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.     

基于ISSR分子标记的叶子花亲缘关系分析和指纹图谱构建

为分析品种遗传多样性和遗传距离并构建品种聚类图和指纹图谱,该研究从DNA模板浓度、引物浓度、退火温度和循环次数等方面优化了叶子花ISSR-PCR反应体系和反应程序,利用11个ISSR引物对131个叶子花品种进行PCR扩增,扩增产物经琼脂糖凝胶电泳检测。结果表明:优化的ISSR-PCR反应体系中DNA模板浓度为0.5 ng·μL^-1,引物浓度为0.5μmol·L^-1,引物UBC813、UBC814、UBC815、UBC823、UBC824、UBC835、UBC840、UBC841、UBC843、UBC844和UBC876的最佳退火温度分别为52.3、55.9、54.3、54.3、53.6、56.2、56.2、51.9、54.4、54、50℃,循环次数为32。用11个ISSR引物对131个叶子花品种扩增出161条带,其中多态性条带156条,多态性比率为96.89%。单个引物的等位基因数、有效等位基因数、Nei’s基因多样性指数和Shannon’s信息指数分别为1.86~2.00、1.33~1.68、0.21~0.39和0.34~0.57,平均值分别为1.969、1.478、0.294和0.447。引物UBC841的鉴别率最高(80.92%),可有效鉴别106个品种,与引物UBC876结合可将131个叶子花品种完全鉴别开,建立了各品种的指纹图谱。叶子花品种的遗传距离范围为0.00~0.60,平均值为0.365,遗传多样性较低,在遗传距离0.58处,131个品种分为6大类群,聚类分析显示同一个种的品种大多聚在一类,但同一个种仍有品种未聚在一类或亚类、也有多个种的品种聚在一类。该研究较为准确地揭示了叶子花种质资源的遗传多样性,建立的指纹图谱为叶子花品种登记、知识产权保护以及品种鉴定提供了可靠技术和有效手段。     

The effectiveness of ISSR profiling for studying genetic diversity of Aspergillus flavus from peanut-cropped soils in China

We used markers based on inter-simple sequence repeats (ISSR) to examine the genetic diversity of Aspergillus flavus from peanut-cropped soils in China. Of the 100 primers, 22 primers produced clear and reproducible ISSR bands, and the di-nucleotide accounted for 73% of those primers. The size of DNA fragments ranged from 100 to 2000 bp. The primer UBC 834 produced the largest number of polymorphic bands (10), followed by UBC 809, UBC 817, UBC 895, and UBC 899, which all amplified 7 polymorphic bands. Using the five primers, the tested strains were clearly separated based on genetic similarity coefficients (GSC). The range of GSC was from 0.59 to 0.90. In unweighted pair-group method with arithmetic averages (UPGMA) analysis, the A. flavus samples grouped in five clusters. The study showed that the ISSR technology is an effective molecular approach for studying diversity of A. flavus from peanut-cropped soils in China.     

Determination of genetic diversity among Saccharina germplasm using ISSR and RAPD markers

Various species of genus Saccharina are economically important brown macroalgae cultivated in China. The genetic background of the conserved Saccharina germplasm was not clear. In this report, DNA-based molecular markers such as inter simple sequence repeats (ISSR) and random amplified polymorphic DNA (RAPD) were used to assess the genetic diversity and phylogenetic relationships among 48 Saccharina germplasms. A total of 50 ISSR and 50 RAPD primers were tested, of which only 33 polymorphic primers (17 ISSR and 16 RAPD) had an amplified clear and reproducible profile, and could be used. Seventeen ISSR primers yielded a total of 262 bands, of which 256 were polymorphic, and 15.06 polymorphic bands per primer were amplified from 48 kelp gametophytes. Sixteen RAPD primers produced 355 bands, of which 352 were polymorphic, and 22 polymorphic bands per primer were observed across 48 individuals. The simple matching coefficient of ISSR, RAPD and pooled ISSR and RAPD dendrograms ranged from 0.568 to 0.885, 0.670 to 0.873, and 0.667 to 0.862, revealing high genetic diversity. Based on the unweighted pair group method with the arithmetic averaging algorithm (UPGMA) cluster analysis and the principal components analysis (PCA) of ISSR data, the 48 gametophytes were divided into three main groups. The Mantel test revealed a similar polymorphism distribution pattern between ISSR and RAPD markers, the correlation coefficient r was 0.62, and the results indicated that both ISSR and RAPD markers were effective to assess the selected gametophytes, while matrix correlation of the ISSR marker system (r = 0.78) was better than that of the RAPD marker system (r = 0.64). Genetic analysis data from this study were helpful in understanding the genetic relationships among the selected 17 kelp varieties (or lines) and provided guidance for molecular-assisted selection for parental gametophytes of hybrid kelp breeding.     

