Informative Simple Sequence Repeat Research Articles

Identification of Informative Simple Sequence Repeat (SSR) Markers forTesting of Hybridity in Maize

Identification of Informative Simple Sequence Repeat (SSR) Markers forTesting of Hybridity in Maize

Development of NBS-LRR gene-specific microsatellite markers for passion fruit Passiflora edulis Sims

The chromosome-level genome assembly of passion fruit (Passiflora edulis Sims) has facilitated computational analyses in developing novel informative SSR markers for genetics and breeding research. In this study, in-silico analysis was conducted to discover potential microsatellite loci and to design specific primers from the sequences of the characterised nucleotide-binding site - leucine-rich repeats (NBS-LRR) gene family of passion fruit, and a genotyping experiment was performed to validate the novel simple sequence repeat (SSR) markers.In total, 497 microsatellite loci were detected from 161 passion fruit NBS-LRR genes, of which 383 loci were qualified for primer generation. Forty-six SSR markers were selected for polymorphism verification, targeting 35 NBS-LRR genes that showed potential gene expression in response to biotic and abiotic stresses. From that, 25 SSR markers showed consistent polymorphic alleles among different passion fruit genotypes via PCR-PAGE analysis. These novel NBS-LRR-specific SSR markers might provide valuable tools for fortifying research on germplasm evaluation, genetic mapping, and molecular breeding for disease-resistant passion fruit varieties.

Genome-Wide Survey and Analysis of Microsatellites in Waterlily, and Potential for Polymorphic Marker Development.

Waterlily (Nymphaeaceae), a diploid dicotyledon, is an ornamental aquatic plant. In 2020, the complete draft genome for the blue-petal waterlily (Nymphaea colorata) was made available in GenBank. To date, the genome-wide mining of microsatellites or simple sequence repeats (SSRs) in waterlily is still absent. In the present study, we investigated the characteristics of genome-wide microsatellites for N. colorata and developed polymorphic SSR markers across tropical and hardy waterlilies. A total of 238,816 SSRs were identified in 14 N. colorata chromosomes with an average density of 662.60 SSRs per Mb, and the largest number of SSRs were present on chromosome 1 (n = 30,426, 705.94 SSRs per Mb). The dinucleotide was the most common type, and AT-rich repeats prevail in the N. colorata genome. The SSR occurrence frequencies decreased as the number of motif repeats increased. Among 2442 protein-coding region SSRs, trinucleotides, accounting for 63.84%, were the most abundant. Gene ontology terms for signal transduction (e.g., GO: 0045859 and GO: 0019887) and the lipoic acid metabolism (ko00785,) were overrepresented in GO and KEGG enrichment analysis, respectively. In addition, 107,152 primer pairs were identified, and 13 novel polymorphism SSR markers were employed to distinguish among nine waterlily cultivars, of which Ny-5.2 and Ny-10.1 were the most informative SSR loci. This study contributes the first detailed characterization of SSRs in N. colorata genomes and delivers 13 novel polymorphism markers, which are useful for the molecular breeding strategies, genetic diversity and population structure analysis of waterlily.

Simple Sequence Repeat Markers Reveal Genetic Diversity and Population Structure of Bolivian Wild and Cultivated Tomatoes (Solanum lycopersicum L.).

The western part of South America is a centre of diversity for tomatoes, but genetic diversity studies are lacking for parts of that region, including Bolivia. We used 11 simple sequence repeat (SSR) markers (including seven novel markers) to evaluate genetic diversity and population structure of 28 accessions (four modern cultivars, four advanced lines, nine landraces, 11 wild populations), and to compare their genetic variation against phenotypic traits, geographical origin and altitude. In total, 33 alleles were detected across all loci, with 2–5 alleles per locus. The top three informative SSRs were SLM6-11, LE20592 and TomSatX11-1, with polymorphism information content (PIC) of 0.65, 0.55 and 0.49, respectively. The genetic diversity of Bolivian tomatoes was low, as shown by mean expected heterozygosity (He) of 0.07. Analysis of molecular variance (AMOVA) revealed that 77.3% of the total variation was due to variation between accessions. Significant genetic differentiation was found for geographical origin, cultivation status, fruit shape, fruit size and growth type, each explaining 16–23% of the total variation. Unweighted Pair Group Method with Arithmetic Mean (UPGMA) tree and principal coordinate analysis (PCoA) scatter plot both revealed differentiation between accessions with determinate flowers and accessions with indeterminate flowers, regardless of cultivation status. The genetic profiles of the accessions suggest that the Bolivian tomato gene pool comprises both strictly self-pollinating and open-pollinating genotypes.

