Genetic diversity in wild sweet cherries (Prunus avium) in Turkey revealed by SSR markers

Wild sweet cherry (Prunus avium) trees are abundant in the northern part of Turkey, including the Coruh Valley. We analyzed 18 wild sweet cherry genotypes collected from diverse environments in the upper Coruh Valley in Turkey to determine genetic variation, using 10 SSR primers. These SSR primers generated 46 alleles; the number of alleles per primer ranged from 3 to 7, with a mean of 4.6. The primer PS12A02 gave the highest number of polymorphic bands (N = 7), while CPSCT010, UDAp-401 and UDAp-404 gave the lowest number (N = 3). Seven groups were separated in the dendrogram, although most of the genotypes did not cluster according to phenological and morphological traits. This level of genetic diversity in these wild sweet cherry genotypes is very high and therefore these trees would be useful as breeders for crosses between cultivated sweet cherry and wild genotypes.     

Assessing Genetic Diversity in <Emphasis Type="Italic">Olea europaea</Emphasis> L. Using ISSR and SSR Markers

Olea europaea L. is one of the most economically important crops in the Mediterranean area, and known for having large genetic variability. In order to assess the genetic diversity, DNA from 41 olive cultivars, present in the protected denomination of origin (PDO) region of Trás-os-Montes, was screened using inter simple sequence repeat (ISSR) and microsatellite (SSR) markers. Eleven ISSR primers amplified 135 reproducible bands of which 108 were polymorphic. The percentage of polymorphic bands detected by ISSR was 79%. The highest number of polymorphic bands was obtained by the use of primers UBC807 (15) and UBC809 (16). A total of 67 alleles were detected by six SSR primers, with an average of 11 alleles per primer. The number of alleles per locus ranged from five (ssrOeUaDCA05) to 15 (ssrOeUaDCA03). The observed heterozygosity ranged from 0.219 (ssrOeUaDCA05) to 0.900 (ssrOeUaDCA04), while the expected heterozygosity varied between 0.426 (ssrOeUaDCA05) and 0.887 (ssrOeUaDCA03). The polymorphism information content (PIC) values ranged from 0.392 (ssrOeUaDCA05) to 0.863 (ssrOeUaDCA03). The collection of primers selected gave a reasonable number of amplification products for the genetic diversity analysis. Based on the results, the genetic diversity among 41 olive cultivars is discussed. This study reveals the great importance of guaranteeing the differentiation of olive cultivars and their application for certification purposes.     

Molecular characterisation of sweet cherry (Prunus avium L.) genotypes using peach [Prunus persica (L.) Batsch] SSR sequences

A total of 76 sweet cherry genotypes were screened with 34 microsatellite primer pairs previously developed in peach. Amplification of SSR loci was obtained for 24 of the microsatellite primer pairs, and 14 of them produced polymorphic amplification patterns. On the basis of polymorphism and quality of amplification, a set of nine primer pairs and the resulting 27 informative alleles were used to identify 72 genotype profiles. Of these, 68 correspond to unique cultivar genotypes, and the remaining four correspond to three cultivars that could not be differentiated from the two original genotypes of which they are mutants, and two very closely related cultivars. The mean number of alleles per locus was 3.7 while the mean heterozygosity over the nine polymorphic loci averaged 0.49. The results demonstrate the usefulness of cross-species transferability of microsatellite sequences allowing the discrimination of different genotypes of a fruit tree species with sequences developed in other species of the same genus. UPGMA cluster analysis of the similarity data divided the ancient genotypes studied into two fairly well-defined groups that reflect their geographic origin, one with genotypes originating in southern Europe and the other with the genotypes from northern Europe and North America.     

