Ivan Pejić

Comparative analysis of genetic similarity among maize inbred lines detected by RFLPs, RAPDs, SSRs, and AFLPs

Abstract DNA-based fingerprinting technologies have proven useful in genetic similarity studies. RFLP is still most commonly used in the estimation of genetic diversity in plant species, but the recently developed PCR-based marker techniques, RAPDs, SSRs and AFLPs, are playing an increasingly important role in these investigations. Using a set of 33 maize inbred lines we report on a comparison of techniques to evaluate their informativeness and applicability for the study of genetic diversity. The four assays differed in the amount of polymorphism detected. The information content, measured by the expected heterozygosity and the average number of alleles, was higher for SSRs, while the lowest level of polymorphism was obtained with AFLPs. However, AFLPs were the most efficient marker system because of their capacity to reveal several bands in a single amplification. In fact, the assay efficiency index was more than ten-fold higher for AFLPs compared to the other methods. Except for RAPDs, the genetic similarity trees were highly correlated. SSR and AFLP technologies can replace RFLP marker in genetic similarity studies because of their comparable accuracy in genotyping inbred lines selected by pedigree. Bootstrap analysis revealed that, in the set of lines analysed, the number of markers used was sufficient for a reliable estimation of genetic similarity and for a meaningful comparison of marker technologies.

Microsatellite variability in grapevine cultivars from different European regions and evaluation of assignment testing to assess the geographic origin of cultivars

Abstract Nine microsatellite markers (VVMD5, VVMD7, VVS2, ssrVrZAG21, ssrVrZAG47, ssrVrZAG62, ssrVrZAG64, ssrVrZAG79 and ssrVrZAG83) were chosen for the analysis of marker information content, the genetic structure of grapevine cultivar gene pools, and differentiation among grapevines sampled from seven European vine-growing regions (Greece, Croatia, North Italy, Austria and Germany, France, Spain and Portugal). The markers were found to be highly informative in all cultivar groups and therefore constitute a useful set for the genetic characterization of European grapevines. Similar and high levels of genetic variability were detected in all investigated grapevine gene pools. Genetic differentiation among cultivars from different regions was significant, even in the case of adjacent groups such as the Spanish and Portuguese cultivars. No genetic differentiation could be detected between vines with blue and white grapes, indicating that they have undergone the processes of cultivar development jointly. The observed genetic differentiation among vine-growing regions suggested that cultivars could possibly be assigned to their regions of origin according to their genotypes. This might allow one to determine the geographical origin of cultivars with an unknown background. The assignment procedure proved to work for cultivars from the higher differentiated regions, as for example from Austria and Portugal.

Discriminating maize inbred lines using molecular and DUS data

Growing numbers of candidate varieties, decrease of their variability for morphological traits, and internationalization of the national list all contribute to excessive increase of the trial costs, thus creating the need for the improvement of current variety evaluation procedures, especially regards their distinctness, Uniformity, and Stability (DUS) component. Due to rapid advancement in molecular techniques, the use of molecular markers in DUS testing as a complement to, or replacement of, morphological observations became the subject of great interest in scientific studies, and consequently topic for discussion within International Union for the Protection of New Varieties of Plants (UPOV). In order to explore the potential of simple sequence repeat (SSR) markers for distinctness tests, present study involved set of 41 maize inbred lines that were scored for 32 DUS characters prescribed by UPOV and genotyped at 28 SSR loci. Results were largely in favor of the use of molecular markers, revealing or confirming their already known advantages over morphological markers like better consistency with the pedigree, and relatively higher discriminative power. However, their integration into DUS testing protocols still depends upon resolving of several important issues.

Genetic diversity of Bradyrhizobium japonicum field population revealed by RAPD fingerprinting

RAPD fingerprinting was used for strain identification and the assessment of genetic diversity within a field population of Bradyrhizobium japonicum. Total genomic DNAs from 13 field isolates and two inoculant strains were amplified using six different 10‐mer primers. Different and informative band patterns were obtained for all strains analysed. Cluster analysis unexpectedly revealed that none of the field isolates was identical to inoculant strains which were regularly used for soybean inoculation. Among field isolates two highly divergent groups were determined. The results indicate that RAPD is a very discriminative and efficient method for differentiating and studying genetic diversity of B. japonicum strains.