Genetic Diversity and DNA Fingerprinting in Broccoli Carrying Multiple Clubroot Resistance Genes Based on SSR Markers

To identify cultivars quickly and accurately, DNA fingerprinting of 10 broccoli varieties was performed by using simple sequence repeat (SSR) marker technology. Highly informative and polymorphic SSR markers were screened using broccoli and rapeseed. Out of the 93 SSR marker pairs, 21 pairs were selected and found to have good polymorphism. Each marker pair generated 1 to 10 polymorphic bands with an average of 4.29. The average polymorphism information content (PIC) was 0.41 with a range from 0.16 to 0.95. Six selected marker pairs established the fingerprinting of the 10 accessions and their unique fingerprints. Cluster analysis of 10 accessions showed that the genetic similarity coefficient was between 0.57 and 0.91. They can be divided into 3 groups at the genetic similarity coefficient (GSC) of 0.73. The above results indicated that DNA fingerprinting could provide a scientific basis for the identification of broccoli polymerized multiple clubroot resistance genes. Research shows that SSR marker-based DNA fingerprinting further ensures plant seed purity.

Genome-Wide Analysis of Simple Sequence Repeats in Cabbage (Brassica oleracea L.).

Cabbage (Brassica oleracea L. var. capitata) accounts for a critical vegetable crop belonging to Brassicaceae family, and it has been extensively planted worldwide. Simple sequence repeats (SSRs), the markers with high polymorphism and co-dominance degrees, offer a crucial genetic research resource. The current work identified totally 64,546 perfect and 93,724 imperfect SSR motifs in the genome of the cabbage ‘TO1000.’ Then, we divided SSRs based on the respective overall length and repeat number into different linkage groups. Later, we characterized cabbage genomes from the perspectives of motif length, motif-type classified and SSR level, and compared them across cruciferous genomes. Furthermore, a large set of 64,546 primer pairs were successfully identified, which generated altogether 1,113 SSR primers, including 916 (82.3%) exhibiting repeated and stable amplification. In addition, there were 32 informative SSR markers screened, which might decide 32 cabbage genotypes for their genetic diversity, with level of polymorphism information of 0.14–0.88. Cultivars were efficiently identified by the new strategy designating manual diagram for identifying cultivars. Lastly, 32 cabbage accessions were clearly separately by five Bol-SSR markers. Besides, we verified whether such SSRs were available and transferable in 10 Brassicaceae relatives. Based on the above findings, those genomic SSR markers identified in the present work may facilitate cabbage research, which lay a certain foundation for further gene tagging and genetic linkage analyses, like marker-assisted selection, genetic mapping, as well as comparative genomic analysis.

Chromosome-specific polymorphic SSR markers in tropical eucalypt species using low coverage whole genome sequences: systematic characterization and validation.

Eucalyptus is one of the major plantation species with wide variety of industrial uses. Polymorphic and informative simple sequence repeats (SSRs) have broad range of applications in genetic analysis. In this study, two individuals of Eucalyptus tereticornis (ET217 and ET86), one individual each from E. camaldulensis (EC17) and E. grandis (EG9) were subjected to whole genome resequencing. Low coverage (10×) genome sequencing was used to find polymorphic SSRs between the individuals. Average number of SSR loci identified was 95,513 and the density of SSRs per Mb was from 157.39 in EG9 to 155.08 in EC17. Among all the SSRs detected, the most abundant repeat motifs were di-nucleotide (59.6%–62.5%), followed by tri- (23.7%–27.2%), tetra- (5.2%–5.6%), penta- (5.0%–5.3%), and hexa-nucleotide (2.7%–2.9%). The predominant SSR motif units were AG/CT and AAG/TTC. Computational genome analysis predicted the SSR length variations between the individuals and identified the gene functions of SSR containing sequences. Selected subset of polymorphic markers was validated in a full-sib family of eucalypts. Additionally, genome-wide characterization of single nucleotide polymorphisms, InDels and transcriptional regulators were carried out. These variations will find their utility in genome-wide association studies as well as understanding of molecular mechanisms involved in key economic traits. The genomic resources generated in this study would provide an impetus to integrate genomics in marker-trait associations and breeding of tropical eucalypts.

