Analysis of genetic diversity in Portuguese Ceratonia siliqua L. cultivars using RAPD and AFLP markers

Although carob (Ceratonia siliqua L.) is of great economic importance little is still known about the pattern of genetic variation within this species. Morphological characteristics based on 31 fruit and seeds of continuous characters determinant for agro-industrial uses, were compared with RAPD and AFLP markers for assessing genetic distances in 68 accessions of carob trees, from different cultivars, varieties and eco-geographic regions of Algarve. Eighteen selected RAPD primers applied to the 68 accessions produced a total of 235 fragments ranging from 200 to 2000 bp, of which 93 (40%) were polymorphic. Four AFLP selective primer combinations generated a total of 346 amplification fragments of which 110 were polymorphic. The average level of polymorphism based on four primer combinations was 31.8%. The phenetic trees based on RAPD and AFLP analyses gave high co-phenetic correlation values, and were found to be consistent in general with the analysis of morphological data, carried out on the same accessions. A number of RAPD and AFLP markers were found to be diagnostic for ‘Canela’ cultivar and 13 wild ungrafted trees.     

Molecular characterisation of Afghan pistachio accessions by amplified fragment length polymorphisms (AFLPs)

Summary

In Afghanistan, pistachio (Pistacia vera L.) is found mainly in the wild in natural forests. In this study, 17 wild Afghan pistachio accessions and three cultivated genotypes were characterised using amplified fragment length polymorphisms (AFLPs). Material was sampled mainly from the Kunduz and Takhar regions. A total of 288 AFLP fragments were generated using eight AFLP primer combinations. The number of amplified fragments varied from 18 – 48 per primer combination, and 136 bands were polymorphic, with an average of 17 polymorphic bands per primer combination. The percentages of polymorphic bands ranged from 26.2 – 79.2 per primer pair. According to UPGMA clustering of the Nei and Li distance matrix, the 17 wild accessions were grouped according to their region of origin and were distinct from the three cultivars. The AFLP technique was found to be effective for characterising wild P. vera material, which was genetically the closest to cultivated pistachio.     

Genetic variation and differentiation in tea (Camellia sinensis) germplasm revealed by RAPD and AFLP variation

Summary

Genetic variation and resulting variable groupings in tea (Camellia sinensis) and its wild Camelia relatives were assessed using Random Amplified Polymorphic DNA (RAPD) and Amplified Fragment Length Polymorphic (AFLP) markers. Numerous polymorphic bands were generated, of which 266 unambiguous ones were scored. The highest level of polymorphism as determined by the expected heterozygosity (H_av) was detected with AFLPs. Three major groups were recognized in the germplasm based on the parsimony method of cluster analysis. Two of the groups corresponded to varieties assamica and sinensis while the third group consisted of a heterogeneous mix of tea cultivars and related wild species. The Taiwan yamacha, unique tea cultivars grown predominantly in the highlands of Taiwan for the production of pan fired semi-fermented (Oolong and Pouchong) tea clustered in this group. Analysis of phenotypic diversity based on the generated RAPD and AFLP profiles revealed that population diversity (H), decreased in the order; Wild Camellia >India > China > Kenya > Sri-lanka > Vietnam > Japan > Taiwan. Analysis of molecular variance (AMOVA) revealed that most variation resided among individuals within populations (72%). Calculation of genetic distances and nested AMOVA further revealed a significant degree of structuring among the populations based on the country of origin.     

Characterization of white sapote (Casimiroa edulis Llave & Lex.) germplasm using floral morphology,RAPD and AFLP markers

Floral morphology, random amplified polymorphic DNA (RAPD), and amplified fragment length polymorphism (AFLP) were used to characterize and verify genetic diversity within a white sapote cultivar collection and to develop molecular markers for germplasm identification. On the basis of floral morphology, the cultivars were classified into three types: type I included 23 cultivars with large ovaries and small anthers; type II included 13 cultivars with small ovaries and large anthers; and type III included one cultivar, named ‘Maltby’, with a large ovary and large anthers. DNA was isolated from 39 cultivars of white sapote and subjected to RAPD and AFLP analysis using 24 and 7 primers, respectively. One hundred and sixty-eight RAPD and 286 AFLP bands were used to assess genetic characterization among white sapote. Sixty percent of the RAPD and 77% of the AFLP amplification products were polymorphic among accessions. RAPD or AFLP markers differentiated all white sapote cultivars effectively. Moreover, each flower type was characterized as specially associated with two RAPD bands. UPGMA dendrograms based on RAPD and AFLP data, showed the majority of the cultivars from flower type I and flower type II clustering together. Finally 101 RAPD markers and 220 AFLP markers were used to construct a neighbor-joining dendrogram. This showed that the 37 cultivars could be classified into six distinct clusters, between which the similarity coefficient was as low as 0.00–0.55, even though the cultivars were morphologically very similar. The remaining two cultivars namely ‘Smathers’ and ‘Maltby’ were found genetically very distant from the other cultivars in RAPD, AFLP or combined RAPD and AFLP based dendrograms. The results suggested that the level of genetic variation among white sapote cultivars is diverse and the morphological and molecular data may lead to representation of the cultivar relationships as well as flower type discrimination.     

