UBC 812 (5'-GA) 8 A-3'; UBC840 (5'-GA) 8 YT-3' and UBC 845 (5'-CT) 8 RG-3'. PCR amplification reactions were carried out in the total volume of 10 μl containing 1× PPP Master Mix (Top-Bio), 12.5 pmol of primer, 1× BSA and 50 ng template DNA. Amplifications were performed using the following programme: pre-denaturation for 2 min at 95°C, 40 cycles of 20 s at 93°C, 1 min 52°C and 20 s at 72°C, finally, 6 min at 72°C. PCR products were dissolved by electropho- resis on 2% agarose gel in 1× TBE buffer using the following programme: 20 min at 40 V followed by 280 min at 80 V and visualized by EtBr staining. All analyses were performed in pairs, and only samples with the same pattern of ISSR markers were scored. Altogether 19 SSR markers were selected according to variability (PIC value between 0.5 and 0.8, number of alleles between 5 and 10) with high stability and low stutter and genome position (one each chromosome to cover all chromosomes present in B. napus). PCR amplification, detection and analysis of microsatellites were performed according to Plieske and Struss (2001). AFLP analysis was carried out as described No. Accession name Type Country of origin registration Year of first
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There are few reports on the use of ISSR markers in Passiflora, however, some authors reported high polymorphism level in their work with species of this genus using such markers. Santos et al. (2011) evaluated 45 Passiflora accessions (three P. alata accessions and 42 P. edulis accessions) using 18 ISSR primers and obtained 227 polymorphic bands with 12.61 bands per primer on average. Costa et al. (2012) characterized 63 genotypes of sour passion fruit vine in Embrapa’s Manioc and Fruit Production program and obtained 22 polymorphic primers generating 266 bands with 11.56 bands per primer on average. Sousa et al. (2015) evaluated 25 wild species of Passiflora from the UESC germplasm bank in Ilheus, Bahia, using ISSR markers and obtained 20 polymorphic primers among the 31 tested, with a total of 331 bands and 16 bands per primer on average.
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A parameter MI, has been widely used to evaluate the overall utility of each marker system . The high MI in the SCoTs results from its high EMR, making these markers appropriate for fingerprinting  or evaluating genetic variation in breeding populations [48, 49]. In addition, the SCoTs performed well in other species. Compared with ISSR and inter-retrotransposon ampli- fied polymorphism (IRAP), SCoT markers were more in- formative than IRAP and ISSR for the assessment of diversity among Persian oak (Quercus brantii Lindl.) in- dividuals . Results from the evaluation on the genetic variation of mango (Mangifera indica L.) cultivars indi- cated that the SCoT analysis represents actual relation- ships better than the ISSR analysis .
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have used 10 EST-SSR markers (developed from EST data of pea available on NCBI), in amplification of 3 legumes plants (Table 2). All 10 EST-SSR markers showed more amplification capability in all 3 legumes, contained better capacity for quantifying the genetic diversity through total number of effective alleles. Around 80% of EST-SSR showed amplification in pea, followed by 60% in chickpea and 50% in mung bean.
based on genomic data and have more polymorphic detection capabilities in comparison to single primer PCR based techniques like RAPD and ISSR. Various studies have used SSR markers to investigate genetic diversity in cultivated hexaploid wheat genotypes of T. aestivum L. (Liu et al.2005; Schuster et al. 2009). The SSR markers have also been successfully used to characterize genetic diversity among wild relatives of wheat. The SSR markers are co-dominant, reproducible and have high polymorphism levels and equal distribution when it compared to other types of molecular markers. The SSRs are more abundant, ubiquitous in presence, hypervariable in nature and have high polymorphic information content. Thus the availability of a large number of molecular markers in wheat suggests their use in intraspecific analysis, comparative analysis and gene introgression studies as well as in wheat breeding (Borner et al., 2000 and Hammer 2000). .