Assessment of Genetic Purity in Rice Using Polymorphic SSR Markers and Its Economic Analysis with Grow-Out-Test

Genetic purity is conventionally performed through grow-out-test (GOT) with morphological characters. Simple sequence repeat (SSR) markers are independent of G X E interaction. Herewith, 16 high yielding varieties of rice were analysed using 55 SSR markers for DNA fingerprinting and identification of genetic impurities: 14 were found to be polymorphic and amplified 48 alleles with an average of 3.43 alleles per each primer pair. The number of alleles amplified ranged from 2 to 6 and the size of the PCR products amplified from these 14 primer pairs ranged from 80 to 450 bp with polymorphic information content (PIC) from 0.14 (RM 346) to 0.99 (RM 5900). PICs at 0.5 or higher are highly informative SSR markers for genetic studies and the study reported (7/14) markers to be highly informative based on PIC values. Besides, the values of effective multiplex ratio, marker index and resolving power for the selected polymorphic primer pairs disclosed that the SSR markers used in the study are highly informative and could be potentially used for distinctness, uniformity and stability (DUS) testing, genetic purity analysis and DNA fingerprinting of rice varieties. Economic analysis of grow-out-test and SSR marker based genetic purity showed that the GOT process incurs Rs. 5.8 (INR) per seed (USD 0.07), while DNA-based marker analysis incurs Rs. 53.75 (INR) or $ 0.71 (USD).

SSR-based evaluation of genetic diversity in populations of Agriophyllum squarrosum L. and Agriophyllum minus Fisch. & Mey. collected in South-East Kazakhstan.

The development of informative polymorphic DNA markers for poorly studied genera is an important step in population analyses of living organisms, including those that play very important ecological roles in harsh environments, such as desert and semi-desert area. Examples of those poorly studied desert species are Agriophyllum squarrosum L. and Agriophyllum minus Fisch. & Mey. However, a recent RNA-sequencing project in A. squarrosum has proposed a large set of hypothetical SSR (simple sequence repeat) markers. In this work, 11 novel polymorphic SSRs were found due to the screening of 24 randomly selected SSRs for three populations of A. squarrosum and one population of A. minus. The analysis of 11 SSRs revealed 16 polymorphic loci in two Agriophyllum species, 8 polymorphic loci within three populations of A. squarrosum, and 6 polymorphic loci in the population of A. minus. Statistical analyses showed high interspecific, but relatively low intraspecific genetic diversity. The phylogenetic clusterization and population structure analysis have demonstrated a clear segregation of A. minus from A. squarrosum, as well as the separation of population 1 from populations 2 and 3 of A. squarrosum. Thus, we identified the set of novel and informative SSR markers suitable for the study of genetic diversity in Agriophyllum.

Genotyping by RAD Sequencing Analysis Assessed the Genetic Distinctiveness of Experimental Lines and Narrowed Down the Genomic Region Responsible for Leaf Shape in Endive (Cichorium endivia L.).