Evaluation of the genetic fidelity of in vitro-propagated gerbera (<Emphasis Type="Italic">Gerbera jamesonii</Emphasis> Bolus) using DNA-based markers

The genetic fidelity of in vitro-raised gerbera clones was assessed by using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. Out of 35 RAPD and 32 ISSR primers screened, only 12 RAPD and 10 ISSR primers produced clear, reproducible and scorable bands. The 12 RAPD primers produced 54 distinct and scorable bands, with an average of 4.5 bands per primer. The number of scorable bands for ISSR primers varied from 3 (ISSR-14) to 9 (ISSR-07), with an average of 5.5 bands per primer. The number of bands generated per primer was greater in ISSR than RAPD. All banding profiles from micropropagated plants were monomorphic and similar to those of the mother plant. A similarity matrix based on Jaccard’s coefficient revealed that the pair-wise value between the mother and the in vitro-raised plantlets was 1, indicating 100% similarity. This confirmed the true-to-type nature of the in vitro-raised clones.     

Molecular genetic diversity of Satureja bachtiarica

Fifty-seven genotypes from eight population of Satureja bachtiarica was evaluated using fifteen ISSR and eleven RAPD markers. DNA profiling using RAPD primers amplified 84 loci, among which 81 were polymorphic with an average of 7.36 polymorphic fragments per locus. Also, using RAPD markers maximum and minimum polymorphic bands observed for Semyrom (77.38 %) and Farsan (40.48 %) populations, respectively. Semyrom population recorded the highest unbiased expected heterozygosity (0.259) and Shannon’s Indices (0.38). While, the lowest values of unbiased expected heterozygosity (0.172) and Shannon’s Index (0.245) were recorded for Eghlid and Farsan populations, respectively. On the other hand, ISSR primers produced 136 bands, from which 134 were polymorphic with an average of 9.06 polymorphic fragments per primer (98.52 %). The ISSR markers evaluation revealed that maximum and minimum polymorphic bands observed for Semyrom (66.18 %) and Farsan (31.62 %), respectively. Shahrekorud population recorded the highest unbiased expected heterozygosity (0.211) and Shannon’s Indices (0.301). While, the lowest value of unbiased expected heterozygosity (0.175) observed for Farsan and Yazd populations and the lowest Shannon’s Index (0.191) recorded by Farsan population. The overall results of the study revealed that both ISSR and RAPD markers were effective for evaluation of genetic variation of S. bachtiarica.     

Construction of a cucumber genetic linkage map with SRAP markers and location of the genes for lateral branch traits

Using SRAP (sequence-related amplified polymorphism) markers a genetic linkage map of cucumber was constructed with a population consisting of 138 F₂ individuals derived from a cross of the two cucumber lines, S06 and S52. In the survey of parental polymorphisms with 182 primer combinations, 64 polymorphism-revealing primer pairs were screened out, which generated totally 108 polymorphic bands with an average of 1.7 bands per primer pair and at most 6 bands from one primer pair. The constructed molecular linkage map included 92 loci, distributed in seven linkage groups and spanning 1164.2 cM in length with an average genetic distance of 12.6 cM between two neighboring loci. Based on this linkage map, the quantitative trait loci (QTL) for the lateral branch number (lbn) and the lateral branch average length (lbl) in cucumber were identified by QTLMapper1.6. A major QTL lbn1 located between ME11SA4B and ME5EM5 in LG2 could explain 10.63% of the total variation with its positively effecting allele from S06. A major QTL lbl1 located between DC1OD3 and DC1EM14 in LG2 could account for 10.38% of the total variation with its positively effecting allele from S₀₆.     