MICROSATELLITE MARKERS FOR GRAPEVINE: TOOLS FOR CULTIVAR IDENTIFICATION & PEDIGREE RECONSTRUCTION

Microsatellites have become a favorite type of DNA marker for identification of grapevine cultivars, and their properties enable a wide range of applications from cultivar identification based on various parts of the grapevine plant to pedigree reconstruction and genome mapping (Sefc et al. 2001). Genetic markers have the advantage that the DNA of a certain plant is identical in all cells of any tissue at any stage of development and is not influenced by environmental or sanitary conditions of the plants. DNA can be obtained from every kind of plant tissue available, e.g. wood, leaves or berries, and analyses can therefore be carried out at any time of the year. Thanks to the PCR (Polymerase Chain Reaction) technology, minute amounts of DNA can be analyzed, which extends the usefulness of the method from plant tissue to grapevine products such as must and wine. The typical microsatellite sequence consists of five to about one hundred tandem repeats of short, simple sequence motives composed of 1 to 6 nucleotides (e.g. (GA)n, (GATA)n (Fig. 1). In eukaryotes, an estimated 104 to 105 microsatellite loci are scattered randomly throughout the genome. This abundance of microsatellite sequences in eukaryote genomes constitutes an almost unlimited source of polymorphic sites that may be exploited as genetic markers. Thomas et al. (1993) first investigated the use of microsatellite DNA for identifying grapevine cultivars. They showed that microsatellite sequences were abundant in grapevine and very informative for identifying V. vinifera cultivars. Moreover primer sequences were conserved across other Vitis species and Muscadinia (Thomas and Scott 1993). It was also demonstrated through pedigree analysis that the microsatellite alleles were inherited in a co-dominant Mendelian manner (Thomas and Scott 1993) confirming their suitability for genetic mapping and investigation of genetic relatedness (Thomas et al. 1994). As other groups throughout the world became interested in grapevine microsatellite genotyping, a large number of markers has been developed by individual groups (e.g. Bowers et al. 1996, 1999b, Sefc et al. 1999, Arroyo-Garcia et al. 2004, Adam-Blondon et al. 2004, Di Gaspero et al. 2005, Merdinoglu et al. 2005, Goto-Yamamoto et al. 2006) and in the framework of the Vitis Microsatellite Consortium (VMC, 1997-2004). The approach used for microsatellite marker development in these studies was the construction of a genomic library from a grapevine cultivar or rootstock, screening of the library with microsatellite probes, sequencing of microsatellite containing clones, design of PCR primers from the sequences flanking the microsatellite, optimization of PCR conditions and characterization of the microsatellite polymorphism. Furthermore, public databases as those compiled from cDNA and EST (Expressed Sequence Tag) type data were recognized as a source of microsatellite loci, and a large grape EST dataset (Ablett et al. 1998) was employed for the development of microsatellite markers (Scott et al. 2000b). Over 100 microsatellites were readily identified, and the further investigation of some of these loci showed enough polymorphism to allow the discrimination of several grapevine and rootstock cultivars, and transferability of the primers to species of the related genera Cissus and Cayratia. To date, many microsatellite markers have been developed and mapped in Vitis, and locus information including primer sequences and mapping position can be retrieved from the UniSTS database (http://www.ncbi.nlm.nih.gov/sites/entrez?db=unists).

Genetic Characterization of Grapevine Cultivars Collected throughout the Dalmatian Region

Eleven SSR loci were used to identify 76 accessions of presumed native Dalmatian grapevine cultivars (Vitis vinifera L.). Plant material was collected throughout the Dalmatian region and used to reestablish a grape germplasm collection in Dalmatia, the Adriatic region of Croatia. The 76 accessions had 63 unique genotypes, of which 35 are published here for the first time. Twelve synonyms were found within the Dalmatian cultivars and 11 Dalmatian cultivars matched previously published genotypes, mainly from neighboring countries, suggesting historic exchange of grapevine cultivars. Several cultivars with similar names had different SSR profiles, making them homonyms. Genetic distance analysis revealed five groups of cultivars and confirmed several distinct grape gene pools in Croatia.

Identification of mega-environments in Europe and effect of allelic variation at maturity E loci on adaptation of European soybean

Soybean cultivation holds great potential for a sustainable agriculture in Europe, but adaptation remains a central issue. In this large mega-environment (MEV) study, 75 European cultivars from five early maturity groups (MGs 000-II) were evaluated for maturity-related traits at 22 locations in 10 countries across Europe. Clustering of the locations based on phenotypic similarity revealed six MEVs in latitudinal direction and suggested several more. Analysis of maturity identified several groups of cultivars with phenotypic similarity that are optimally adapted to the different growing regions in Europe. We identified several haplotypes for the allelic variants at the E1, E2, E3 and E4 genes, with each E haplotype comprising cultivars from different MGs. Cultivars with the same E haplotype can exhibit different flowering and maturity characteristics, suggesting that the genetic control of these traits is more complex and that adaptation involves additional genetic pathways, for example temperature requirement. Taken together, our study allowed the first unified assessment of soybean-growing regions in Europe and illustrates the strong effect of photoperiod on soybean adaptation and MEV classification, as well as the effects of the E maturity loci for soybean adaptation in Europe.