The Polymerase Chain Reaction (PCR) based molecular markers such as RAPD, SSR and ISSR have successfully been utilized to assess the genetic diversity among the genotypes of several crop plants. Out of these, SSR are highly informative and locus specific genetic markers which are co- dominant in nature with high information content (Danin-Poleg et al., 2000). SSR has facilitated the studies of genetic diversity (Plaschke et al., 1995), gene mapping (Pestsova et al., 2002) and testing of authenticity of genetic stocks (Pestsova et al., 2002). It is typically multi-allelic marker (Matsuoka et al., 2002) with heterozygosity values much higher than
(r = 0.95, P < 0.01), suggesting that the cluster analysis strongly represents the similarity matrix. The accessions studied had similarity values rang- ing from 0.79 to 1.00. Pummelo and grapefruit groups were separated clearly (Group A and B). Group A consisted of 5 pummelos with a simi- larity value of 0.79 to grapefruits. All pummelos were distinguished clearly from each other. Yong et al. (2006) also distinguished pummelo acces- sions using SSR markers. Genetic similarities of pummelos used in our study were equal or higher than 0.85. Corazza-Nunes et al. (2002) found a similarity level of three pummelos ~0.90 based on RAPD and SSR data. This report was consist- ent with our results. Pink, Kao Panne and Red pummelos nested in the same subcluster whereas Reinking and Pummelo WN were included in another subcluster.
In addition, rootstocks obtained from desirable species of Prunus are needed for producing commercial orchards. In Iran, the cultivation of this genus as rootstocks is common among commercial orchards because of the ease of the interspecific hybridization and many advantages that are related to parents such as substantial resistance or tolerance to biotic and abiotic stresses (9). On the other hand, cultivar mislabeling (e.g. synonym or homonym trees) and none targeted interspecific crosses within Prunus species can dramatically influence on the orchard performance. However, an accurate characterization and discrimination of Prunus rootstocks is required in the effective control and utilization of the materials in breeding programs. Traditionally, rootstocks identification relied on morphological traits which were very difficult task because of its high susceptibility to environmental factors and the developmental stage of plants (4). Thus, molecular fingerprinting procedures are becoming practical necessities for the recognition of promising Prunus rootstocks and the preparation of genetic identities as a reference to establish more efficient orchards. In the last decades, many different molecular markers such as RAPD (14, 21); ISSR (3, 8); SCAR (12, 14); AFLP (13, 17, 18) and SSR (5, 6, 7, 10, 12, 22, 23) have been extensively used in Prunus rootstocks in order to the germplasm identification and evaluation of the genetic variability and relationship, Among these molecular markers, microsatellites are considered as the most reliable molecular marker systems which are used for management and diversity analysis in fruit trees due to their high levels of polymorphism suitable for DNA fingerprinting analysis, high degrees of transferability and reproducibility as well as the co-dominant mode of inheritance (20). However, there is no accurate report on generating the molecular identities of Prunus rootstocks. With regard to the phylogenetic studies of Prunus rootstocks, the relationships among 29 Prunus spp. rootstocks were evaluated resulting in 2 subgroups of Prunophora or Amygdalus using SSR markers (1). The genetic relationships among 44 clones of
Pratik et al. (2016) assessed confamiliar transferability of SSR markers from cotton (Gossypium hirsutum) and jute (Corchorus olitorius) to 22 species distributed in different taxonomic groups of Malvaceae. Of the 14 cotton SSR loci tested, 13 (92.86 %) amplified in G. arboreum and 71.43 % exhibited cross-genera transferability. Nine out of 11 jute SSRs (81.81 %) showed cross-transferability across genera. At tribe level, transferability of jute SSRs (41.04 %) was higher than that of cotton SSRs (33.74 %). Transferability of wheat SSR markers to rye was 17%, whereas 25% of rye markers were amplifiable in wheat. In triticale, 58% and 39% transferability was achieved for wheat and rye markers, respectively (Kuleung et al., 2004). In the process of identification of molecular markers linked to economically important traits, use of heterologous EST markers like markers derived from disease resistant analogues will be most useful. Therefore in this study, markers developed from disease resistant analogues from pulses and safflower was tried in cotton.
