Comparison of SSR polymorphisms using automated capillary sequencers,and polyacrylamide and agarose gel electrophoresis: Implications for the assessment of genetic diversity and relatedness in almond

In this work we have performed a comparative study of the utilization of three electrophoresis separation methods for the analysis of peach SSR polymorphism in almond and its implications in the assessment of genetic diversity and relatedness. Automated SSR detection and polyacrylamide gel electrophoresis were the most efficient methods and would be able to resolve allelic variation at a finer scale than the electrophoresis in MetaPhor^® agarose. Moreover, automated electrophoresis detection is much more expensive in terms of cost of equipment and cost of the analysis. In addition, SSR detection using polyacrylamide gel electrophoresis showed similar results than automated sequencing, although it is more time-consuming and toxic than electrophoresis in agarose gels. Discrepancies among polyacrylamide and automated capillary, and MetaPhor^® were observed when differences in SSR polymorphisms were between 1 and 5 bp. While the use of MetaPhor^® agarose appears less indicated for genotype characterization, this technique may be the most convenient in other applications, i.e. mapping of population, due to its lower cost and easier routine application. The utilization MetaPhor^® agarose produced a slightly different clustering of genotypes in spite of the big differences observed in the DNA fingerprinting. However, bootstrap values using MetaPhor^® agarose were lower indicating a lower accuracy of this SSR polymorphism analysis method.     

The origin and dissemination of the cultivated almond as determined by nuclear and chloroplast SSR marker analysis

Sixteen nuclear and 10 chloroplast SSR markers were evaluated for 40 almond genotypes including cultivated almond, 18 related species and 5 interspecific-hybrid populations. Results establish the value of SSR (nuclear and chloroplast) markers for distinguishing different genetic lineages and characterize an extensive gene pool available to almond genetic improvement. Hierarchical analysis using integrated nuclear and chloroplast DNA markers support Prunus fenzliana, a species native to the northeast Iran, as a probable ancestor of the cultivated almond. Results also established the importance of interspecific hybridization and subsequent genetic introgression in the development of cultivated almond and demonstrate continuing value of an interspecific gene pool for modern cultivar improvement. Molecular results implicate a dissemination of the cultivated almond from Asia to the Eastern Mediterranean and subsequently the Western Mediterranean and the New World is supported by the molecular analysis of regional germplasm.     

Genetic characterization of different demes in Prunus persica revealed by RAPD markers

DNAs of 180 accessions in 10 demes in Prunus persica were amplified with twenty-two, 10-base primers selected from 200 arbitrary primers using Randomly Amplified Polymorphic DNA (RAPD) technology. One hundred and eighty loci were observed and recorded. With statistical analyses of the data from the study, genetic diversity of the demes was expressed as follow: yellow peach group > honey peach group > flat peach group > red leaf peach group > crisp peach group > bitao group and juicy peach group > nectarine group > shouxingtao group > weeping peach group. Genetic variations among and within groups by AMOVA analyses were 11.9, 88.1%, respectively. Demes clustered by UPGMA modified from NEIGHBOR procedure of PHYLIP Version 3.5, the edible peaches of which were combined as a section, while the ornamental species were classified into separate sections. Through analyses of genetic diversity and genetic structure, the results could provide molecular biological evidence for conservation and utilization of P. persica germplasm.     

Molecular characterization and analysis of somaclonal variation in chrysanthemum cultivars using RAPD markers

Molecular characterization using RAPD analysis was carried out in eight cut flowers and two pot plant cultivars of chrysanthemum. Three of them (‘Refocus’, ‘Red Reagan’, and ‘Sheena Select’) were established in vitro and the occurrence of somaclonal variation was studied using the same molecular technique. Two induction media (MS + 0.1 mg l⁻¹ NAA + 0.1 mg l⁻¹ BA, and MS + 2.0 mg l⁻¹ IAA + 0.5 mg l⁻¹ Kinetin), and two proliferation media (MS + 0.1 mg l⁻¹ NAA + 0.2 mg l⁻¹ BA, and MS + 4.0 mg l⁻¹ IAA + 2.0 mg l⁻¹ Kinetin) were employed in order to evaluate the effect of the medium composition in the shoots’ stability. Likewise, the effect of the culture age was considered in assessing genetic stability. Monthly subcultures were carried out, identifying the origin and history of the shoots, throughout a nine-month proliferation period followed by acclimatization. Molecular markers were obtained in every subculture cycle and from the acclimatized plants. Only one shoot from the 7th subculture of the cultivar ‘Refocus’ showed a different band pattern. The use of RAPD for chrysanthemum cultivar characterization and somaclonal variation detection is discussed.     

