SSR Primer Pairs Research Articles

Construction of DNA Fingerprinting and Analysis of Genetic Diversity for Xinluzao Cotton Varieties

Fifty-one Xinluzao conventional cotton varieties authorized before 2013 were detected with 75 pairs of primers with high polymorphism, good repeatability, and even distribution on 26 chromosomes(2–3 pairs from each chromosome) selected from 5000 pairs of SSR primers. A total of 226 polymorphic loci were obtained, and each marker detected 2–12 polymorphic loci with an average of 3.01; the polymorphism information content ranged from 0.0799 to 0.8752, with an average of 0.6624. The results showed that 21 varieties were differentiated by characteristic primers, and the rest 30 varieties could be identified by primer conbinations. In the end, 40 specific primers and combinated primers could completely differentiate the 51 Xinluzao cotton varieties from each other. DNA fingerprinting of the 51 Xinluzao conventional varieties were constructed with the 40 pairs of SSR markers. NTSYS-pc V2.10 software was used to analyze genetic diversity of the 51 conventional varieties, the results indicated that genetic similarity coefficient for the varieties ranged from 0.4269 to 0.9873, with an average of 0.7071, showing the narrow genetic diversity in Xinluzao cotton varieties. The 51 Xinluzao cotton varieties were divided into four types with the genetic similarity coefficient matrix and cluster analysis, which were strongly consistent with their pedigrees.

Scope of novel and rare bulbiferous coconut palms (Cocos nucifera L.)

A naturally occurring, rare bulbiferous coconut palm was identified at the Central Plantation Crops Research Institute, Regional Station, Vittal in Karnataka State, India, among West Coast Tall population. The palm produces only bulbil shoots in leaf axils in place of normal inflorescence. The identified palm happened to be twins in which one member was more vigorous than the other, but both of them produced only bulbil shoots instead of floral parts. Morphological and molecular studies on these palms revealed the main palm and their bulbil progenies are genetically uniform. The genetic uniformity of the twin mother palms and their bulbil progenies was confirmed through microsatellite analysis using 10 polymorphic SSR primer pairs specific to coconut and has been well demonstrated to differentiate coconut cultivars. The primary and secondary bulbil shoots were found to be capable of growing into independent plants making it possible to use them as propagules to develop a homogeneous clonal population hitherto unavailable in coconut. The bulbils showed axillary growth in 6–12th leaf axil which further again develop as secondary bulbils indicating the complete vegetative state of the palm. Comparison of shoot apices of a normal seedling with bulbil shoot revealed variation in cell growth pattern. Conservation of bulbiferous palms as a unique genetic resource needs to be taken up to utilize these rare sources for future breeding programmes, provided their seed-fertility can be restored.

Characterization of masson pine (Pinus massoniana Lamb.) microsatellite DNA by 454 genome shotgun sequencing

Microsatellites (simple sequence repeats, SSRs) are important genetic markers in tree breeding and conservation. Here we utilized high-throughput 454 sequencing technology to mine microsatellites from masson pine (MP) genomic DNA. First, we analyzed the characteristics of SSRs in all nonredundant MP reads (genome survey sequences, GSSs) and compared them with loblolly pine (LP) GSSs and BACs (bacterial artificial chromosome clone sequences), and three other nonconiferous species GSSs. Second, a set of MP GSS–SSR primer pairs were designed. There were extremely low overall GSS–SSR densities (28 SSR/Mb) in MP when compared with LP (48 SSR/Mb) and the other species. AT, AAT, AAAT, and AAAAAT were the richest motifs in di-, tri-, tetra-, and hexanucleotides, respectively. Two hundred forty GSS–SSR primer pairs were designed in total, and 20 novel polymorphic markers were identified using three populations (two natural and one clonal seed orchard) as evaluating samples. These markers should be useful for future MP population genetics studies.

Optimization of PCR Conditions to Amplify SSR Markers in Sweet Cherry Cultivars

In all Romanian national fruit collections, all accessions holding a phenotypic, biochemical and genetic evaluation, but less a molecular level assessment of their….. For introduction of this material (accessions) into the breeding programs is necessary and important to know the level of characterization. Considering this fact, in the present study was optimization of the PCR protocol, specially annealing temperature of fifteen SSR primer pairs to characterize and study the genetic similarity and to determine the polymorphism between 36 sweet cherry accessions from I.C.D.P. Iasi, Romania. In the optimization process of annealing temperature for primers, was started with 5oC less them the temperature recommendated by the manufacturer, gradually ascending with 1-2oC/PCR cycle, up to 10-11oC, above the recommended value. For every primers pairs it was 6-7 steps for optimization of annealing temperature, obtaining satisfactory results to the following primers pairs: UCDCH17, UDP96-001 and PceGA25 at 65oC, UCDCH21: 64oC, UCDCH31: 66oC,:UPD97-402: 60oC, PMS3: 68oC. The primer pairs UDP96-008 obtained at 65oC, monomorphic bands, so it was excluded from the analysis. The other reagents used in PCR and temperature regimes (denaturation and extension temperature) were kept constant. From these results we conclude the transferability and applicability of SSR markers for genotyping and phylogenetic studies in the genus Prunus.

