Ampelographic and genetic characterisation of ancestral grapevine (Vitis vinifera L.) accessions present in the Umbria Region (Central Italy)

Summary

During an ongoing effort to recover and preserve local germplasm, 14 accessions of indigenous minor grapevine (Vitis vinifera L.) cultivars from the Umbria Region, Central Italy, were chosen because they had been neglected and were threatened with extinction. Their phenotypic and genetic characteristics were evaluated through an ampelographic study of their shoots, mature leaves, bunches, and berries and by genomic analysis using an international set of nine microsatellite (simple sequence repeat; SSR) markers (VVS2, VVMD5, VVMD7, VVMD27, VrZAG62, VrZAG79, VVMD25, VVMD28, and VVMD32). Comparisons of the SSR profiles of all 14 accessions with grapevine accessions in several databases permitted the identification of unique genotypes, as well as possible synonyms. Information on these older, neglected cultivars will help to reduce the genetic erosion of grapevine germplasm, improve conservation and possible recovery, and assist in the future production of new, distinctive wines.     

SSR-based DNA fingerprints reveal the genetic diversity of Sicilian olive (Olea europaea L.) germplasm

Summary

Twelve published simple sequence repeat (SSR; microsatellite) markers, belonging to the ssrOeUA-DCA, GAPU and UDO series, were tested in a panel of 46 accessions of olive germplasm belonging to 30 unique cultivars collected in seven Provinces of Sicily. Four well-known reference olive cultivars were also added. The analysis was carried out on an automatic capillary sequencer using fluorescent dyes, and fragment sizes were determined using internal standards. The results allowed us to rank the SSRs assayed according to their information content and reproducibility. Up to 115 alleles were identified (119, if those unique to sport mutations were included), the frequency of which allowed genetic relationships among accessions to be investigated. The probability that two unrelated genotypes displayed the same SSR pattern at all loci examined was calculated to be as low as 1.18 10^–11. Sixteen accessions were identified as synonyms. Of these, eight matched perfectly with another accession at all SSR loci examined. The others showed one or two allelic differences from the reference accession. These were interpreted as mutations. Otherwise, all accessions were clearly separated from each other. Two likely parentages were also identified (‘Giarfara’ = ‘Nocellara del Belice’

‘Cacaridduni’; and ‘Pizzo di Corvo’ = ‘Nocellara Etnea’ ‘Tonda Iblea’). The genetic diversity of the pool represented by the unique accessions was very high, reflecting the richness of the olive germplasm accumulated in Sicily. A database of the accessions is available to the scientific community (http://www.unipa.it/germolive/ssr.html) to facilitate comparisons of data.     

Assessment of genetic relationships among 29 introduced and 49 local sweet cherry accessions in Turkey using AFLP and SSR markers

Summary

The characterisation of sweet cherry (Prunus avium L.) genetic resources in Turkey may help to increase their use in breeding programmes worldwide, as Turkey is the centre of origin of sweet cherry. Amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers were therefore used to analyse genetic diversity among a total of 78 local and introduced sweet cherry cultivars. Four AFLP primer combinations, and six SSR primer pairs for sweet cherry were used for genetic diversity analysis. A genetic similarity matrix was calculated using the combined data from AFLP and SSR analyses with simple matching coefficient. Genetic similarities among the sweet cherry genotypes studied were higher than 42%. No two accessions had an identical AFLP and SSR marker profile, indicating that all 78 genotypes were unique. An UPGMA dendrogram, based on the similarity matrix, revealed 18 separate Groups at or above the 70% similarity level. While some Groups consisted of both introduced and local genotypes, other Groups had only local genotypes. This result suggests that there was broad genetic diversity among the local Turkish sweet cherry genotypes, which was not present in the introduced sweet cherry accessions. The genetic variation present in local Turkish sweet cherry genotypes may be useful for future breeding programmes. We found that the use of both SSR and AFLP marker systems was effective for distinguishing between genetically-close sweet cherry genotypes. These marker systems can be used to complement pomological and morphological markers during the characterisation and identification of sweet cherry genotypes.     