新疆甜高粱种质资源遗传多样性的SSR分析

选用50对SSR引物对新疆现有72份甜高粱种质资源进行遗传多样性分析。结果表明:有20对引物在72份甜高粱种质资源中表现为多态性。共检测到91个等位基因,每对引物可检测到的等位基因数目为2～5个,平均为3.45个。多态性信息量(PIC)的变动范围为0.2859～0.6652,平均为0.5057。72份甜高粱种质间的遗传相似系数变化范围为0.2001～1.000,平均值为0.5599。UPGMA聚类分析将72份材料划分为A、B两大类群,A群包括69份材料,而A群又被分成从Ⅰ到Ⅺ共11个亚群,B群包括3份材料,农艺性状近似的大多被聚到同一类群。     

Genetic variation and identification of promising sour cherries inferred from microsatellite markers

The aim of this study was to identify the group of highly polymorphic microsatellite markers for identification of promising sour cherries. From among 30 tested microsatellite (SSR) markers, 19 were selected to profile genetic variation in sour cherries due to high polymorphisms. Results indicated a high level of polymorphism of the accessions based on these markers. Totally 148 alleles were generated at 19 SSR loci which 122 alleles were polymorphic. The number of total alleles per locus ranged from 2 to 15 with an average of 7.78 and polymorphism percentage varied from 50 to 100% with an average of 78.76%. Also, PIC varied from 0.47 to 0.89 with an average of 0.79 and heterozygosity ranged from 0.35 to 0.55 with a mean of 0.45. According to these results, these markers specially PMS3, PS12A02, PceGA34, BPPCT021, EMPA004, EMPA018, and Pchgms3 produced good and various levels of amplifications and showed high heterozygosity levels. By the way, the genetic similarity showed a high diversity among the sour cherries. Cluster analysis separated improved cultivars from promising sour cherries, and the PCoA supported the cluster analysis results. Since the studied sour cherries were superior to the improved cultivars and were separated from them in most groups, these sour cherries can be considered as distinct genotypes for further evaluations in the framework of breeding programs and new cultivar identification in cherries. Results also confirmed that the set of microsatellite markers employed in this study demonstrated usefulness of microsatellite markers for the identification of sour cherry genotypes.     

Characterization of microsatellites in wild and sweet cherry (Prunus avium L.)--markers for individual identification and reproductive processes.

Nuclear microsatellites were characterized in Prunus avium and validated as markers for individual and cultivar identification, as well as for studies of pollen- and seed-mediated gene flow. We used 20 primer pairs from a simple sequence repeat (SSR) library of Prunus persica and identified 7 loci harboring polymorphic microsatellite sequences in P. avium. In a natural population of 75 wild cherry trees, the number of alleles per locus ranged from 4 to 9 and expected heterozygosity from 0.39 to 0.77. The variability of the SSR markers allowed an unambiguous identification of individual trees and potential root suckers. Additionally, we analyzed 13 sweet cherry cultivars and differentiated 12 of them. An exclusion probability of 0.984 was calculated, which indicates that the seven loci are suitable markers for paternity analysis. The woody endocarp was successfully used for resolution of all microsatellite loci and exhibited the same multilocus genotype as the mother tree, as shown in a single seed progeny. Hence, SSR fingerprinting of the purely maternal endocarp was also successful in this Prunus species, allowing the identification of the mother tree of the dispersed seeds. The linkage of microsatellite loci with PCR-amplified alleles of the self-incompatibility locus was tested in two full-sib families of sweet cherry cultivars. From low recombination frequencies, we inferred that two loci are linked with the S locus. The present study provides markers that will significantly facilitate studies of spatial genetic variation and gene flow in wild cherry, as well as breeding programs in sweet cherry.     