SRAP在检测黄瓜基因组多态性中的特征

将SRAP (Sequence-Related Amplified Polymorphism)应用于黄瓜(Cucumis sativus L.)遗传图谱和耐高温QTL的定位过程中, 发现SRAP在检测黄瓜亲本基因组多态性中呈现出一些特征。对于每个正向引物, 在与12个不同的反向引物组合时, 产生多态性条带的引物组合数均在5~8个之间; 而对于每个反向引物, 在与11个不同的正向引物组合时, 产生多态性的引物组合数则在2~11个之间, 差异较大。反向引物SA4或EM6与研究的所有正向引物组合时产生的多态性条带分子量完全相同, 这些条带可能是由反向单引物扩增而来的。引物组合OD3ME11扩增出的多态性条带存在共分离现象。同时对利用SRAP的这些特征指导我们的研究进行了讨论。     

Assessment of genetic diversity of Czech sweet cherry cultivars using microsatellite markers

We analyzed 24 sweet and wild cherry genotypes collected in Czech Republic to determine genetic variation, using previously described 16 SSR primers to adapt a fast, reliable method for preliminary screening and comparison of sweet cherry germplasm collections. All SSRs were polymorphic and they were able all together to distinguish unambiguously the genotypes. These SSR primers generated 70 alleles; the number of alleles per primer ranged from 2 to 7, with a mean of 4.4 putative alleles per primer combination. The primer UDP-98-412 gave the highest number of polymorphic bands (totally 7), while Empa2 and Empa3 gave the lowest number (2). The allele frequency varied from 2.1% to 87.5%. We observed 10% of unique alleles at different loci. The observed heterozygosity value ranged from 0.25 to 0.96 with an average of 0.72 while expected heterozygosity value varied from 0.22 to 0.75 with an average of 0.59. The PIC value ranged from 0.21 to 0.71 with a mean value of 0.523. Cluster analysis separated the investigated cultivars in two groups. High level of genetic diversity obtained in the collection and proved to be sufficiently genetically diverse and therefore these genotypes would be useful to breeders for the development of new cherry cultivars.     

红掌品种亲缘关系SRAP分析

利用相关序列扩增多态性（SRAP）分子标记,从100对引物组合中筛选出 26对多态性高、条带清晰的SRAP引物,对33个红掌品种进行遗传多样性和亲缘关系分析。结果如下：（1）26对引物共扩增出366条条带,其中有314条多态性条带,多态性比率为85.79%。引物组合产生的条带数在9～23之间,平均每对引物组合扩增出14.1条和12.1条多态性条带。（2）根据SRAP扩增结果,利用UPGMA法进行聚类分析,33份材料的遗传相似系数在0.55～0.94之间,在遗传相似系数0.786处可将33个红掌品种分为5个类群。结果表明,供试品种遗传多样性丰富,本研究为品种鉴定和杂交育种提供了参考信息。     

Start codon targeted polymorphism for evaluation of functional genetic variation and relationships in cultivated peanut (<Emphasis Type="Italic">Arachis</Emphasis><Emphasis Type="Italic">hypogaea</Emphasis> L.) genotypes

Cultivated peanut possesses an extremely narrow genetic basis. Polymorphism is considerably difficult to identify with the use of conventional biochemical and molecular tools. For the purpose of obtaining considerable DNA polymorphisms and fingerprinting cultivated peanut genotypes in a convenient manner, start codon targeted polymorphism technique was used to study genetic diversity and relatedness among 20 accessions of four major botanical varieties of peanut. Of 36 primers screened, 18 primers could produce unambiguous and reproducible bands. All 18 primers generated a total of 157 fragments, with a mean of 8.72 ranging from 4 to 17 per primer. Of 157 bands, 60 (38.22%) were polymorphic. One to seven polymorphic bands were amplified per primer, with 3.33 polymorphic bands on average. Polymorphism per primer ranged from 14.29 to 66.67%, with an average of 36.76%. The results revealed that not all accessions of the same variety were grouped together and high genetic similarity was detected among the tested genotypes based on cluster analysis and genetic distance analysis, respectively. Further, accession-specific markers were observed in several accessions. All these results demonstrated the following: (1) start codon targeted polymorphism technique can be utilized to identify DNA polymorphisms and fingerprint cultivars in domesticated peanut, and (2) it possesses considerable potential for studying genetic diversity and relationships among peanut accessions.     