Correlations between quantitative tree and fruit almond traits and their implications for breeding

Correlations between 29 quantitative tree and fruit traits were studied in 46 almond genotypes and some inter-specific crosses. Results reflected a significant diversity in the assayed almond germplasm. In addition, the obtained cluster demonstrated varying degrees of relationships, illustrating higher correlation values for the flowering traits and lower correlation values for the nut and kernel traits. To determine the importance of the traits and the levels of similarity existing between the various studied traits, a discriminate analysis was carried out. According to the results obtained from these analyses, the different traits were discriminated on basis of the characteristics of the presence of double kernels, the nut and the kernel size, the flowering date, kernel yield and shelling percentage. Finally, this study evidenced correlations between most of the agronomic traits in almond, although the correlations coefficient (r) value was found to be higher than 0.5 only in some cases. This analysis can help breeders for choosing the most favorable entries to build a core subset of the almond collection for the purpose of breeding.     

Evaluation of genetic relationships among Iranian pistachios using microsatellite markers developed from Pistacia khinjuk Stocks

To evaluate the genetic relationships among wild and cultivated Pistacia species grown in Iran and the analysis of genetic variation among Iranian pistachio genotypes, two DNA libraries enriched for dinucleotide (AG)_n and trinucleotide (ATG)_n microsatellite motifs were developed from Pistacia khinjuk genome. Following screening of clones by colony PCR technique, 44 clones were sequenced and 27 pairs of primers designed from flanking regions of the repeats. The examination of primer pairs, designed from P. khinjuk sequences, showed successful cross-species amplification within the genus Pistacia. A dendrogram constructed on the basis of the Minimum Evolution clustering algorithm revealed that Pistacia vera has closer relationships with P. khinjuk, than with Pistacia integerrima, Pistacia palaestina, Pistacia atlantica and Pistacia mutica. The dendrogram further distinguished the wild Sarakhs pistachio from the rest of P. vera genotypes suggesting that the domesticated genotypes of P. vera are evolved from P. vera var. Sarakhs and then this wild genotype likely develops to other local pistachios. Hence, it seems that the wild Sarakhs pistachio plays an important role in evolutionary trend of the edible pistachios in Iran. The results indicated that microsatellites developed in P. khinjuk are distributed in the genome of indigenous pistachio species including P. vera genotypes and therefore they will be useful in characterization of Iranian pistachio genotypes.     

Comparative analysis of genetic diversity in Prunus L. as revealed by RAPD and SSR markers

RAPD and SSR markers were used for genetic diversity evaluations among 15 genotypes selected from the genus Prunus L. Altogether 40 RAPD primers and 21 primer pairs designated for microsatellite loci were applied on the whole group of genotypes.     

Genetic diversity of apricot revealed by a set of SSR markers from linkage group G1