用SSR和AFLP技术分析花生抗青枯病种质遗传多样性的比较

由Ralstonia solanacearum E.F.Smith引起的青枯病是若干亚洲和非洲国家花生生产的重要限制因子,利用抗病品种是防治这一病害最好的措施。虽然一大批抗青枯病花生种质资源材料已被鉴定出来,但对其遗传多样性没有足够的研究,限制了在育种中的有效利用。本研究以31份对青枯病具有不同抗性的栽培种花生种质为材料,通过简单序列重复(SSR)和扩增片段长度多态性(AFLP)技术分析了它们的遗传多样性。通过78对SSR引物和126对AFLP引物的鉴定,筛选出能显示抗青枯病种质多态性的SSR引物29对和AFLP引物32对。所选用的29对多态性SSR引物共扩增91条多态性带,平均每对引物扩增3.14条多态性带;32对多态性AFLP引物共扩增72条多态性带,平均扩增2.25条多态性带。在所筛选引物中,4对SSR引物(14H06,7G02,3A8,16C6)和1对AFLP引物(P1M62)检测花生多态性的效果优于其他引物。SSR分析获得的31个花生种质的遗传距离为0.12-0.94,平均为0.53,而AFLP分析获得的遗传距离为0.06～0.57,平均为0.25,基于SSR分析的遗传距离大于基于AFLP分析的遗传距离,疏枝亚种组的遗传分化相对大于密枝亚种组。基于两种分析方法所获得的聚类结果基本一致,但SSR数据聚类结果与栽培种花生的形态分类系统更为吻合。根据分析结果,对构建青枯病抗性遗传图谱群体的核心亲本和抗性育种策略提出了建议。     

Development and use of anchored‐SSRs to study DNA polymorphism in bread wheat (Triticum aestivum L.)

In bread wheat, 21 anchored simple sequence repeat (SSR) primer pairs detecting SSR length polymorphism and 42 anchored SSR primers detecting microsatellite‐anchored fragment length polymorphisms (MFLPs) are reported. Eight bread wheat genotypes were used for detecting polymorphism. The number of alleles in SSR analysis ranged from two to six, with a mean of 2.9 alleles per SSR. The number of polymorphic bands in MFLP ranged from two to 40, with a mean of 12.74 polymorphic bands/primer combination, the SSRs with CT/GA motifs giving the highest level of polymorphism (a mean of 18.37 bands). The average value of polymorphic information content (PIC) was 0.473 for SSRs and 0.061 for MFLP.     

Genetic diversity of some Iranian sweet cherry (<Emphasis Type="Italic">Prunus avium</Emphasis>) cultivars using microsatellite markers and morphological traits

The aim of this study was to characterize 23 important Iranian sweet cherry (Prunus avium) cultivars collected from different provinces of Iran and 1 foreign cultivar, which was used as control, considered for breeding programs by using 21 microsatellite markers and 27 morphological traits. In sweet cherry (Prunus avium) accessions, leaf, fruit, and stone morphological characters were evaluated during two consecutive years. The study revealed a high variability in the set of evaluated sweet cherry accessions. The majority of important correlations were determined among variables representing fruit and leaf size and variables related to color. Cluster analysis distinguished sweet cherry accessions into two distinct groups. Principal component analysis (PCA) of qualitative and quantitative morphological parameters explained over 86.59% of total variability in the first seven axes. In PCA, leaf traits such as leaf length and width, and fruit traits such as length, width, and weight, and fruit flesh and juice color were predominant in the first two components, indicating that they were useful for the assessment of sweet cherry germplasm characterization. Out of 21 SSR markers, 16 were polymorphic, producing 177 alleles that varied from 4 to 16 alleles (9.35 on average) with a mean heterozygosity value of 0.82 that produced successful amplifications and revealed DNA polymorphisms. Allele size varied from 95 to 290 bp. Cluster analyses showed that the studied sweet cherry genotypes were classified into five main groups based mainly on their species characteristics and SSR data. In general, our results did not show a clear structuring of genetic variability within the Iranian diffusion area of sweet cherry, so it was not possible to draw any indications on regions of provenance delimitation. The results of this study contribute to a better understanding of sweet cherry genetic variations in Iran, thus making for more efficient programs aimed at preserving biodiversity and more rational planning of the management of reproductive material.     