黄瓜种质资源遗传多样性及其亲缘关系的AFLP分析

 采用AFLP分子标记技术，对中国黄瓜种质资源遗传多样性及其与外来种质的关系进行了分析， 结果表明8对AFLP引物在70份黄瓜种质中共扩增出425条带，多态性带的比例为66％。供试黄瓜种质的平 均期望杂合度为0．376，中国种质的平均期望杂合度为0．387，明显高于国外种质的n 291。西双版纳黄瓜和印度野生黄瓜具有一些栽培种质没有的特异位点，中国栽培种质的特异位点多于外来栽培种质，后者也有一些中国栽培种质没有的特异位点。聚类分析将70份种质分为三大组群，即西双版纳黄瓜(Cucumis sativus L.vaF．xishuangbannanesis Qi et Yuan)组群，e sativus var．hardwickii野生黄瓜组群和栽培黄瓜组群。西双版纳黄瓜与栽培黄瓜的距离最远，与野生黄瓜次之。按一定的遗传距离可以将中国和外来栽培种质分开。大多数 华南型和华北型种质归属于不同的亚组。这些结果有助于有目的地利用这些变异拓宽育种材料的遗传背景。     

Detection of variation among clonal selections of grapevine (Vitis vinifera L.) ‘Kishmish Chernyi’ by AFLP analysis

Summary

Clonal selection is an important method for varietal improvement in grapevine. Ampelometric and morphological markers fail to differentiate clones from their parent genotype. Molecular markers offer the opportunity to identify the clonal material. In this study, five clones of the grapevine variety ‘Kishmish Chernyi’ were analysed using microsatellite (SSR) and AFLP markers. These clones differed significantly in their bunch characteristics including berry size, shape, and colour. Microsatellite (SSR) analysis using 24 primers could not distinguish between these clones. The allele profiles of the clones and the parent variety were identical. AFLP analysis using 13 primer pair combinations yielded 592 markers ranging in size from 50 – 500 bp. Of these, 79 markers (13%) were polymorphic. The majority of the polymorphic markers (75/79) were detected in the clone ‘Sharad Seedless’. Three AFLP primer combinations detected unique markers in three clones which could be useful for future identification.     

Further Work on Plant Injection for Diagnostic and Curative Purposes

Summary

Randomly Amplified Polymorphic DNA (RAPD) markers were used to evaluate genetic similarity and inter-relationship among31 acid citrus species and cultivars, including sour oranges (six accessions); ‘Yuzu’ (four accessions) andits relatives (21 accessions). Out of the 60 decamer primers screened, 27 were selected which produced 108 markers; 76 of which were polymorphic. Species or cultivar-specific RAPD markers were also found. A dendrogram based on genetic distance implied that sour oranges were very distinct from ‘Yuzu’ and its relatives. ‘Yuzu’ accessions were very closely linked to each other, however; for the other specimens genetic polymorphism could easily be detected by RAPDs and the genetic variation between accessions was quite high and revealed their different origins. In this study some RAPDs allowed the distinction of very close cultivars, for instance ‘Kabosu’ from ‘Aka kabosu’.     

SSR markers reveal the uniqueness of olive cultivars from the Italian region of Liguria

Twenty-three important Ligurian olive accessions corresponding to 16 cultivars were studied using 12 SSR markers and 40 Mediterranean cultivars were included in the study in order to investigate the relationships between Ligurian and Mediterranean germplasm. All SSRs produced polymorphic amplifications. One hundred and forty-nine alleles were found in the 63 accessions analysed. Twenty-two alleles were specific to germplasm from Liguria and of these 12 were unique to single cultivars. Heterozygosity and discriminating power calculated in this regional germplasm were high on average (0.70 and 0.74) and not so much lower than the values in the total sample that includes cultivars from different Mediterranean countries (0.77 and 0.88 respectively). No cases of genetic identities were found between Ligurian and Mediterranean accessions. Several cases of homonyms and synonyms within the Ligurian germplasm were explained. Cluster analysis generally revealed a clear discrimination of the profiles from Liguria and Italy with respect to the cultivars from other Mediterranean countries. Only one Ligurian cultivar, “Negrea”, appeared to have a different origin, grouping with the Mediterranean cultivars.     

Morphological and molecular analysis of genetic diversity in jackfruit

Summary

RAPD markers were used to estimate genetic diversity in 12 high-yielding jackfruit (Artocarpus heterophyllus Lamk.) accessions obtained from different locations in southern India. Marker data were compared with morphological data obtained over three successive seasons. PCR-amplifiable DNA was isolated using the CTAB method and 171 amplified fragments were obtained using 23 random primers. The genetic dissimilarity matrix was calculated based on Squared Euclidian Distances, which revealed a maximum genetic distance of 7.9% between a clone of ‘Mottavarica’ (‘M₀’), and ‘Chandrahalasu’ from distant locations, while the minimum genetic distance (5%) was between the genotypes (‘M₀’) and ‘Kerala’, indicating their similar geographical origin. Ward's method of cluster analysis grouped all individuals on the dendrogram into two major clusters according to their geographical location. The present study showed low-to-moderate genetic diversity among the 12 jackfruit accessions, which will assist in the identification and management of jackfruit germplasm for breeding purposes.     