Analysis of genetic diversity of Guizotia abyssinica from Ethiopia using inter simple sequence repeat markers

Within and among population genetic diversity of 37 Guizotia abyssinica populations from Ethiopia were analyzed using inter simple sequence repeats (ISSRs). Five primers amplified a total of 118 genomic DNA fragments across a total of 370 individuals of which 106 were polymorphic (89.83%). The average number of polymorphic bands per primer was 21.2. More bands were generated by primer UBC 888 (BDB(CA)(7.) The total genetic diversity (Ht) and the coefficient of genetic differentiation (Gst) were 0.4115 and 0.0918 respectively, while the within population genetic diversity (Hs) and the among population genetic diversity(Dst) were 0.3738 and 0.03776 respectively suggesting more variability within the populations than among them. The standard genetic distances between the G. abyssinica populations of the eight regions ranged from 0.0281 (between Wollo and Gojam) to 0.1148 (between Jimma and Hararghe). Generally, the standard genetic distances are smaller between populations of neighboring regions and highest between those of Jimma and the other regions, ranging from 0.0696 (between Jimma and Shewa) to 0.1148 (between Jimma and Hararghe). The ISSR based UPGMA clustering using the standardized genetic distances matrix also placed populations from neighboring regions closer than those from farther apart areas, while the UPGMA clustering by regions based on the standard genetic distances produced three clusters following the proximity and the contiguity of the regions. The mean Shannon Weaver diversity indices for the populations of the eight regions ranged from 0.8197 (Jimma) to 0.9176 (Hararghe), with a mean of 0.8841 for the whole material.     

转基因抗虫棉SSR和AFLP遗传变异研究

利用SSR和AFLP两种分子标记技术,分析了52份转基因抗虫棉品种（系）的遗传多样性。结果表明：在61对SSR引物中,有4对引物在供试材料中表现出多态性,共扩增出102个标记,其中多态性标记25个,多态性百分率为24．51％,每对引物的扩增带数变化在17～30之间;在100对AFLP引物中,有9对引物在供试材料中产生多态性,共扩增出618个标记,多态性标记33个,占总数的5．34％,每对引物组合扩增的标记数分布于47～81之间。成对品种的欧式距离变化在2．00～5．57之间,平均值为4．21,单一品种欧氏距离的平均值分布在3．73～4．75之间,表明不同品种之间遗传差异不大。基于SSRs和AFLPs多态性数据的聚类分析,可以将供试材料划分为3个类群（SAGs）,但类群划分与品种地理来源不十分吻合。     

Molecular Characterization of <Emphasis Type="Italic">Cyclamen</Emphasis> Species Collected from Different Parts of Turkey by RAPD and SRAP Markers

The genus Cyclamen (family Myrsinaceae) contains about 20 species, most of which occur in the Mediterranean region. Turkey has critically important Cyclamen genetic resources. Molecular characterization of plant materials collected from different regions of Turkey in which Cyclamen species grow naturally, namely Adana, Antalya, Ayd?n, Mu?la, ?zmir, Denizli, Kahramanmara?, Osmaniye, Eski?ehir, Trabzon, and Rize provinces, was performed using RAPD and SRAP markers. DNA was successfully amplified by 30 RAPD primers and 14 SRAP primer pairs. Among the 470 bands generated by the RAPD primers, 467 were polymorphic. The number of bands detected by a single primer set ranged from 11 to 22 (average of 15.6). The percentage polymorphism was 99.3 % based on the RAPD data. In the SRAP analysis, a total of 216 bands were generated, showing 100 % polymorphism. The number of bands detected by a single primer set ranged from 9 to 22 (average of 15.4). All data were scored and UPGMA dendrograms were constructed with similar results in both marker systems, i.e., different species from nine provinces of Turkey were separated from each other in the dendrograms with the same species being clustered together.     