Microsatellite marker-based identification and genetic relationships of olive cultivars from the Extremadura region of Spain

Seventy-seven olive accessions corresponding to 25 cultivars from the Extremadura region of Spain were studied using four microsatellite or SSR markers in order to fingerprint them, and evaluate genetic similarity and relationships between local and introduced olive cultivars. The number of alleles per locus ranged from 4 to 8, with a mean of 6.25 alleles per primer pair (a total of 25 alleles). The observed heterozygosity ranged from 0.58 to 0.95, while the expected heterozygosity varied between 0.68 and 0.83. The polymorphism information content values ranged from 0.63 to 0.79. The mean polymorphism information content value of 0.70 for the SSR loci provided sufficient discriminating ability to evaluate the genetic diversity among the cultivars. The SSR data allowed unequivocal identification of all the cultivars; a combination of three SSR markers was sufficient to discriminate all 25 olive cultivars. A dendrogram was prepared, using the unweighted pair-group method with arithmetic mean clustering algorithm; it depicted the pattern of relationships between the cultivars. Most of the local cultivars grouped according to their geographic origin. No clear clustering trends were observed when the morphological traits of fruit endocarps or fruit use of cultivars were employed as analysis criteria. We conclude that there is a high level of variability among local olive cultivars from the Extremadura region at both the morphological and molecular levels; these data should be useful for identifying and distinguishing local germplasm.     

Development of EST-derived SSR markers using next-generation sequencing to reveal the genetic diversity of 50 chrysanthemum cultivars

Chrysanthemum plants are popular worldwide as cut flowers, potted, and in gardens. Several hundred cultivars have been commercialized, indicating that there is substantial genetic variations that can be manipulated under cultivations to produce a wide array of phenotypic variation. To study the genetic diversity of chrysanthemum cultivars in Korea, we first identified simple sequence repeats from chrysanthemum expressed sequence tags generated by FLX 454 sequencing. A total of 1109 ESTs out of 18,226 chrysanthemum ESTs were identified to carry SSRs. A total of 16 out of 46 primer pairs exhibited several polymorphisms among 50 chrysanthemum cultivars. The number of alleles per locus varied from 1 to 15, with an average of 6.25 alleles. The expected heterozygosity ranged from 0 to 0.8958, whereas polymorphism information content ranged from 0 to 0.8872. Based on polymorphisms using 16 SSR markers, a phylogenetic tree was generated revealing four groups within the 50 cultivars showing various levels of genetic diversity. The 16 polymorphic chrysanthemum SSR markers generated in this study would be useful for studies of the genetic conservation, diversity, and population structure of commercial chrysanthemum cultivars as well as closely related species.     

黄淮麦区以1B/1R类品种为抗源主要育成小麦品种的遗传多样性分析

采用SSR技术对黄淮麦区以1B／1R类品种为抗源育成的38个小麦品种进行聚类分析。39个SSR引物共扩增出186条谱带,其中143务为多态性条带,占76．9％。每个引物可扩增出1～9条多态性条带,平均3．7条。位点多态性信息含量PIC变幅为0．320-0．857,平均为0．634。聚类分析表明,在遗传距离GD值0．32水平上38个小麦品种可聚成六大类。品种间遗传距离GD变幅为0．10769～0．48571。SSR标记揭示出这38个具有黑麦血缘的小麦品种遗传变异较小,遗传基础比较狭窄。     

Identification and genetic diversity of plum cultivars grown in Belarus

A study of the collection of sour cherry, sweet cherry, common plum, diploid and tetraploid types of plums, and apricots grown in Belarus carried out using 20 SSR markers showed that they are characterized by high genetic diversity. Among 106 genotypes, 524 polymorphic alleles were identified. The average number of alleles was 15.4 in common plum samples, 11.3 in diploid and tetraploid plum, 9.3 in sour cherry, 6.0 in apricot, and 4.9 in sweet cherry. The greatest genetic diversity is characteristic of common plum cultivars (PD = 0.811). The genetic diversity decreases as follows: diploid plum (PD = 0.741), sour cherry (PD = 0.721), apricot (PD = 0.673), and sweet cherry (PD = 0.655). Cluster analysis shows that the degree of intraspecific divergence in sour cherry and sweet cherry cultivars is less than that of common plum, diploid plum, and apricot plum. Although apricots and plums belong to the subgenus Prunophora, according to the results of SSR analysis, apricot cultivars form a cluster that is more distant from both Cerasus and Prunophora. A set of seven SSR markers (EMPA001, EMPA005, EMPA018, EMPA026 and BPPCT025, BPPCT026, BPPCT039) was selected for DNA identification of cultivars of sour cherry, sweet cherry, common plum, diploid plum, and apricot, as well as species and interspecies hybrids.     