Eight polymorphic simple sequence repeat (SSR) markers located in the G1 linkage group of apricot (Prunus armeniaca L.) were previously developed and evaluated in a small set of cultivars. Those primers were used for studying variability in 77 apricot cultivars belonging to five different geographical groups, such as Chinese, Asian (Irano-Caucasian and Central Asian), North American, Mediterranean and Western European as well as Middle European cultivars. Six of the markers were polymorphic and revealed a total of 71 alleles ranging from 5 (aprigms11) to 20 (aprigms1) alleles per locus with a mean value of 11.83 alleles per locus. In conclusion, the SSR loci located in the G1 linkage group show a level of polymorphism which is similar to loci dispersed throughout the entire genome. The total number of alleles and the number of unique alleles were the highest in Chinese apricots and the lowest in Middle European cultivars. Heterozygosity also showed a decrease from Asia and China to Middle Europe. No association could have been observed between any SSR markers tested and plum pox virus (PPV) resistant phenotype of cultivars. PPV resistant cultivars did not form a separate clade on the dendrogram obtained by UPGMA cluster analysis. Middle European and Chinese cultivars formed separate clusters while other genotypes formed smaller multiple sub-groups or scattered among different clusters. Our results support previous hypotheses on the origin of PPV resistance in North American apricots. The allele data was also presented in a form that allowed the easy observation of allele frequencies in each geographical group at each locus. Using this data field, differences and similarities between cultivar groups can be easily assessed. The analysis demonstrated the links between the North American and Mediterranean apricot germplasm and confirmed that the Chinese and Eastern European cultivars are distantly related.     

Cross-transferable polymorphic SSR loci in Prunus species

This work reports the transferability and polymorphism of previously reported SSRs in 10 Prunus species. The availability of a large number of SSRs in the genus Prunus makes marker choice random, while preventing comparison of results in fingerprinting studies. The availability of SSR markers, polymorphic in a wide sample of Prunus species, would facilitate marker choice, while allowing the comparison of results. In this work, microsatellite markers useful for analyzing 10 different Prunus species (P. persica, P. dulcis, P. armeniaca, P. domestica, P. insititia, P. salicina, P. cerasifera, P. avium, P. cersus and P. mahaleb) were searched through screening SSRs previously reported to be conserved and/or polymorphic in more than one Prunus species. A selected group of 13 SSRs, transferable to the 10 species, was analyzed in terms of their usefulness for analyzing these species. The amplification range, polymorphism and variability detected by these loci are reported. The information provided will be useful for Prunus genetic studies as well as conservation and management of Prunus germplasm resources.     

Comparative analysis of genetic diversity in Tunisian apricot germplasm using AFLP and SSR markers

Eighty-one accessions representing apricot germplasm in Tunisia were collected from different areas of cultivation and fingerprinted using amplified fragment length polymorphism (AFLP) and microsatellites (SSR) markers. A total of 339 polymorphic markers were revealed using 5 AFLP primers combinations and 24 SSR loci. AFLP and SSR markers expressed a high level of polymorphism allowing the distinction of the accessions with an efficiency coefficient of discrimination of 100% for AFLP and 97% for SSR markers. Genetic diversity structure was assessed with AFLPs and SSRs markers separately then with combined matrix data by the help of hierarchical clustering elaborated using Wards method based on Nei and Li (1979) distances. Comparison of the obtained dendrograms revealed a phylogeographic structure into two major groups with significant conservation between the observed subgroups in relation with the geographic origin of the accessions. The relative efficiency of the markers in determining the genetic relationships among apricot accessions has been assessed and a combination of AFLPs and SSRs markers was the most effective. In addition, Mantel test based on genetic distances indicated highly significant correlation between AFLP-SSR data and each of the AFLP and SSR ones, with Pearson correlation values of r = 0.873 and r = 0.692, respectively, revealing the higher efficiency of the combination of both molecular techniques (AFLP and SSR) to estimate the levels of genetic variability among apricot germplasm.     

Molecular and physiological identification of new S-alleles associated with self-(in)compatibility in local Spanish almond cultivars

The S-allele characterisation of ‘Alzina’ and ‘Garondès’, two local almond cultivars from the island of Majorca, by a multidimensional approach has allowed the confirmation of the presence of the S_f-allele and the identification of a new allele not previously described in almond, S₃₆. When these cultivars were phenotypically evaluated, both showed a self-incompatible phenotype and were cross-incompatible, as assessed by artificial self- and cross-pollinations and fruit sets after field pollinations, confirming that their S_f-allele is in its active form, S_fa. Thus a new CGI group in almond is proposed and named XXVIII. These results confirm the wide diversity of S-alleles in almond both at genotypic and phenotypic levels, as well as their similarity with the S-alleles from other close Prunus species. This similarity suggests the possibility of allele introgression between species or allele identity by descent from a common ancestor.     