Resolving the Parentage of the Apple Cultivar ‘Meran’

In the mid-1970s, a new apple variety named ‘Meran’ was discovered in South Tyrol (northern Italy), which harbours the largest continuous apple growing area in Europe. The cultivar was registered for varietal protection and patented in several countries, and was declared to be a cross of the varieties ‘Golden Delicious’ and ‘Morgenduft’ (synonym ‘Rome Beauty’). The parentage of ‘Meran’ has, however, been questioned, and the present study aimed to assess the descent of this cultivar by the combined use of molecular genetic and bioinformatic tools. Five accessions of ‘Meran’ were collected from three different European germplasm collections and analysed at 14 variable microsatellite DNA loci. Subsequently, computer software was used to allocate the most likely parent pair from a set of cultivars representative for the apple growing area of South Tyrol in 1975. The molecular genetic data clearly excluded ‘Morgenduft’ as a gene donor to ‘Meran’ and provided strong evidence that ‘Meran’ is a cross of the cultivars ‘Golden Delicious’ and ‘Jonathan’, confirming previous assumptions based on morphological traits of the tree and fruit.     

红肉猕猴桃种质资源果实性状及AFLP 遗传多样性分析

 对中国红肉猕猴桃种质资源进行收集和调查,并对其进行果实性状变异分析和AFLP 遗传多 样性及遗传关系分析。结果表明,红肉猕猴桃野生资源主要分布于湖南省、湖北省、河南省、江西省、 四川省和陕西省等地,共采集到52 份野生资源和2 份品种资源（包括软枣猕猴桃红肉类型、中华猕猴桃 红肉类型和美味猕猴桃红肉类型）。红肉猕猴桃种质资源在果实性状和DNA 分子水平上都存在丰富的变 异和较高的遗传多样性水平,4 对AFLP 引物共扩增出259 个多态性位点,多态性位点百分率为90.56%, Nei’s 基因多样性和Shannon’s 信息指数分别为0.318 和0.477;资源间遗传相似性系数介于0.568 ~ 0.883 之间,平均为0.714。聚类分析和主坐标分析将54 份资源划分为4 个组,软枣猕猴桃红肉类型单独聚为 一类;中华猕猴桃和美味猕猴桃红肉类型亲缘关系较近且有按地理来源优先聚类的趋势。果实性状数据 和AFLP 数据之间具有极显著的相关性,二者可结合用于红肉猕猴桃资源评价和保护利用工作中。     

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.     

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.     

Microsatellite characterization of grapevine (Vitis vinifera L.) genetic diversity in Asturias (Northern Spain)

The genetic heritage of the Asturian grapevine (Vitis vinifera L.) has been declining over the past century due to the phylloxera attack and the further abandonment of this culture. In addition, efforts in recent years to restore the Asturian vineyard with the pulling-up of old vineyards and replanting with cultivars endorsed by Cangas Quality Wine regulations are contributing even more to this genetic erosion. The aim of this study was the evaluation and identification of the phytogenetic resources of the Asturian grapevine. A total of 293 accessions were collected in old vineyards and analyzed through nine microsatellite markers. Forty-two different genotypes were obtained, including six profiles with allelic variations. Only 27 cultivars were identified when compared with national and international databases; some of them had not been found in this region before. Homonymies and synonymies have also been detected. These results provide an overview of the status of current grapevine phytogenetic resources in Asturias. Despite the substantial genetic erosion that the Asturian vineyard has suffered, a higher variability than expected has been detected. The finding of new grapevine genotypes is a fact of great importance. The genetic grapevine resources are being drastically reduced all over the world, so this new genetic material has to be included in germplasm banks for its conservation and further agronomical and enological evaluation.     

Analysis of genetic diversity among Indian short-day onion (Allium cepa L.) cultivars using RAPD markers.