Transferability and Level of Heterozygosity of Microsatellite Markers in <Emphasis Type="Italic">Citrus</Emphasis> Species

Microsatellite markers are a powerful tool for genetic studies, including germplasm conservation, cultivar identification, and integration of linkage maps. Several works have shown that primer pairs designed for one species can be used in related species to facilitate wider application because it reduces the costs for primer development. The objective of this study was to evaluate the transferability of microsatellite primers which was previously developed from the genomic library of Pêra sweet orange (Citrus sinensis L. Osbeck) and to determine the level of heterozygosity between citrus accessions and related genera. Twenty-four microsatellite loci were evaluated on 12 genotypes of Citrus, Poncirus, and an intergeneric hybrid. All analyzed markers were transferable across all genotypes. Seventeen loci were polymorphic, and the number of alleles per loci ranged from one to six. The lowest level of heterozygosity was observed for Poncirus trifoliata (L.) Raf. cultivars while the highest level was for Swingle citrumelo. In general, microsatellite markers showed wide genetic variation and demonstrated that they can be useful in citrus breeding programs.     

中国主栽香菇品种SSR指纹图谱的构建

以商业栽培的25个香菇(Lentinula edodes)品种为材料,应用SSR分子标记技术进行区别性分析。本研究使用14对引物,引物的多态性为100%,每对引物产生的等位基因数为2～9个,平均5.0个,基因型数为2～12个,平均6.3个。预期杂合度为0.1151～0.8131,平均预期杂合度为0.6126;PIC值为0.1064～0.7736,平均PIC值为0.5541。25个品种中,除申香10号和申香12号不能区分外,对其他23个品种清晰鉴别,为构建香菇栽培品种的SSR分子指纹图谱提供了依据和方法。本方法获得的数据可以成为重复性良好、实验室间可比对的香菇栽培品种标准指纹图谱,在品种特异性鉴定中不再需要已有所有品种做参照,较RAPD、ISSR、SRAP等鉴定方法工作量大大减少。     

基于转录组测序信息的水生植物莕菜SSR标记开发

利用软件MISA对水生植物莕菜转录组测序所获得的79536条EST序列进行分析,共检测出12319个EST-SSR位点。在莕菜的EST-SSR中,二核苷酸和三核苷酸重复单元是主导类型,分别占总SSR的57.31%和30.87%,其中AG/CT、AAG/CTT分别是二、三重复单元类型的优势重复基元,分别占总SSR的29.76%和8.66%。随机挑选了130对EST-SSR引物对莕菜两个居群进行遗传多样性检测,结果发现：78对引物能扩增出清晰可分辨的条带,其中37对能成功检测出多态性,引物多态率为 47.44%。这些多态性引物共检测出114个等位基因,每个位点2~6个,平均3.08个。观测杂合度（H_o）及预期杂合度（H_e）分别在0.229~1.000和0.351~0.756之间,多态信息含量PIC值在0.286~0.698之间,平均达0.495。以上研究结果表明,通过莕菜转录组测序产生的EST数据来开发SSR标记是一种简单而高效的途径,这些新的SSR分子标记为研究莕菜的居群遗传多样性及其遗传结构提供了工具。     

利用SSR分子标记进行海岛棉遗传多样性研究

利用SSR分子标记,对20世纪50年代我国引入海岛棉以来培育的45个国内品种(系)及8个国外品种的遗传多样性进行研究.通过256对SSR引物的筛选,选择24对扩增效果好的引物对53个海岛棉种质资源进行遗传多样性的检测分析,共检测出106个等位位点,每对引物等位位点数在2～8之间,平均为4.4.其中多态性等位基因变异97个,占91.5%.位点多态性信息含量平均为0.688,最高为0.848,最低为0.245.利用NTSYSpc2.1软件,分别计算农艺经济性状的欧氏距离(Euclid)和分子标记数据的Jaccard系数矩阵,采用UPGMA法对所选材料进行聚类分析.结果表明,两个树状聚类图基本吻合,53个品种被分为两大类,与系谱来源一致.实验证明SSR分子标记在鉴别品种和品种遗传多样性研究方面具有重要作用.     