diversity of yacon was verified for the first time in the State of Espírito Santo to understand the relationships between the accessions cultivated in the State. Of the total fragments generated using ISSR, only 39.6% presented polymorphism. However, the association between the observed low polymorphic level and the genetic dissimilarity values and cophenetic correlation coefficient confirmed that the ISSR markers used in this study were efficient in grouping the accessions.
found in the Novo Segredo population, and 27 alleles were found in the Formoso population, ranging from two to five alleles per locus, with a mean of three alleles per locus. The expected heterozygosity in the Formoso population varied from 0.100 to 0.668, with an average of 0.421. In the Novo Segredo population, a variation between 0.100 and 0.710, with a mean of 0.418, was found. The observed heterozygosity values ranged from 0.100 to 0.800, with averages of 0.333 and 0.267 for Formoso and Novo Segredo, respectively. This set of markers will support further studies on the molecular characterization of the natural populations of E. precatoria and assist with the recommendation of sustainable management practices and strategies for the conservation
The polymorphism identified by the markers was used to construct a genetic distance matrix. Based on the Jaccard coefficient, two main clusters were identified in the UPGMA analysis (Figure 2). Most genotypes were allocated in a main cluster with several subclusters. The genotypes Pacovã, Prata-Anã, Enxerto, FHIA-18, PA94-01, Japira, Tropical YB42-47, Bucaneiro, and Grand Naine presented higher similarities, and thus, were allocated to the same subcluster. The tetraploid genotype FHIA-23 (Gros Michel) was more isolated from the other genotypes, despite being present in the main cluster. The second main cluster, which was composed of the genotypes ‘Garantida’ (tetraploid) and ‘Thap Maeo’ (triploid), both from the cluster Prata, were allocated with high similarity, and were more isolated from the other genotypes.
When assessing genetic diversity in local varieties of Algerian cowpea using ISSR markers, Ghalmi et al. (2010) found genetic distances ranging from approximately 0.025 to 0.325. Ali et al. (2015), when studying 252 genotypes of the species collected in the Sudan using codominant markers, which present a large amount of information, saw a variation in genetic distance of 0.031 to 0.303. Both results are very close to those found in our study, and this shows a possible relationship between the genotypes, which may share a common origin. This is because the varieties grown in Brazil are believed to have been introduced from Africa, which according to Tan et al. (2012) is the probable origin of the species.
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Total genomic DNA was isolated from young leaf material following the modified CTAB method described by Keim et al. (1988). To avoid co-isolation of phenolics and polysaccharides 2% polyvinyl pyrrolidone (PVP) was added in DNA extraction buffer. The quality and quantity were estimated by measuring O.D. at 260/280 nm and 260 nm, respectively, in Nano drop spectrophotometer. Intactness of genomic DNA was checked on 0.8 % agarose gel. The genomic DNA was amplified using random primers of OPE, OPF, OPG, OPH, OPI and OPJ (Operon Tech., California, USA) and 10 UBC ISSR primers. Twenty five decamers (Table 1) were selected after screening a set of 120 primers from OPE, OPF, OPG, OPH, OPI and OPJ to obtain RAPD profile. Three ISSR primers UBC-807, UBC-843 and UBC-873 were selected after screening of 15 UBC primers.
Appreciable intra cultivar variation has warranted clonal selections, which has emerged as an important tool in mango breeding. This important source of morphological variability manifested in altered fruit and quality attributes has yielded improved clones in India and abroad, few of which were exposed to SSR based analysis. Statistical parameters viz., Polymorphic Information Content (0.319 in MiIIHR12 to 0.819 for MiIIHR26) and Gene Diversity (0.399 in MiIIHR12 to 0.839 for MiIIHR26), defined the ability of the chosen SSR markers to discriminate the intra cultivar variability, besides highlighting the extent of diversity captured by the improved clones. Furthermore, genetic relationship among the clones derived by Wards minimum variance, placed Himsagar and Langra clones in Cluster I and II respectively, Himsagar recording high genetic heterogeneity within its cluster, intra cultivar variability being 0.16-0.916, thus showing suitability for breeding by clonal selections. Even the use of limited SSR marker loci (6), could reveal and document the genomic variations accounting for the variations in the Dashehari clones as well as placing land race ‘Suraiyya’, as an out-group. The sampled clones of elite variety Chausa did not show any variation at the studied marker loci, thus exposing limited heterogeneity in the clones and demanding more explorations for breeding superior types targeting regularity in bearing.