Plum germplasm in Croatia and neighboring countries assessed by microsatellites and DUS descriptors

At a certain period during the last century, former Yugoslavia (which among others used to include Bosnia and Herzegovina, Croatia, and Serbia) was the biggest producer of plums in the world. Traditional plum cultivars, still grown in this region, represent a mixture of several species including: European plums (Prunus domestica L.), mirabelles (Prunus insititia var. syriaca (Borkh.) Koehne), and damsons (P. insititia L.). The basic problem with the utilization of this plum germplasm, either for cultivation or breeding purposes, is a lack of reliable pomology data or reference repositories that would enable positive identification of cultivars. In this study, 62 plum accessions (42 traditional Croatian accessions, six well-known traditional accessions collected from Serbia and Bosnia, and 14 international, reference cultivars) were assessed using microsatellite markers and distinctness, uniformity, and stability (DUS) plum descriptors. Nine primer pairs amplified 168 distinct alleles, or on average 18.7 alleles per locus. A significant differentiation between the traditional plum cultivars and international reference cultivars, was detected through Fst (Fst = 0.022; P < 0.0001), analyses of molecular variance (AMOVA; fCT = 0.054; P < 0.05) and later confirmed by a factorial correspondence analysis (FCA). Bayesian method enabled the classification of mirabelle, damson, and European plum genotypes. Principal component analyses, based on 22 morphologic traits, managed to separate mirabelle accession from the European plum and damson accessions, but there was a general lack of correlation between the observed morphologic traits and the molecular data. Results of this study indicate that traditional Croatian accessions represent a diverse and underutilized plant genetic material, which should be conserved.

Whole genome amplification and microsatellite genotyping of herbarium DNA revealed the identity of an ancient grapevine cultivar

Reconstruction of the grapevine cultivation history has advanced tremendously during the last decade. Identification of grapevine cultivars by using microsatellite DNA markers has mostly become a routine. The parentage of several renowned grapevine cultivars, like Cabernet Sauvignon and Chardonnay, has been elucidated. However, the assembly of a complete grapevine genealogy is not yet possible because missing links might no longer be in cultivation or are even extinct. This problem could be overcome by analyzing ancient DNA from grapevine herbarium specimens and other historical remnants of once cultivated varieties. Here, we present the first successful genotyping of a grapevine herbarium specimen and the identification of the corresponding grapevine cultivar. Using a set of nine grapevine microsatellite markers, in combination with a whole genome amplification procedure, we found the 90-year-old Tribidrag herbarium specimen to display the same microsatellite profile as the popular American cultivar Zinfandel. This work, together with information from several historical documents, provides a new clue of Zinfandel cultivation in Croatia as early as the beginning of fifteenth century, under the native name Tribidrag. Moreover, it emphasizes substantial information potential of existing grapevine and other herbarium collections worldwide.

Originality of M3S maize population and changes in allele frequencies revealed by SSR markers after two cycles of selfed progeny recurrent selection

Maksimir 3 Synthetic (M3S) maize population was developed at the Faculty of Agriculture University of Zagreb by intercrossing inbred lines, whose origins trace back to several open-pollinated varieties and local populations from different regions of the former Yugoslavia. The population was subjected to two cycles of selfed progeny recurrent selection for grain yield. The objectives of this study were: (i) to determine genetic distances among the parental inbred lines of the M3S population (M3S progenitors), the M3S population before and after two cycles of recurrent selection, and elite inbred lines representing the BSSS and Lancaster heterotic group; and (ii) to examine the effect of two cycles of recurrent selection on allele frequency changes in the population. Nine M3S progenitors, three BSSS lines, and three Lancaster lines were genotyped at 24 SSR loci, out of which nine randomly chosen loci were used for genotyping 96 individuals from both C0 (the M3S population before selection) and from C2 (M3S population after two cycles of selection). A total of 101 alleles were detected across 24 loci in the 15 lines, whereas 83 alleles were found in the nine M3S progenitors. Among the latter 83 alleles 31 were unique, i.e. found only in one of the progenitors. Mean genetic distance among nine M3S progenitors was 0.61 indicating a broad genetic base of the M3S population. High mean genetic distance was found between M3S progenitors and BSSS lines (0.69) and M3S progenitors and Lancaster lines (0.71). This indicates that the M3S population represents a germplasm source unrelated to both the BSSS and Lancaster germplasm. Mean genetic distance between the M3S population and BSSS as well as Lancaster lines decreased slightly after two cycles of recurrent selection suggesting the need to introduce testers from both groups in future selection in the M3S population in order to maintain heterotic complementarity of the M3S population to these groups. A test of selective neutrality identified several non-neutral loci in the population whose allele frequency changes from the C0 to the C2 cannot be explained by genetic drift. The majority of non-neutral alleles, whose frequency increased after two cycles of selection, were present in at least one line from the BSSS or Lancaster heterotic group.