Assessing Rosa persica genetic diversity using amplified fragment length polymorphisms analysis

The genetic diversity among 128 Iranian Rosa persica (R. persica) accessions in the different populations was analyzed. Amplified fragment length polymorphisms (AFLP) technique was used to produce 171 polymorphic fragments. The number of polymorphic loci ranged from 101 to 147 and the polymorphism information content (PIC) varied from 0.289 to 0.073, with an average of 0.16. This shows extreme variability and genetic diversity among the studied R. persica populations. An indirect estimate of the number of migrants per generation (Nm = 0.376) indicated that gene flow was relatively low among populations of the species. Cluster analysis using the UPGMA method grouped all accessions into six clusters. The results did not show relative agreement with the genotypes’ region of origin. Based on an analysis of molecular variance, 48% of the genetic variation of R. persica was within population and 52% was among populations. The present analysis revealed that Iranian R. persica genotypes are highly variable and genetically distinct from their origins. The apparent unique nature of the R. persica genotypes revealed by our results supports the case for the implementation of more intense characterization and conservation strategies, and provides useful information to address breeding programmes and germplasm resource management in Rosa spp.     

Characterization of Tunisian pomegranate (Punica granatum L.) cultivars using amplified fragment length polymorphism analysis

The amplified fragment length polymorphism (AFLP) analysis of DNA was used to characterize 34 pomegranate cultivars. By using a combination of six primers, a total of 327 markers were scored with a mean of 57.5. The high percentage of polymorphic bands (ppb) of 94.7 and the resolving power (R_p) collective rate value of 129.14 were scored. Data proved that the tested primers were informative to discriminate among cultivars and to survey the genetic diversity in this fruit crop. It has been assumed that the local pomegranate germplasm is characterized by a typically continuous genetic diversity. The derived dendrogram proved that cultivars are clustered independently from their geographical origin and their denomination. In addition, AFLP permitted the generation of a nearly unlimited number of molecular markers that are reliable in differentiating the cultivars and/or the polyclonal varieties.     

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.     

栽培番茄品种指纹图谱的AFLP分析

以62份栽培番茄为材料,利用65对AFLP引物进行选择性扩增,选出5对多态性丰富、带型清晰的引物进行扩增,共获得85个多态性位点,占总扩增位点的35.41%;选出清晰的16个位点构建了62份栽培番茄的AFLP指纹鉴别图谱。结果表明,由引物组合P55M48、P58M68、P21M49、P77M51、P58M60所构建的DNA指纹鉴别图谱可将供试材料完全区分开。     

利用AFLP鉴定柑橘变异

利用AFLP分析鉴定了7个柑橘变异类型,从64对引物组合中筛选了8对引物组合,共得到334个扩增位点2099条带数据。7个变异类型与亲本品种之间多态性带为36至92条,变异类型的多态性带数与亲本品种总带数的比例为0.1721至0.4182。说明变异类型的遗传基础已经发生了不同程度的改变,7个柑橘变异类型应是芽变,具备了成为新品种的遗传基础,特别是红皮冰糖脐橙选育成功将填补高糖脐橙空白。研究同时表明,AFLP具有扩增谱带多,谱带清晰,扩增信息量大的特点。根据谱带位点差异即可鉴定柑橘变异是否遗传,多态性位点数也反映了性状变异程度。AFLP方法1次可同时鉴定多个变异类型,是柑橘新品种选育早期鉴定灵敏、可靠、有效的方法。     

梨新品种及其亲本的AFLP分析

利用荧光AFLP技术, 对20个梨新品种及其23个亲本(共43个品种) 进行研究。从64对引物组合中筛选出7对用于扩增, 共获得784条带, 其中多态性带699条, 多态性为89.2%。扩增结果显示, 7对引物组合在29个品种中扩增出特征带, 每对引物组合均能将所有品种鉴别开, 表明荧光AFLP技术用于梨品种鉴定的效率很高。通过聚类, 从分子水平对梨新品种及其亲本的遗传关系进行分析, 并对20个梨新品种进行分类, 为梨杂交育种的亲本选配提供了理论参考。     

Genetic relationships of gladiolus cultivars inferred from fluorescence based AFLP markers