The characterization of genetic diversity in elite breeding stocks is crucial for the registration and protection of new varieties. Moreover, experimental population structure analysis and information about the genetic distinctiveness of commercial materials are essential for crop breeding programs. The purpose of our research was to assess the genetic relationships of 32 endive (Cichorium endivia L.) breeding lines, 18 from var. latifolium (escarole) and 14 from var. crispum (curly), using heterologous Cichorium intybus-derived simple sequence repeats (SSR) markers and single-nucleotide polymorphisms (SNP) markers. We found that 14 out of 29 SSR markers were successfully amplified, but only 8 of them were related to polymorphic loci. To overcome the limitation of the low number of informative SSR marker loci, an alternative SNP-based approach was employed. The 4621 SNPs produced by a restriction site-associated DNA marker sequencing approach were able to fully discriminate the 32 endive accessions; most importantly, as many as 50 marker loci were found to distinguish the curly group from the escarole group. Interestingly, 24 of the marker loci mapped within a peripheral segment of chromosome 8 of lettuce (Lactuca sativa L.), spanning a chromosomal region of 49.6 Mb. Following Sanger sequencing-based validation, three genes were determined to carry nonsynonymous SNPs, and one of them matched a putative ortholog of AtELP1, subunit 1 of the Elongator complex. Considering that several previously characterized Elongator complex subunit mutants exhibited elongated and/or curly leaf phenotypes, this gene should be taken into consideration for a better understanding of the underlying mechanism controlling leaf shape in endive.

Genome-wide characterization and development of simple sequence repeat markers for genetic studies in pomegranate (Punica granatum L.)

Genome-wide characterization and development of first set of polymorphic Class I SSRs markers in pomegranate (Punica granatum L.) through in silico analysis using the draft genome sequence of pomegranate cv. Dabenzi (296 Mb) as reported by Qin et al. (2017). The availability of the draft genome sequence of pomegranate cv. Dabenzi presents unprecedented opportunities for the development of largescale genomic resources, such as DNA markers for genotyping applications. In this study, we identify a new set of highly polymorphic simple sequence repeat (SSR) markers by targeting the SSR motif lengths of ≥ 24 bp. A total of 1,73,633 SSRs were identified in the 296-Mb pomegranate genome assembly, reflecting an average density of 527.97 SSRs/Mb. Of these, 43,853 SSRs belong to Class I category (> 20 bp). Concerning the abundance of repeat types in the current dataset, dinucleotide (NN) repeats (31.19%) were the dominant class among all SSRs identified in the genome, followed by tetranucleotide (NNNN: 20.5%) and trinucleotide repeats (NNN: 16.8%). The top two SSR motifs in NN category were AT/AT (64.90%) and AG/CT (28.51%), whereas AAT/ATT (34.66%) and AAG/CTT (28.91%) were the most abundant among NNN repeats. Primer pairs were designed for a total of 2856 Class I SSRs and 110 primers were then assayed initially on eight pomegranate genotypes for polymorphism survey. Polymorphic fragments were obtained for 82 SSRs (77.36%), of which a subset of 13 informative SSRs was further employed to investigate genetic diversity among 46 pomegranate genotypes. Approaches, such as population structure, cluster and PCA elucidated genetic relationships among 46 diverse pomegranate genotypes. In summary, here we developed the first set of genome-wide SSRs in pomegranate that will serve as a powerful genomic tool for future genetic studies. These SSRs have widespread applications in QTL mapping and marker-assisted selection for breeding.

Genome-wide characterization and analysis of microsatellite sequences in camelid species

Microsatellites or simple sequence repeats (SSRs) are among the genetic markers most widely utilized in research. This includes applications in numerous fields such as genetic conservation, paternity testing, and molecular breeding. Though ordered draft genome assemblies of camels have been announced, including for the Arabian camel, systemic analysis of camel SSRs is still limited. The identification and development of informative and robust molecular SSR markers are essential for marker assisted breeding programs and paternity testing. Here we searched and compared perfect SSRs with 1–6 bp nucleotide motifs to characterize microsatellites for draft genome sequences of the Camelidae. We analyzed and compared the occurrence, relative abundance, relative density, and guanine-cytosine (GC) content in four taxonomically different camelid species: Camelus dromedarius, C. bactrianus, C. ferus, and Vicugna pacos. A total of 546762, 544494, 547974, and 437815 SSRs were mined, respectively. Mononucleotide SSRs were the most frequent in the four genomes, followed in descending order by di-, tetra-, tri-, penta-, and hexanucleotide SSRs. GC content was highest in dinucleotide SSRs and lowest in mononucleotide SSRs. Our results provide further evidence that SSRs are more abundant in noncoding regions than in coding regions. Similar distributions of microsatellites were found in all four species, which indicates that the pattern of microsatellites is conserved in family Camelidae.