云南莲瓣兰主栽品种SSR指纹图谱的构建和遗传差异分析

为了解云南莲瓣兰(Cymbidium tortisepalum)的遗传多样性,利用SSR技术对32个莲瓣兰主栽品种进行遗传变异分析,并构建莲瓣兰栽培品种的指纹图谱。结果表明,筛选出的12对多态性高、稳定性好的引物共检测到95个等位基因,每对引物检测到4~18个等位基因,有效等位基因数(N E)为61.489,平均有效等位基因数(NA)为5.124,Shannon信息指数(I)和多态性信息含量(PIC)分别为0.806~2.624和0.789~0.953。12对引物中,以引物SSR03的等位基因数、NE、观测杂合度、I和PIC最高。32个品种在12对引物上都具有不同的特异性条带,可以彼此区别。从12对引物中筛选出3对核心引物SSR02、SSR03和SSR12构建了莲瓣兰主栽品种SSR分子指纹图谱,这3对核心引物组合即可鉴定32个莲瓣兰栽培品种。这为莲瓣兰的品种鉴定、遗传多样性分析和分子育种研究提供理论基础和技术支持。     

Genetic diversity of different apricot geographical groups determined by SSR markers.

Forty apricot cultivars with different geographic origins belonging to the germplasm collections of St. Istvan University (Budapest, Hungary) and the Instituto Valenciano de Investigaciones Agrarias (IVIA) (Valencia, Spain) were studied by means of SSR markers. The aim of the study was to determine the genetic relationships among genotypes from different eco-geographical groups. Sixteen primer pairs flanking microsatellite sequences in the peach genome were assayed. Eleven of them were polymorphic in the set of cultivars studied and allowed every genotype to be unambiguously distinguished. Genetic diversity in the population studied was analyzed using several variability parameters. A total of 34 alleles were detected with a mean value of 3.1 alleles/locus. The expected heterozygosity mean was 0.46 and the observed heterozygosity was 32% on an average leading to a high value of the Wright's fixation index (0.32). Additionally, UPGMA cluster analysis based on Nei's genetic distance grouped genotypes according to their geographic origins and pedigrees. SSR markers have proved to be an efficient tool for fingerprinting cultivars and conducting genetic-diversity studies in apricot.     

基于转录组测序信息的水生植物蕃菜SSR标记开发

利用软件MISA对水生植物蓄菜转录组测序所获得的79536条EST序列进行分析,共检测出12319个EST—SSR位点。在蓄菜的EST—SSR中,二核苷酸和三核苷酸重复单元是主导类型,分别占总SSR的57．31％和30．87％,其中AG／CT、AAG／C1_r分别是二、三重复单元类型的优势重复基元,分别占总SSR的29．76％和8．66％。随机挑选了130对EST-SSR引物对蓄菜两个居群进行遗传多样性检测,结果发现：78对引物能扩增出清晰可分辨的条带,其中37对能成功检测出多态性,引物多态率为47．44％。这些多态性引物共检测出114个等位基因,每个位点2—6个,平均3．08个。观测杂合度（心）及预期杂合度（He）分别在0．229～1．000和0．351-0．756之间,多态信息含量PIC值在0．286～O．698之间,平均达0．495。以上研究结果表明,通过萏菜转录组测序产生的EsT数据来开发SSR标记是一种简单而高效的途径,这些新的SSR分子标记为研究苔菜的居群遗传多样性及其遗传结构提供了工具。