Assessment of genetic diversity among mango (Mangifera indica L.) genotypes using RAPD markers

Knowledge about the extent of genetic diversity/relatedness in mango germplasm is vital for developing coherent strategies for future gains in productivity. The genetic diversity/relatedness among mango cultivars/genotypes developed in Pakistan has not been investigated previously. We have assessed the genetic diversity among 25 mango genotypes/cultivars using randomly amplified polymorphic DNA (RAPD). Sixty random ten-mer primers were surveyed, out of which 45 yielded amplicons in all the genotypes. Genetic similarity between genotypes/cultivars was in the range of 64–89% with an average of 74%. Similarly, the genetic relatedness among all variants derived from a mango cultivar Chaunsa was in the range of 81.18–88.63%. These coefficients were utilized to construct a dendrogram using the unweighted pair group of arithmetic means (UPGMA). The genotypes were grouped into three (A, B, C) clusters. Generally, genotypes originating from Pakistan were grouped in cluster ‘A’ while cluster ‘B’ primarily composed of southern India as well as Florida cultivars. Kensington Pride was the most distantly related genotype which grouped with Maya and Yakta, forming a distinct cluster ‘C’.     

Analysis of genetic diversity among some Persian walnut genotypes (Juglans regia L.) using morphological traits and SSRs markers

There is a high diversity among cultivated walnut trees in Iran due to its long time of seed propagation and vast area of cultivation. In this study some morphological characters as well as Simple Sequence Repeat (SSRs) markers were used to analyze the genetic diversity and relationships among 31 Iranian walnut genotypes along with four foreign cultivars. The nut weight ranged from 7.52 to 17.73 g, kernel weight from 4.00 to 9.83 g, and kernel percentage ratio from 38.78 to 67.05% among studied genotypes. In SSRs analysis, nine primer pairs were tested that produced 39 alleles ranging from 2 to 8, with a mean value of 5.10 allele per primer. The Iranian genotypes showed relatively high diversity both for their SSRs loci and morphological traits. Although the foreign cultivars (‘Serr’, ‘Vina’, ‘Franquette’ and ‘Lara’) clustered with each other, they also laid close and within the Iranian genotype. The results of the study provided us with valuable diversity among our genotypes which could be used for breeding studies and also showed the power of genetic markers for analysis and evaluation of this diversity.     

EST-PCR markers developed for highbush blueberry are also useful for genetic fingerprinting and relationship studies in rabbiteye blueberry

The pedigrees of most rabbiteye blueberry (Vaccinium virgatum) cultivars can be traced back to four wild selections, ‘Ethel’, ‘Clara’, ‘Myers’, and ‘Black Giant’; thus, they result from a very narrow germplasm base and are highly related. Until now randomly amplified polymorphic DNA (RAPD) has been the only type of molecular marker used in rabbiteye blueberry. Here we have tested whether a type of sequence-tagged site (STS) marker which utilizes specific ∼20-mer primers from expressed sequence tags (ESTs) of highbush blueberry (V. corymbosum), called EST-PCR markers, are useful for genetic fingerprinting and relationship studies in rabbiteye blueberry. Of 44 EST-PCR primer pairs, from an assortment of genes expressed in flower buds of cold acclimated and non-acclimated plants, and shown to amplify polymorphic fragments among a collection of highbush genotypes, 40 (91%) resulted in successful amplification, and 33 of those (83%) amplified polymorphic fragments among the rabbiteye genotypes. The average number of scorable bands per primer pair was two. A dendrogram constructed from genetic similarity values, based on the EST-PCR marker data, tended to group siblings and parent/progeny together, generally agreeing with pedigree information. A group of 20 markers from five EST-PCR primer pairs distinguished all the genotypes in this study. These markers are as easy to generate and as affordable as RAPDs, but are based on actual gene sequences, and should have general utility for DNA fingerprinting, genetic diversity, and mapping studies.     

Rose (Rosa hybrida L.) EST-derived microsatellite markers and their transferability to strawberry (Fragaria spp.)