Exploring genetic diversity of tomato (Solanum lycopersicum L.) germplasm of genebank collection employing SSR and SCAR markers

Α wide selection of tomato (Solanum lycopersicum L.) genotypes with diverse origin and breeding history (14 modern varieties, 71 landraces and 22 commercial hybrids) has been initially genotyped with a selection of highly informative simple sequence repeat (SSR) markers and two SCAR markers originally developed for resistance against two main fungal tomato diseases. Our data revealed a high level of genetic diversity across the selection, with an average number of alleles per locus (NA) equal to 9.6, and the average polymorphism information content (PIC) equal to 0.74. Further, the selected SSRs have been verified as highly polymorphic and able to discriminate different patterns within our collection, amplifying a total of 56 alleles. Cluster analysis indicated that the collection could be grouped into three clades, with most of landraces and modern varieties being clearly distinguished from hybrids, but also with each other. Breeding involve the selection of specific phenotypes, limiting the genetic variation of the population. Herein, a notable genetic loss due to breeding was detected in the modern tomato gene pool.

Transcriptome profiling reveals genetic basis of disease resistance against Corynespora cassiicola in rubber tree (Hevea brasiliensis)

Corynespora leaf disease caused by Corynespora cassiicola (Berk. & Curt.) is one of the major diseases responsible for significant yield loss in rubber trees (Hevea brasiliensis). Next-generation sequencing based transcriptomic study of two rubber clones: RRII 105 (susceptible) and GT 1 (moderately resistant) were performed to understand the molecular basis of host tolerance to fungal diseases. Genes encoding disease resistance proteins, leucine-rich repeat proteins and genes involved in carbohydrate metabolic processes were significantly up-regulated in GT 1 upon infection, but were either completely suppressed or down-regulated in RRII 105. Transcription factor activity was a major molecular function triggered in both inoculated clones. Gene Ontology analysis revealed that majority of the transcripts was enriched for defense response, response to stimulus and stress. Higher expression of 118 transcripts with complete ORFs was identified in inoculated GT 1, indicating their possible role in disease resistance. In addition, both unique and common simple sequence repeats (SSRs) were identified. In silico analysis revealed 191 informative SSRs differentiating the two clones. Variant calling in control and disease GT 1 transcriptomes with reference to RRII 105 revealed over one lakh putative base substitutions. Microarray was used to validate the results obtained on transcriptional responses. Biotic stress overview from MapMan analysis revealed stronger activation of defense-related genes, receptor-like kinases and transcription factors. This study presents the first comprehensive transcriptome of resistant and susceptible rubber clones in response to C. cassiicola. The newly identified differentially regulated genes and sequence variation provide critical knowledge for understanding the genetic basis of disease resistance and marker development.

Genetic identity based on simple sequence repeat (SSR) markers for Quinoa (Chenopodium quinoa Willd.)

Molecular markers based on simple sequence repeats (SSRs) constitute a highly effective instrument in the identification of quinoa genotypes ( Chenopodium quinoa ), and they are very useful in the management and conservation of germplasm banks. The present study was carried out in the Molecular Biology Laboratories of the Megalaboratory of the National University of the Altiplano and the National Agrarian University la Molina. With the objective of determining a minimum group of highly informative initiators for the cultivation of quinoa to study and identify the obtained alleles and implementing and incorporating this technology into research of genetic identity, the molecular analysis of nine loci located by microsatellite markers (SSRs) was performed on a sample of 26 varieties of quinoa: Ayrampo, Amarilla de Marangani, Choclito, Chullpi, Huariponcho, Pandela, Sajama, Witulla, Kcancolla, Negra Collana, Salcedo, Pasankalla, Blanca de Juli, and Chenopodium petiolare from the CIP-Camacani and Blanca de Juli, Kcancolla, Negra Collana, Pasankalla, Altiplano, Illpa INIA, Salcedo, Ayara Blanca de Juli, Ayara Blanca de Arequipa, Ayara Cancolla, Ayara Pasankalla, and Ayara Salcedo from the INIA. Genomic DNA was extracted by PCR (GeneJET Plant Genomic DNA Purification), and 20 microsatellite regions were amplified. The amplified fragments were loaded on polyacrylamide gels to determine their size in base pairs, of which only nine showed products with reading quality (QCA012, QCA015, QCA021, QCA029, QCA034, QCA040, QCA053, QCA055 and QCA067). The fragments were evaluated for their allelic richness, heterozygosity (H) and polymorphic information content (PIC). The data were processed with Gen Alex software ver. 3.5 A total of 67 alleles were detected among the different regions analyzed, with an average of 7 alleles for loci ranging from 142 to 240 bp and an effective number of alleles (ENA) of 5.36. The mean heterozygosity was 0.80, and the mean Polymorphic Information Content (PIC) was 0.81. The markers were highly polymorphic; therefore, the most informative SSR primers in the present study would be made up of three markers with PIC, QCA053 (0.87), QCA015 (0.86) and QCA034 (0.86), for determining the genetic identity of Chenopodium quinoa Willd. These markers can be easily interpreted and are useful for the molecular characterization of quinoa varieties. Analysis of the hierarchical clusters using UPGMA (Unweighted Pair Group Method) clustering identified 5 groups at a similarity coefficient of 0.77 among the quinoa varieties studied in this research.