Gladiolus is one of the important commercial flowers with a large number of cultivars. However, genetic relationships among its genotypes have not been reported. This study analyzed genetic relatedness of 54 gladiolus cultivars using amplified fragment length polymorphism (AFLP) markers. A total of 24 AFLP primer pairs with three samples were initially screened, from which 9 primer sets that showed clear scorable and highly polymorphic bands were selected for AFLP reactions. Fluorescence-labeled amplification products were subjected to electrophoresis and then analyzed using an automated sequencer. A dendrogram was constructed by the unweighted pair group method using the arithmetic average (UPGMA). The number of AFLP fragments generated per primer set ranged from 10 to 151 with fragment sizes varying from 50 to 450 bp. A total of 660 AFLP fragments were detected, of which 658 (99.70%) were polymorphic. All the primers except E-AGG/M-CTA displayed 100% polymorphism. All cultivars were clearly differentiated by their AFLP profiles. The AFLP data were compared with previously obtained RAPD data and combined to generate a common dendrogram. The first cluster was dominated with indigenously bred cultivars while the second was dominated with exotic cultivars. This shows that most of the exotic cultivars as well as indigenous cultivars are closely related with each other. However, two indigenous cultivars viz., Pusa Suhagin and Pusa Archana share genetic similarity with exotic cultivars. Among the genotypes selected for the investigation, Pusa Gunjan was identified as the most distinct genotype. The AFLP markers developed will help future Gladiolus cultivar identification, germplasm conservation and new cultivar development. The assessed genetic relationships among gladiolus cultivars may enhance the efficiency of breeding program by selecting desirable parents with reduced breeding cycle.     

杧果主要品种遗传多态性的AFLP标记研究

 采用扩增片段长度多态性AFLP标记对国内外31个杧果主要品种进行分类与亲缘关系研究。结果表明: 筛选出的14对引物组合在31份杧果种质中共扩增出了1 761条带, 其中多态性带的比例为97%。平均每对引物产生125.8条带和121.6条多态性带, 总的多态性带率为97%; 基于AFLP标记, 以0.48的相似系数为阈值, 所有供试　果材料被分为7大组群, 第一组群内以0.52为阈值, 又可分为6组。与形态标记相比, 基于AFLP标记的杧果分类体系更能反映杧果品种间的亲缘关系。     

枣树品种品系的AFLP分析

为建立完善枣种质资源分类鉴定技术体系,采用了基因组DNA-AFLP分子标记技术,从32对引物中筛选出了E-AAC/M-CCT、E-AAC/M-CAT、E-ACT/M-CCG、E-AAG/M-CCC和E-ACC/M-CAT等5个多态性好、分辨率高的引物组合,共扩增213条带,其中多态性带172条,多态性百分率76.16%。通过计算品种间单匹配系数,不加权算术平均值法绘制聚类图,可将28个供试品种和品系全部区分开;通过观察指纹图谱,也可明显看出新品系与其原种的差异带的多少,可快速准确地区分和确定新变异品系;另外,还可消除枣种质资源同名异物或同物异名现象。从而表明,AFLP技术是鉴定枣品种的有效方法。探讨了基因组DNA-AFLP分析技术的各关键步骤,并展望AFLP技术在枣品种鉴定上的应用前景。     

Sequence analysis of the internal transcribed spacers (ITSs) region of the nuclear ribosomal DNA (nrDNA) in fig cultivars (Ficus carica L.)

Sequences of the internal transcribed spacers (ITSs) of nuclear ribosomal DNA (nrDNA) were analysed in a set of Tunisian fig (Ficus carica L.) cultivars. The size of the spacers sequences ranged from 200 to 279 bases for ITS1 and from 253 to 314 bases for ITS2. Variation of GC contents has been also observed and scored as 59–68% and 55–68% for ITS1 and ITS2, respectively. This data exhibited the presence of polymorphism among cultivars. The intra-specific variability level of the ITS sequences proved a variation both in the length and in the sequences studied. In fact, ITS1 and ITS2 sequences were considered as a useful tool to establish genetic relationships among cultivars. Our results indicate that the diversity detected among closely related genotypes supported strongly the efficiency of ITS sequences for establishing relationships between cultivars. ITS2 seems to be relatively more informative than ITS1 regarding length or GC contents. Considerable genetic diversity was observed among fig at intra and inter-cultivars levels. Two polyclonal varieties were identified. In addition, data proved that a typically continuous genetic diversity characterizes the local fig germplasm. The topology of the derived dendrogram strongly supported this assumption. In fact, genotypes are clustered independently from their geographical origin or the sex of trees suggesting a narrow genetic basis among the ecotypes studied in spite of their phenotypic distinctiveness. Implications of these results for management of fig germplasm collections are discussed.     