The ‘Genome database for Rosaceae (GDR)’ provides a large collection of expressed sequence tags (ESTs) harboring simple sequence repeats (SSRs) from several Rosaceae genera, including Rosa (rose). Primer pairs flanking SSR were designed for 312 unique Rosa ESTs based on GDR database. Eight rose (Rosa hybrida L.) genotypes were tested for PCR amplification, and 287 (92%) of the primer pairs generated allele-specific PCR bands that were readily scored. From 183 (63.7%) primer pairs that evidenced polymorphic alleles among the eight rose cultivars, 20 pairs evidencing EST sequence homology to known gene functions and high levels of polymorphism were selected and utilized for DNA fingerprinting and genetic diversity assessments of 47 rose hybrids. A total of 202 polymorphic bands were scored and generated unique fingerprints for each rose hybrid. The Nei–Li genetic similarity coefficients among 1081 pair-wise comparisons of 47 cultivars exhibited a broad range of genetic variations from 0.30 (‘Grand King’ and ‘Carnival’) to 0.99 (‘First Red’ and ‘Red Champ’). UPGMA cluster analysis divided 47 hybrids into five major groups and two sub-groups. The cross-species transferability of 273 EST-SSR primer pairs was evaluated using four genotypes of the strawberry, a genus member of the Rosaceae family. PCRs on the DNA samples of strawberry were successful for 165 primer pairs; among these, 123 pairs amplified 243 polymorphic bands. As surrogates of the marker transfer, the phenetic relationship among the four strawberry genotypes was evaluated. Genetic similarity coefficients varied from 0.78 (‘Maehyang’ and ‘Janghyee’) to 0.64 (‘Janghyee’ and ‘Pragana’). The results of cluster analysis showed that the three octaploid strawberry cultivars were quite similar, whereas the diploid ‘Pragana’ was related distantly at the genomic DNA level. The EST-SSR markers developed in the present study can be efficiently utilized for genetic diversity studies in Rosaceae.     

Intra-cultivar variability of three major olive cultivars grown in different areas of central-southern Italy and studied using microsatellite markers

A collection of 70 olive samples, originating from diverse areas in central-southern Italy (Abruzzo, Apulia, Calabria, and Umbria) and corresponding to 3 major cultivars denominations (‘Carolea’, ‘Coratina’ and ‘Frantoio’), was genotyped at 10 microsatellite loci. In total, 44 alleles with a mean number of 4.4 alleles per locus were detected. The molecular analysis, allowed the study to show a clear genetic diversity between the three cultivars ‘Carolea’, ‘Coratina’ and ‘Frantoio’ and to state that ‘Carolea’ is a polyclonal cultivar, while ‘Coratina’ and ‘Frantoio’, are probably monoclonal ones. The analysis of intra-varietal polymorphism, through the SSR analysis, proved to be very useful both for varietal identification and for intra-varietal ones. Our work shows that the current designations of olive cultivars fall short of describing the genetic variability among economically important plant material. A thorough investigation of the existing variability will prove of major importance for both management and economic production of olive trees.     

SSR fingerprinting Chinese peach cultivars and landraces (Prunus persica) and analysis of their genetic relationships

Thirty-two Chinese peach landraces/cultivars, a major subset of the core Chinese peach collection, were fingerprinted using seven pairs of SSR primers to assess their genetic diversity and relatedness. The seven primer pairs detected eight loci and revealed an allele richness of 3.125 (average alleles per locus), an expected heterozygosity (He) of 0.450, and a Shannon index of 0.728 among the landraces/cultivars. This level of genetic diversity is lower compared to other fruit trees and Prunus congenus species (cherry and apricot), but it is comparable to previous reports in peaches. A greater level of genetic diversity was observed in landraces than in cultivars, indicating that peach landraces are valuable for germplasm collection. All cultivars and landraces, except two, were unambiguously identified based on multi-locus genotypes. Eight unique alleles were detected among this group of Chinese peaches. UPGMA clustering analysis separated the 32 cultivars/landraces into two distinct groups, which is generally in accordance with the known pedigree information. The results provide accurate genetic information for defined acquisition policy in the repositories, improving the integrity and efficiency of germplasm management and giving evidences for protection of breeder's intellectual rights.     