Identification of high-efficiency SSR markers for assessing watermelon genetic purity

Genomic simple sequence repeat (SSR) markers were used to fingerprint and determine genetic similarity (GS) of the watermelon breeding lines, as well as the purity of their hybrid derivatives. Cluster analysis and Jaccard's distance coefficients using the unweighted pair group method with arithmetic mean (UPGMA) have classified these lines into three major groups. Notwithstanding,the genetic background of these lines is narrow as revealed by the restricted GS coefficients. Fifty-five sets of SSR markers were employed in this study. Fourteen of these markers were polymorphic between the breeding lines and were used for assessing hybrid purity. Cross-checking assay validated nine SSR markers as informative SSR markers for purity detection of these hybrids. To confirm the accuracy and efficiency of these markers, their derived PCR products were further sequenced, and ClSSR09643, ClSSR18153 and ClSSR01623 were selected as high-efficiency SSR markers. Interestingly, SSR markers ClSSR09643 and ClSSR18153 were broadly applied for purity detection of more than two different hybrids, while SSR marker ClSSR01623 behaved as a specific marker forpurity detection in this study. Genetic purity of six commercial watermelon hybrids was definitely evaluated using these SSR markers. Genetic purity of all tested hybrids exceeded 96% while the field purity was above 98%. Genetic purity test was an emergency for identifying off-types and selfed female in a lot of hybrid seeds. Here, we elucidated the potential of nine SSR markers including threewith higher breeding selection efficiency. We recommended them to seed company for purity improvement of watermelon commercial hybrid varieties.

Development of New SSR (Simple Sequence Repeat) Markers for Lentils (Lens culinaris Medik.) from Genomic Library Enriched with AG and AC Microsatellites.

Simple sequence repeat (SSR) markers are the major molecular tools for genetic and genomic researches that have been extensively developed and used in major crops. However, few are available for lentils (Lens culinaris M.), economically an important cool-season legume. The lack of informative simple sequence repeat (SSR) markers in lentil has been a major limitation for lentil molecular breeding studies. Therefore, in order to develop SSR markers for lentil, an enriched genomic libraries for AC and AG repeats were constructed from the Lens culinaris cv Kafkas. A total of 350 clones were inquired for the detection of SSRs. Of 350 clones, 68 had SSR motifs. In polymorphism analysis using 53 newly developed SSRs, a total of 144 alleles across 24 lentil cultivars were detected with an average of 4.64 per locus. The average heterozygosity was 0.588 and polymorphism information contents ranged from 0.194 to 0.895 with an average value of 0.520. These newly developed SSRs will constitute useful tools for molecular breeding, mapping, and assessments of genetic diversity and population structure of lentils.