Construction of a cucumber genetic linkage map with SRAP markers and location of the genes for lateral branch traits

Cucumber (Cucumis sativus L. 2n = 2x = 14), thatbelongs to Cucurbitaceae family, is one of majorvegetables with a planting area second to that of to-mato in the world[1]. Due to its economical importanceplant breeders and geneticists have paid much atten-tion to the genetic study on this important vegetablecrop, but the research progress in cucumber is muchless than that in tomato. In 1990, Pierce[2] reviewed allthe reported genes of cucumber that had been geneti-cally analyzed since the 1930…     

Evaluation of clonal integrity in desert date tree (Balanites aegyptiaca Del.) by inter-simple sequence repeat marker assay

Axillary shoot bud multiplication as a safest mode of micropropagation to obtain clonal progeny was revealed through the application of molecular marker technique in Balanites aegyptiaca. Inter-simple sequence repeat (ISSR) markers were used to evaluate the genetic constancy of micropropagated plantlets chosen from a clonal collection of shoots that originated from mature nodal explants (mother plant). Out of 20 ISSR primers screened, ten primers yielded reliable and reproducible patterns of amplified products in all the tested plants. In this study, on an average, 11.7 bands were amplified per primer. A total of 117 bands were scored for the tissue culture-raised plantlets; 115 amplification products were monomorphic and 2 bands were polymorphic. Based on the ISSR band data, 98.2 % genetic uniformity was detected among the regenerants. Thus, the amplification products validated that the plantlets were true-to-type in morphological or growth characteristics when compared with the mother plant.     

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。     

利用RAPD标记分析东亚地区桔梗的亲缘关系

对取自中国、日本、韩国和朝鲜等东亚地区的24个桔梗种质资源,利用RAPD-PCR标记方法,分析了其遗传变异和遗传关系,旨在为桔梗的品种鉴定和杂交育种的亲本选择提供理论依据。结果表明： （1）用11个引物扩增出131条谱带,平均每个引物扩增出11.9条谱带,并扩增出61个多态性谱带,平均每个引物扩增出5.5条多态性谱带,显示出了相对高的多态率（46.6%）。（2）得到的RAPD数据的遗传相似性范围为0.668～0.994,并以遗传相似系数0.78为标准,将24个桔梗种质资源分为7个类群。     

Qualitative and quantitative characterization of RAPD variation among snap bean (Phaseolus vulgaris) genotypes

Ten snap bean (Phaseolus vulgaris) genotypes were screened for polymorphism with 400 RAPD (random amplified polymorphic DNA) primers. Polymorphic RAPDs were scored and classified into three categories based on ethidium bromide staining intensity. An average of 5.19 RAPD bands were scored per primer for the 364 primers that gave scorable amplification products. An average of 2.15 polymorphic RAPDs were detected per primer. The results show that primer screening may reduce the number of RAPD reactions required for the analysis of genetic relationships among snap-bean genotypes by over 60%. Based on the analysis of the distribution of RAPD amplification, the same number of polymorphic RAPDs were amplified from different genotypes for all RAPD band intensity levels. A comparison of RAPD band amplification frequency among genotypes for the three categories of bands classified by amplification strength revealed a measurable difference in the frequencies of RAPDs classified as faint (weakly amplifying) compared to RAPD bands classified as bold (strongly amplifying) indicating a possible scoring error due to the underscoring of faint bands. Correlation analysis showed that RAPD bands amplified by the same primer are not more closely correlated then RAPD bands amplified by different primers but are more highly correlated then expected by chance. Pairwise comparisons of RAPD bands indicate that the distribution of RAPD amplification among genotypes will be a useful criterion for establishing RAPD band identity. For the average pairwise comparison of genotypes, 50% of primers tested and 15.8% of all scored RAPDs detected polymorphism. Based on RAPD data Nei's average gene diversity at a locus was 0.158 based on all scorable RAPD bands and 0.388 if only polymorphic RAPD loci were considered. RAPD-derived 1 relationships among genotypes are reported for the ten genotypes included in this study. The data presented here demonstrate that many informative, polymorphic RAPDs can be found among snap bean cultivars. These RAPDs may be useful for the unique identification of bean varieties, the organization of bean germplasm, and applications of molecular markers to bean breeding.