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.     

DNA fingerprinting of olive varieties in Istria (Croatia) by microsatellite markers

The Istria region, where olives have been cultivated for many centuries, is characterized by a considerable variety of microclimates. The study of varieties traditionally cultivated in Croatian Istria and their relationships with varieties in historically and geographically connected regions is very important in order to identify native olive germplasm, well adapted to local conditions, and to characterize the oil of regional origin. Twelve olive microsatellite markers were used for identification and differentiation of a set of 27 olive accessions grown in Istria (Croatia). Among the 27 accessions, 18 different SSR profiles were discriminated. All 12 microsatellite markers analysed were polymorphic, revealing a total of 81 alleles. The number of alleles per locus ranged from four to nine. This is the first molecular characterization of olive germplasm in Croatian Istria. The analysis clarified the genetic relationships of varieties native to Croatian Istria with introduced olive varieties, as well as with varieties in the neighbouring Slovene Istria region. Numerous varieties in neighbouring regions showed high similarity and a few cases of synonymy (‘Bilica’-‘Bjankera’; ‘Buga’-‘?rna’) and one Croatian-Slovenian homonymy (‘Bu?a’-‘Buga’) were observed. The results provide useful information for a native germplasm survey and can be used for the construction of a unique database comprising all olive varieties in the Istrian region of Croatia and Slovenia.     

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.     

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.     

Study of Genetic Diversity of Pear Genotypes and Cultivars (<Emphasis Type="Italic">Pyrus communis</Emphasis> L.) Using Inter<Emphasis Type="Italic">-</Emphasis>Simple Sequence Repeat Markers (ISSR)

ISSR molecular marker was used to investigate genetic diversity of ‘Dare Gazi’ genotypes of Mashhad Esteghlal orchard and its relationship with other commercial and native cultivars of pear. Among ‘Dare Gazi’ genotypes of Mashhad Esteghlal orchard 23 genotypes were selected base on difference in tree vigor, leaf color, shape and color of fruit and also 33 other commercial and native pear cultivars from Esteghlal orchard and other Mashhad commercial orchards were studied. A total number of 230 DNA fragments were obtained using 11 primers of which 225 were polymorphic. On average, each primer produced 20.9 bands. Dice similarity coefficient ranged from 0.27 (between ‘Dom Kaj’ and Asian pear) to 1 (between ‘Dare Gazi’ 1 and 2 genotypes). Sample cluster dendrogram indicated that 56 genotypes were divided into 12 distinct clusters. The dendogram generated on the principle of Unweight Pair Wise Method using Arithmetic Average (UPGMA) was constructed by Dice coefficient and the genotypes were grouped into 12 clusters. ‘Dare Gazi’ genotypes did not show 100% similarity due to seed propagation or mutation, as ‘Dare Gazi’ 3 and 18 genotypes had the lowest similarity coefficient (0.64). Asian pears were placed in a separate group from European pears. And ‘Dare Gazi’ genotypes from different orchards were grouped separately, but all of them are called as ‘Dare Gazi’ pear for convenience. ISSR molecular marker can well identify the genetic variability among genotypes and cultivars and found suitable for grouping them.     

Genetic variation in tomato populations from four breeding programs revealed by single nucleotide polymorphism and simple sequence repeat markers

Genetic variability in modern crops is limited due to domestication and breeding. To investigate genetic variation in different populations, 216 tomato (Solanum lycopersicum L.) cultivars, hybrids, and elite breeding lines from four breeding programs were genotyped using single nucleotide polymorphism and simple sequence repeat markers. Of 47 markers analyzed, 72.3% were polymorphic in the whole collection of 216 genotypes and 51.06–59.57% showed polymorphisms in individual populations. However, genetic variation was narrow in all four populations. Nei's genetic distance varied from 0.0422 to 0.1135 between populations and from 0.0085 to 0.3187 between lines in individual populations. Cluster and principal coordinate analysis indicated that the four populations could be grouped into three clades. Lines from Shenyang Agricultural University and China Agricultural University population formed the first clade, lines from Beijing Vegetable Research Center were in the second clade, and lines from Nunhems were in the third clade. This was further supported by population structure analysis using STRUCTURE2.2, and suggested that a lack of germplasm exchange might exist among breeding programs. It might be the reason that the progress of developing new varieties with significant improvement of horticultural traits in China is slow in recent years.     