Comparison of the use of morphological,protein and DNA markers in the genetic characterization of Iranian wild Prunus species

In this study, the genetic diversity of four Iranian wild Prunus species including Prunus eleagnifolia, Prunus hauskenchtii, Prunus scoparia and Prunus lycioides were investigated using morphological, protein and DNA markers. DNA markers included nuclear and chloroplast SSRs and self-incompatibility (S) allele amplification. At the morphological level, leaf width showed significant differences between the four wild Prunus species. Concerning fruit and kernel characters, their values are relatively similar indicating the high degree of homoplasy described in Prunus. Nuclear SSR markers have been the most abundant markers with a higher polymorphism in comparison with morphological, protein and chloroplast SSR markers. Results also indicated the high variability present in the S locus. On the other hand, the correlation between the clustering based on DNA markers and protein were in general low. Dendogram performed using nuclear and chloroplast SSR indicated a more diffuse clustering between the wild almond species probably due to the natural introgression of genes observed in these wild almond species. Data from the analysis of the total protein seems to be more accurate to establish taxonomy relationships in these very close wild species.     

Inter- and intra-specific genetic diversity among cyclamen accessions investigated by RAPD markers

Random amplified polymorphic DNA (RAPD) analysis was used to characterize genetic diversity of 26 Cyclamen persicum and Cyclamen com accessions. Eighty-four arbitrary primers tested, among which nine primers showed reliable polymorphic banding patterns and yielded 104 polymorphic markers. Jaccard's similarity coefficient among accessions ranged from 0.99 to 0.08. At a similarity of 68%, accessions were divided into three clusters. The cophenetic correlation coefficient between the similarity matrix and the dendrogram was relatively high (r = 0.9) showing the goodness of fit of the dendrogram. The RAPD analysis offered a rapid and reliable tool for the estimation of inter- and intra-specific variability in cyclamens. The wide genetic variation observed for cyclamens within Iran guarantees a promising future of breeding.     

Assessment of genetic diversity of Latvian and Swedish sweet cherry (Prunus avium L.) genetic resources collections by using SSR (microsatellite) markers

Three previously described highly polymorphic SSR (microsatellite) primer pairs were tested on 126 sweet cherry (Prunus avium L.) accessions to adapt a fast, reliable method for preliminary screening of sweet cherry germplasm collections and to compare two sweet cherry germplasm collections: at the Latvia State Institute of Fruit-Growing, Dobele (LIFG-Dobele) and at the Division of Horticultural Genetics and Plant Breeding at Balsgård, Department of Crop Sciences, Swedish University of Agricultural Sciences (SLU-Balsgård). The SSR loci were highly polymorphic with 4–10 different alleles and 5–18 genotypes. Heterozygosity values ranged from 0.431 to 0.809, gene diversity (PIC) values ranged from 0.400 to 0.753, and the discriminating power of each locus varied from 0.631 to 0.894. The combined discriminating power of all loci was highly effective (0.996). Sixteen identical accession groups with the same allele profile were discovered in both collections. This study demonstrated that SSR fingerprinting with the three primer pairs tested, can be used for preliminary characterization of sweet cherry germplasm collections.     

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.     

Self-fertilization in homozygous and heterozygous self-compatible almonds

In homozygous self-compatible genotypes 100% of the pollen grains are potentially able to grow through their own pistil, and thus the rate of self-fertilization could be higher than in heterozygous self-compatible genotypes. To evaluate the advantages of growing homozygous self-compatible almonds, pollen tube growth along the pistil at different times following self-pollination, and fruit set were studied in four homozygous and four heterozygous self-compatible seedlings. The results showed important differences between homozygous and heterozygous individuals for the percentages of pollen tubes in the third section of the style at 24 and 48 h, the pollen tube growth rate being higher in the homozygous. Twenty-four hours following self-pollination only the homozygous individuals showed pollen tubes in the ovary. However, at 72 and 96 h those values were similar for both genotypes, suggesting that space and availability of nutrients become the main limiting factors, overcoming the genetic interactions between pollen and pistil. In general, fruit set was similar in homozygous and heterozygous individuals. Interestingly, one of the homozygous individuals showed problems of fruit development, which might be explained by its inbred origin.     