Development of potential dbEST-derived microsatellite markers for genetic evaluation of sugarcane and related cereal grasses

Sugarcane expressed sequence tags (ESTs) database (db) offers a great opportunity for developing functional molecular markers associated with the desirable agronomic traits. The aim of the present study was to develop informative simple sequence repeat (SSR) markers in sugarcane using sugarcane expressed sequence tags (ESTs), characterization with respect to their abundance, distribution, variability, and to test their competence in genetic diversity. A total of 361 EST-SSR markers were developed and screened for their ability to detect polymorphism across the wide range of sugarcane germplasm. Among the developed SSRs, TNRs containing motifs were predominant with a frequency of 55%, which followed by TtNRs (22%), PNRs (11%), DNRs (7%), and HNRs (5%). Gene Ontology and homology search was conducted to establish the relevancy of SSRs in functional genomics. Resulting, ∼39% SSRs were found to be functional and confirmed homology with different proteins and transcription factors involved in gene expression. A set of 96 polymorphic primer pairs was used to evaluate genetic diversity among Saccharum species, related genera and cross amplification in other cereal grasses. A total of 710 alleles were generated which ranged from 2 to 15 with an average of 7.40 alleles per locus and showed 100% polymorphism. Polymorphism information content (PIC) value was ranged from 0.35 to 0.91 with an average of 0.71. Majority of the markers developed in current study were related to sucrose metabolism, stress physiology, plant immune responses, signal transduction, biosynthesis of secondary metabolites, ATP generation, growth and other biochemical process. The high degree of polymorphism, cross-species amplification and genetic diversity assessed with these SSR markers suggested their utility for genome mapping, QTL analysis, comparative genomics, gene-based association studies, and marker-assisted selection in sugarcane. Moreover, these informative EST-SSRs described here would enrich existing repertoire of genetic resources for sugarcane and also other related taxa constitute the important secondary genepool for sugarcane genetic improvement.

Molecular Verification of Putative Zygotic Seedlings in Different Intra-Specific Crosses in Mandarins (Citrus reticulata) by SSR Markers

The molecular verification of different intra-specific mandarin hybrids was explored during 2014–2015. Simple sequence repeat (SSR) markers were employed to identify putative zygotic seedlings from hybrid progeny produced from different crosses in mandarin. The most informative SSR marker in the F1 hybrids of ‘Daisy × Kinnow’ cross was CCSM170 which identified 73% of zygotic seedlings followed by CMS4, AG14, CiBE6006 and CiBE3397 which identified 38.5%, 31.5%, 27% and 24% of zygotic seedlings, respectively. Most efficient SSR marker in ‘Kinnow × Daisy’ cross was CiBE6006 which identified 22.5% zygotic seedlings followed by CCSM170, CiBE3397 and CMS4 which identified 17.5%, 12.5% and 5.5% of zygotic seedlings, respectively. In ‘W. Murcott × Kinnow’ hybridization, CMS31 marker identified 28.5% zygotic seedlings in F1 progeny followed by CiBE3298 which identified 14.5% zygotic seedlings. The SSR marker CiBE3397 identified 19.5% zygotic seedlings in ‘Kinnow × W. Murcott’ cross. The results show that the SSR markers can differentiate the zygotic seedlings of F1 progeny in different intra-specific crosses in mandarins.

Highly informative SSR genotyping reveals large genetic diversity and limited differentiation in European larch (Larixdecidua) populations from Romania

European larch ( Larix decidua ) is naturally distributed at high elevations in Central Europe. Romanian populations of L. decidua are scattered in different areas of the country. In this study, we used 12 highly informative genomic simple sequence repeat (SSR) markers for genotyping seven populations from different areas of Romania. The SSR markers were highly variable, with up to 11 alleles per SSR locus and an average polymorphic information content of 0.713. High values of observed ( H o = 0.542) and expected ( H e = 0.738) heterozygosities were observed. Cluster analysis of populations did not group them according to geographical distance, but some clusters contained populations from a similar altitudinal range. The partition of genetic variation revealed that two-thirds of the genetic variation was found within individuals (due to high H o levels), while only one-sixth of the total genetic variation corresponded to variation among populations. A population structure analysis identified four genetic clusters, and in most cases individuals from a single population were assigned to several genetic clusters. Multivariate principal coordinates analysis confirmed the population structure analysis. SSR markers are a powerful tool for evaluating diversity, relationships, and the genetic structure of Romanian L. decidua populations, which have high levels of diversity and low genetic structuration.