Genetic diversity in local Tunisian pears (Pyrus communis L.) studied with SSR markers

Few records are available about local Tunisian pear cultivars characterized by low chilling requirements and adaptation to dry conditions. In this work, seven SSRs derived from apple were successfully transferred to 25 local Tunisian pear genotypes and 6 common varieties of Pyrus communis cultivated in Europe. The 7 SSRs used amplified a total of 36 fragments. All the microsatellites except one seem to amplify more than one locus in some of the genotypes studied. Only 12 different fingerprinting patterns could be distinguished among the 25 Tunisian cultivars studied indicating a high number of synonymies. The mean expected and observed heterozygosities in the 25 Tunisian cultivars analyzed averaged 0.71 indicating a high level of genetic diversity among the local Tunisian pear germplasm. These markers will be useful to optimize the conservation of this highly threatened germplasm.     

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.     

利用SSR研究不同国家桃育成品种的遗传多样性

利用34对SSR分子标记对来自不同国家的56份桃育成品种进行遗传多样性分析。筛选的13对SSR引物共检测出226个等位基因,其中多态性等位基因为222个。桃群体的平均Nei’s基因多样度为0.224,Shannon遗传多样性表型指数为0.367,说明桃总群体遗传变异较低;基因分化系数为0.081,与AMOVA分析结果8.13%相近,说明2者遗传变异以群体内遗传变异为主;基因流值为5.657,则说明不同国家间桃育成品种交流比较频繁。根据Nei’s基因多样度和Shannon遗传多样性表型指数2指标所得,欧美品种群遗传变异最高,其次为中国,最后为日本。UP-GMA聚类分析结果表明,品种间的遗传距离与系谱关系基本吻合。     

核桃种质资源研究进展

核桃是世界范围内的重要果树,无论面积和产量在各类干果中居首位。种质资源是核桃品种选育的基础,对国内外核桃种质资源研究工作进行了全面综述,包括国内外种质资源研究现状、分类和利用现状、品种类型适应性研究等方面,并对美国国家种质资源圃收集及利用情况进行了介绍。提出在今后的核桃种质资源研究工作中,应充分发挥我国资源丰富的优势,加强和利用不同特异类型及生物技术开展核桃的种质资源研究。     

Genetic diversity of Iranian and some European grapes as revealed by nuclear and chloroplast microsatellite and SNP molecular markers

Genetic and chloroplast haplotype variations of 35 Iranian genotypes and 10 European grape cultivars were investigated using 9 nuclear simple sequence repeats (nSSRs), 4 chloroplast simple sequence repeats (cpSSRs) and 46 single nucleotide polymorphism (SNP) markers. In total, 83 alleles were detected at nine nSSRs, giving a mean of 15.66 alleles per locus and polymorphism information content (PIC) values ≥0.75 ranged from 0.75 to 0.90. For SNP markers, PIC values varied from 0.30 to 0.39 with an average of 0.34. Analysis of molecular variance revealed 97 and 93% of partitioned genetic diversity within populations using nSSRs and SNPs markers, respectively. Un-weighted neighbour-joining (NJ) cluster analysis grouped grapes into 10 and 9 major clusters using SSR and SNP markers, respectively. Synonyms and homonyms were identified among the Iranian genotypes. Close genetic relationship among Farkhi and Bidane-Sefid genotypes may probably propose a common ancestor and mutational evolution. Most European cultivars were differentiated from Iranian genotypes, however, clustering of some Iranian genotypes with European cultivars in the same clusters suggests that clonally propagated materials have probably been exchanged from the Middle East to West or vice versa. C and D chloroplast haplotypes were the most frequent within the Iranian genotypes, while A chloroplast haplotype was exclusively observed among European cultivars.