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.     

Influence of self-pollination in fruit quality of autogamous almonds

Due to the several advantages expected from growing self-compatible almond cultivars, breeding programmes have recently introduced self-compatibility as an important trait. However, doubts have arisen regarding the capability of the newly obtained self-compatible almonds to produce good yields in monovarietal plantations, and the commercial quality of the fruits from self-pollination. For this reason, in the last years several studies have been conducted to evaluate fruit set and fruit traits following self- and cross-pollination of self-compatible almonds. Their results showed inconsistencies for fruit set and for some of the fruit traits studied. Thus, while some studies showed no differences between both pollination types for any of the fruit traits, others clearly showed important differences with the detriment of self-pollination for commercially important fruit traits. The fact that some of these studies evaluated a low number of individuals and/or fruit traits, implies the need of a more complete study. In this work, 10 fruit traits were studied in 26 self-compatible almonds following self-pollination by bagging and open pollination treatments. In general, and despite a few individuals showed differences between both pollination types for two of the traits, the results showed no influence of self-pollination in fruit quality.     

Comparative analysis of genetic diversity in Citrus germplasm collection using AFLP,SSAP, SAMPL and SSR markers

In this study we evaluate the informativeness and efficiency of Amplified Fragment Length Polymorphism (AFLP), Sequence-Specific Amplified Polymorphism (S-SAP), Selectively Amplified Microsatellite Polymorphic Loci (SAMPL) and Simple Sequence Repeat (SSR) markers for genetic diversity, phylogenetic relationship among the Citrus species and mapping ability of the marker system. The SSR exhibited relatively higher level of polymorphism information content in terms of the expected heterozygosity, than that of the AFLPs, SSAPs and SAMPLs. For each marker system, average level of the discriminating potential was very close to the actual discriminating potential. Similarity matrices showed weak, yet significant correlations when Mantel's test was applied. The highest positive (0.72) correlation was found between the AFLP and SSAP markers. The SSR and SAMPL markers were poorly correlated. The dendrogram topology among the four marker systems had high similarity. Taken together, the SSAP and SAMPL were highly efficient in detecting genetic similarity in Citrus, while the SSR may be more useful for segregation studies and genome mapping in Citrus. The SSAP and SAMPL markers could be useful for Citrus genome mapping in combination with AFLP and SSR markers. To our knowledge, this was the first detail report of a comparison of performances among AFLP, SSR and retrotrasposon based molecular marker technique on a set of samples of Citrus. Our result provides guidance for future efficient use of these molecular methods in genetic analysis of Citrus sp. and its relatives.     

Comparative analysis of autochthonous almond (Prunus amigdalus Bastch) material and commercial varieties in the Castilla-La Mancha Region (Spain)

The Castilla-La Mancha Region is characterized by a large variety of microclimates, allowing the cultivation of species with different climate requirements. While the districts of “Sierra del Segura” and “Campos de Hellín” account for the largest almond growing area in the region. This comparative study is based on the existence of native almond material (local names De Santos and Daniel) which shows kernel morphology and nut quality quite similar to the two most well known commercial varieties of almonds: Marcona and Desmayo Largueta. The identification of this native almond material is of great importance due to its adaptation to local environmental conditions (low temperatures, drought, hot and dry summers) and its morphological similarities with these two almond commercial varieties. A set of 6 Prunus SSR markers were used to identify and differentiate a total of 27 almond samples taken in the “Sierra del Segura” district, including local plant material and material from commercial varieties as a reference. Ten different SSR profiles were discriminated. The number of alleles per locus ranged from seven to twelve with a total of 52 alleles for all loci and an average of 9 alleles per locus. The analysis showed that they share 58% of the studied alleles with one common allele size in both cases for all the SSRs studied. This is the first molecular characterization of native almond germplasm in the Castilla-La Mancha Region. The results provide useful information that could be included in the future almond germplasm bank.