Summary

Randomly amplified polymorphic DNA (RAPD) markers were used to estimate genetic diversity among 24 cultivars of short-day onions. Total genomic DNA was extracted and subjected to RAPD analysis using 90 arbitrary 10-mer primers. Of these, 15 primers were selected which yielded 137 bands, 91.24% of which were polymorphic. None of the primers produced a unique banding pattern for each cultivar. RAPD data were used to calculate a Squared-Euclidian Distance matrix which revealed a minimum genetic distance between cultivars ‘AFLR-722’ and ‘PBR-140’, and a maximum genetic distance between cultivars ‘PBR-139’ and ‘A.Kalyan’, and ‘MS-48’ and ‘A.Kalyan’. Based on the distance matrix, cluster analysis was done using a minimum variance algorithm.The dendrogram thus generated, based on Ward’s method, grouped the 24 onion cultivars into two major clusters. The first cluster consisted of cultivars from the northern region, and the second of cultivars from the southern region of India. The present study shows that there is high diversity among the onion cultivars selected and indicates the potential of RAPD markers for identification and maintenance of onion germplasm for crop improvement purposes.     

杧果种质遗传多样性的表型分析和AFLP分析

 利用表型和AFLP标记,对中国热带农业科学院南亚热带作物研究所杧果种质资源圃的56份国内外杧果种质进行遗传多样性分析。结果表明：这些种质的表型性状表现出较大的差异,多样性指数（H′）在0.56 ~ 1.64之间,平均为1.05,表型性状欧氏距离系数在0.02 ~ 0.45之间。8对AFLP引物共扩增出713条谱带,其中多态性带为630条,占88.36%,遗传相似性系数0.38 ~ 0.85之间。Mantal测量结果表明表型和基因型距离矩阵间存在显著的正相关（r = 0.72,P = 0.05）。表型性状和AFLP分子标记分析的结果相对一致,56份杧果种质具有较丰富的遗传多样性。     

Microsatellite markers based assessment of genetic diversity in Iranian olive (Olea europaea L.) collections

To study the genetic variation in Iranian olive collections and some foreign olive cultivars, 47 accessions of 18 local cultivars from 6 olive collections of Iran (Roudbar, Zanjan, Ahvaz, Dezful, Kazeroon and Shiraz), were analyzed along with 30 imported cultivars using 16 microsatellite primer pairs. All the used microsatellite loci revealed polymorphism in the studied genotypes, except GAPU14 and GAPU113 markers. Fourteen microsatellite primers amplified 126 polymorphic alleles in the 87 selected olive accessions. The average number of alleles per locus was 9, ranging from 3 to 14. Polymorphic information content (PIC) was 0.85. The genetic similarity based on Jaccard coefficient ranged from 0.15 to 1. The genetic relationships among accessions were investigated using cluster analysis and principal component analysis (PCA). Most of the accessions with the same name were grouped together; some exceptions were also observed. As expected, close relationship was observed among accessions within same cultivar. Most of the Iranian olive accessions were clustered to a main distinct group. Two-dimensional scatter plot of principal component analysis revealed a clear separation of most of the Iranian olives from Syrian and other introduced cultivars. These suggest that Iranian cultivars have different origin related to West Mediterranean basin cultivars and have evolved independently from the others. Between and within Iranian and foreign cultivars (cultivars including three or more accessions) genetic diversity was analyzed using analysis of molecular variance (AMOVA). AMOVA revealed higher within cultivar genetic variation (62.76%) as compare to that between cultivar variations (37.24%). The intra- and inter-cultivar variance tested by permutation test showed significant genetic variation at both levels. The high level within cultivar genetic variance could be due to mislabeling and presence of homonyms in cultivars produced by vegetative propagation from original plants.     

Commercial Iranian olive cultivars: morphological traits,molecular diversity,and genetic structure

Iran is considered to have a unique gene pool of different fruit and nut species including olive (Olea europaea L.). In this study, we used 22 previously developed microsatellite (simple sequence repeat; SSR) markers for olive to evaluate the level of genetic variation and to produce identification keys for 63 Iranian accessions of olive belonging to 17 groups of cultivars. Based on morphological features, the number of flowers per inflorescence, fruit weights, endocarp weights, oil percentages, and flesh weights per endocarp had the highest coefficient of variation values, indicating the large extent of morphological variability among the 63 Iranian olive cultivars studied. All 22 microsatellite (SSR) markers revealed a high level of polymorphism, with a mean polymorphic information content (PIC) value of 0.511. Analyses of genetic structure among the 63 olive accessions were carried out using model-based methods, which showed a tendency for geographical clustering. Ten SSRs out of the 22 were successful for the identification of unique ID keys for 52 of the 63 accessions. In most cases, there was disagreement between the molecular data and the morphological data. These results could be used to reconstruct and maintain a collection of olive for future breeding programmes.     

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。