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.     

Molecular assessment of polymorphism among local Jordanian genotypes of the common fig (Ficus carica L.)

This research was conducted to assess polymorphism among local genotypes of common fig available in Jordan (one of countries of origin). Leaf samples were collected in spring for DNA isolation from 20 different local genotypes (5 cultivars and 15 landraces). Two more wild types and one foreign cultivar were included. The genotypes were assessed using the randomly amplified polymorphic DNA (RAPD) technique. Six of the 19 screened primers showed reproducible polymeric profiles. Out of 62 amplified bands, 48 were polymorphic (77%). They generated 1104 data entries (591 for present and 513 for absent bands). After determining Jaccard similarity index, some genotypes showed high genetic similarity (90% between F20 and F22), while other were less similar (3–18% between F11 and all other genotypes). Moreover, the primers were evaluated for their discriminating power, where primer RAPD06 showed the weakest power (0.431), while highest values of 0.989 and 0.996 were achieved for primers RAPD02 and RAPD13, respectively.     

Variation in cytosine methylation in Clementine mandarin cultivars

Summary

Clementines are an important group of citrus cultivars. However, little is known about their genetic diversity. In this study, methylation-sensitive amplification polymorphism (MSAP), based on the application of isoschizomers (Hpa II and Msp I), was used to analyse cytosine methylation patterns in 18 Clementine cultivars. Conventional AFLP analysis showed only two polymorphic bands out of a total of 1,822 AFLP bands generated using 28 primer pairs. Three types of bands were generated by MSAP using 27 pairs of primers. Type I were present in both Eco RI + Hpa II and Eco RI + Msp I gels. Type II or Type III were present only in Eco RI + Hpa II, or Eco RI + Msp I gels, respectively. The total numbers of these three Types of bands were 1,377, 98, and 93, respectively. Among these three Types of bands, the number of polymorphic bands were 76 (5.5%), 30 (30.6%), and 15 (16.1%), respectively. Of these, non-reproducible polymorphic bands in duplicated samples were 27 (35.5%), 18 (60.0%), and 9 (60.0%). Each Clementine cultivar had specific and relatively fixed methylated CCGG sites, which could be revealed from their specific MSAP patterns. The reproducible polymorphic bands in duplicated samples were used to analyse the extent of variation in methylation. The diversity of the cultivars revealed by the resulting dendrogram suggests that DNA methylation may be one of the main factors accounting for the difference in agronomic traits between Clementine cultivars.     

Genetic diversity and phylogenic relationships in date-palms (Phoenix dactylifera L.) as assessed by random amplified microsatellite polymorphism markers (RAMPOs)

Random amplified microsatellite polymorphisms (RAMPOs) were used to assess genetic diversity among 30 date-palm cultivars and 10 male trees. Using 18 primers combinations, 197 bands were scored and 186 were polymorphic suggesting the high level of polymorphism among studied cultivars. Moreover, taking into account the high percentage of polymorphic bands (ppb), the resolving power (R_p) together with the polymorphism information content (PIC) scored values, all the tested primer sets contribute strongly in the discrimination of date-palm genotypes. In addition, the topology of the derived UPGMA dendrogram exhibited cultivars’ clustering made independently both from the geographical origin and/or from the sex of trees. The present data support the Mesopotamian origin of the date-palm domestication. Thus we assume that the used method is efficient to assess genetic diversity within date-palm cultivars. Data are discussed in relation with the opportunity of the RAMPO method to provide additional molecular markers suitable in the improvement of the date-palms germplasm characterisation.     

青菜品种亲缘关系AFLP分析

利用AFLP技术对市场上主栽的10个青菜主要品种进行了遗传多样性及聚类分析.结果表明:最终筛选出的5对引物共扩增出多态性位点454个,多态性位点占总扩增位点的比例平均为57.5%,系统聚类分析将供试材料分为4组,基于分子标记的分类与材料的表现基本吻合,为以后品种的鉴定和分子辅助育种提供一定的参考.     

部分板栗品种遗传多样性的AFLP分析

利用荧光标记AFLP技术,采用7对M+3和E+3引物组合对30份板栗和日本栗栽培品种进行了总基因组DNA水平上的多态性检测,共获得962条可统计的条带,其中852条呈多态性,多态性带百分率达89％。揭示了板栗丰富的遗传多样性。7组引物在30个品种中检测到数目不等的品种特异带型,对供试板栗品种具有一定的鉴别价值。7对引物能将30个板栗和日本栗品种完全区分开。聚类分析结果表明,多数来源地相同的板栗品种资源表现出较为密切的亲缘关系。