Despite its commercial potential, cajuí remains underexploited since, in the absence of a sustainable agricultural system, production relies almost entirely on extractivism. Moreover, little information regarding the biodiversity of cajuí is available. Considering that knowledge of the genetic variability within a species is a prerequisite for agricultural development, it is important to create and characterize active germplasm banks for the purpose of plant breeding, conservation and other research goals. In this context, inter-simple sequence repeat (ISSR) markers are among the most powerful molecular tools employed in the study of plant genetic variability, and the technique is both simple to use and cost efficient. Furthermore, ISSR-based methods allow the detection of polymorphism in microsatellite and intermicrosatellite loci without prior knowledge of the DNA sequence, and a large number of loci can be analyzed simultaneously even when working with different species in the same assay (Gupta et al. 1994; Reddy et al. 2002; Thimmappaiah et al.2009).
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For each sample, each fragment / band that was amplified using ISSR primers was treated as a unit rearrangement in genome. The primers which were given scorable and consistently reproducible amplicons were considered. The gel pictures were taken and documented to computer by using Alpha Imager gel documentation system and size of each amplicon was measured by using Alpha Imager Software with respect to standard molecular weight DNA ladder and molecular weight of each of the potential specific bands was calculated using the software program Alpha Imager.
There are numerous published reports on cotton genetic mapping using restriction fragment length polymorphisms (RFLPs) (Brubaker et al., 1999; Jiang et al., 2000; Paterson et al., 2003; Reinisch et al., 1994; 1999; Saha et al., 1998; Shappley et al., 1996, 1998; Ulloa and Meredith, 2000; Wright et al., 1998), but the RFLP technique is labor intensive, time consuming, and expensive. Scientists from different countries initiated the International Cot- ton Genome Initiative (ICGI) to coordinate future cotton genomics research (Brubaker et al., 2000). ICGI has stressed the need for more portable, pub- licly available, PCR-based framework markers (e.g. SSRs) to expedite genomic research in cotton. Given that the genome size of cotton is 2,200 Mb/1C (Ar- muganathan and Earle, 1991) with an approximate total genetic map length of 5,000 cM (Paterson and Smith, 1999; Reinisch et al., 1994), several hundred markers are needed to achieve a saturated genetic map of cotton.
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Amplification profiles generated from RAPD and ISSR were photographed under UV light, screened, and compiled into a binary data matrix. Only distinct, reproducible and well-resolved fragments (bands) were recorded numerically as (1) when present or (0) when absent. Fragments with the same mobility were considered as identical, irrespective of fragment intensity. Genetic distances were estimated using Jaccard’s genetic similarity index . A dendrogram was generated by cluster analysis using the unweighted pair group method of the arithmetic averages (UPGMA). Principal coordinated analysis (PCA) was also carried out to display multiple dimension of the distribution of the accessions in a scatter-pot by PAST software version 2.17 . The RAPD and ISSR matrices were subjected to the Mantel Test to verify the level of conformity between the data generated.
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fact that the mutation frequency of EST sequences is lower than that of genomic DNA sequences. These results demonstrate the potential value of EST-SSR mar- kers for the development of genetic maps, assessment of genetic diversity, and marker-assisted selection (MAS) breeding in J. curcas , all of which would benefit com- parative mapping and analysis of the comparative func- tions of genes among the economic species in the Euphorbiaceae family. Our finding that the majority of transferable EST-SSRs were trinucleotide repeats agreed with previous studies [21,30-32], and can be explained by the suppression of non-trimeric SSRs in coding regions due to the risk of frameshift mutations, which might occur with non-trimeric microsatellites [33-36].