination of 100% for AFLP and 97% for SSR markers. Genetic diversity structure was assessed with AFLPs and SSRs markers separately then with combined matrix data by the help of hierarchical clustering elaborated using Wards method based on Nei and Li (1979) distances. Comparison of the obtained dendrograms revealed a phylogeographic structure into two major groups with significant conservation between the observed subgroups in relation with the geographic origin of the accessions. The relative efficiency of the markers in determining the genetic relationships among apricot accessions has been assessed and a combination of AFLPs and SSRs markers was the most effective. In addition, Mantel test based on genetic distances indicated highly significant correlation between AFLP-SSR data and each of the AFLP and SSR ones, with Pearson correlation values of r = 0.873 and r = 0.692, respectively, revealing the higher efficiency of the combination of both molecular techniques (AFLP and SSR) to estimate the levels of genetic variability among apricot germplasm.     

野生毛花猕猴桃果实表型性状及SSR遗传多样性分析

以江西省武功山境内的70份野生毛花猕猴桃种质资源为试材,对其果实表型性状、SSR遗传多样性及其亲缘关系进行分析和评价。根据UPOV（国际植物新品种保护联盟）公布的猕猴桃属品种测定标准对供试材料的果实表型性状进行观测。参照猕猴桃遗传连锁图谱,选用分布于中华猕猴桃基因组中的70对SSR引物对供试材料进行多态性分析。结果表明,在70对引物中,21对引物成功扩增出多态性片段。随机采集的70份野生毛花猕猴桃种质资源中,其果实表型性状和DNA分子水平均存在丰富的遗传多样性。基于UPGMA聚类分析将供试的野生毛花猕猴桃资源分为果实圆形和椭圆形混合组、果实椭圆形组以及果实圆柱形组。21对多态性高的SSR引物共检测出127个等位变异位点,变异范围在2 ~ 12之间,平均每对SSR引物可检测到6.04个等位位点。遗传相似系数（GS）变异范围为0.5306 ~ 0.9252。GS值在0.65水平上UPGMA聚类分析可将供试的野生毛花猕猴桃种质资源划分成Ⅰ、Ⅱ和 Ⅲ 共3个组,这与果实表型性状分析的结果基本一致。这些遗传变异丰富的种质资源可为猕猴桃育种提供有价值的材料。     

Identification of 57 sweet orange cultivars using AFLP markers

Summary

Sweet orange (Citrus sinensis) represents an important group of Citrus fruit; however, the identification of sweet orange cultivars during vegetative growth can be difficult. A study on the genetic identification of sweet orange cultivars may be significant for the sweet orange nursery industry, for cultivar-rights protection, and is important for the genetic evaluation and conservation of these orange cultivars. In this study, amplified fragment length polymorphism (AFLP) markers were used to genotype 57 sweet orange cultivars. Ten PCR primer pairs generated 629 unique AFLP bands, with a size range of 50 ? 500 bp. Seventy-four bands (11.8%) were polymorphic. On average, each primer pair produced 62.9 fragments, with 7.4 polymorphic fragments. A dendrogram of the 57 sweet orange cultivars was constructed based on an UPGMA analysis using Jaccard?s coefficients of similarity. This provided a clear comparison of the genetic variation between cultivars and an ability to identify them. From Jaccard?s coefficients, 56 of the 57 cultivars examined were genetically close, with coefficient values ≥ 0.985. ?Variegated Navel? was less closely-related, with a much lower coefficient value (0.94). Among the 57 cultivars, 28 sub-groups, some consisting of only one cultivar, could be separated by their AFLP fingerprints. Compared to ISSR and SSR markers, AFLP seemed to be the preferential marker technique for the identification of sweet orange cultivars.     

Genetic divergence among gerbera accessions evaluated by RAPD

Genetic diversity was evaluated by RAPD markers and morpho-agronomic characters for a total of 42 accessions of Barberton daisy (Gerbera jamesonii) consisting of 29 commercial and 13 wild accessions. A total of 74 polymorphic bands were obtained employing a set of 12 primer pairs. The average genetic similarity coefficient for the 42 accessions, evaluated by Jaccard index was 0.55 ranging from 0.28 to 1.00. The genetic structure found among Barberton daisy accessions was evaluated by hierarchic classification analyses and UPGMA modeling, revealing six clusters of genotypes where two of them include the wild accessions and the remaining four including commercial material, except for wild genotype number 9. Shannon (H′) index was calculated using the molecular markers to investigate the genetic variation among the Gerbera accessions and showed higher values for the commercial cluster in comparison to the values obtained for the individuals from the non-commercial cluster, namely 0.34 versus 0.27, respectively. Therefore, both calculated indices (Jaccard and Shannon) indicated the presence of higher genetic variation among commercial accessions in comparison to the cluster representing non-commercial accessions, suggesting that genetic breeding programs may focus on commercial accessions to recombine interesting genotypes with commercially important and marketing-desired characteristics.     

厚皮甜瓜遗传多样性的SSR分析

采用61对SSR特异引物对46份厚皮甜瓜材料进行分析,其中52对扩增出条带,47对引物具有多样性,扩增带分子量在150～1500bp,187条谱带中154条谱带具有多态性,多态率占82.35%,平均每个引物可扩增出3.60条带。同时,对材料网纹、果形、单果质量、有无条带、果皮颜色、果肉颜色、肉质、种子、有无麝香味、裂叶、是否自落和花性型等12个性状进行了田间调查,经过phylip软件和NTSYS软件分析后,计算出材料间遗传距离,并做出SSR分子标记和性状2个聚类图。结果显示,46份材料的遗传距离在0.0188～1.2119,而单一性状气味、果皮颜色、果肉颜色和表面网纹的遗传距离都小于0.1,十分接近;2种聚类分析结果存在较大差异,这与供试材料的亲缘关系十分复杂,大部分材料来源相近,具有相同的亲本,和回交亲本存在一定关系。