Desenho e validação de iniciadores microssatélites SSR para mamoneira

O objetivo deste trabalho foi desenhar, validar e otimizar pares de iniciadores microssatélites SSR para mamoneira. O desenho dos pares de iniciadores foi feito por meio do aplicativo Websat, a partir de sequências depositadas no GenBank do National Center for Biotechnology Information (GenBank/NCBI), e a sua qualidade foi aferida com uso do aplicativo web NetPrimer. Foram utilizadas diferentes concentrações de DNA, cloreto de magnésio, pares de iniciadores, dNTPs e temperatura de anelamento para otimização das condições de PCR. Um total de 30 pares de iniciadores SSR foi desenhado, sintetizado e otimizado. O gel de agarose foi utilizado para detecção dos produtos amplificados, e o gel desnaturante de poliacrilamida, na otimização das condições de PCR e na identificação de polimorfismo. Os pares de iniciadores apresentaram percentagem média de guanina/citosina (GC) igual a 47,29% e produtos amplificados com tamanhos entre 128 e 381 pb. Vinte e nove pares de iniciadores SSR (96,7%) foram validados, dos quais nove foram polimórficos (23,3%). As concentrações otimizadas para amplificação são: DNA, 25 ng; cloreto de magnésio, 1,2 mmol L-1; iniciadores Forward e Reverse, 0,4 mmol L-1; dNTPs, 0,1 mmol L-1; e temperatura de anelamento, 62 a 64ºC. As ferramentas de bioinformática Websat e Net Primer podem ser utilizadas para desenvolver iniciadores microssatélites de qualidade, para a mamoneira, a partir de sequências depositadas no GenBank/NCBI.

Genetic diversity in Swedish and Finnish heirloom apple cultivars revealed with SSR markers

A set of 85 heirloom apple cultivars aimed for long-term preservation in two germplasm collections in Sweden and Finland was evaluated with 8 SSR primer pairs to evaluate genetic diversity and genetic relatedness. An additional set of 16 European cultivars was included for comparison. The eight SSR primer pairs amplified 9 loci and 105 alleles. Genetic analyses performed by MDS indicated some differentiation between Swedish and Finnish cultivars, with European cultivars intermixed with the Swedish. The existence of three groups was, however, indicated by a Bayesian model-based clustering. One of the groups was clearly dominated by Swedish cultivars and another by Finnish. The third group included almost equal proportions of representatives from all three areas. The obtained results confirmed the genetic distinctness of Finnish apple cultivars, which can be explained by climate adaptation and admixture with a Russian gene pool.

Development of genomic simple sequence repeat markers from an enriched genomic library of grass pea (Lathyrus sativus L.)

AbstractSimple sequence repeat (SSR) or microsatellite markers are a valuable tool for several purposes such as evaluation of genetic diversity, fingerprinting, marker‐assisted selection and breeding. In this study, a SSR genomic enriched library was developed in Lathyrus sativus (grass pea) by affinity capture of restriction fragments to biotinylated microsatellite oligonucleotides. About 400 randomly selected clones were sequenced, and SSRs were present in approximately 30% of them. Clones contained 75%, 9% and 16% of simple, interrupted and compound SSRs, respectively. Of the 10 SSRs tested, 7 primer pairs produced clearly distinguishable DNA banding patterns. Successively, SSR primer pairs were successfully tested to reveal polymorphism in a set of four different grass pea germplasm accessions. The transferability of SSR markers was high among three related species of Lathyrus, namely Lathyrus cicera, Lathyrus ochrus and Lathyrus tingitanus, and the legume crop, Pisum sativum. These results indicate that the novel SSR markers are informative and will be useful and convenient for genetic analysis in grass pea and related species.

Genetic Diversity and Population Structure of Germplasm Resources in Sesame (<I>Sesamum indicum</I> L.) by SSR Markers

A total of 545 sesame accessions, including domestic and foreign germplasm resources, were analyzed systematically with 42 SSR primer pairs. The results showed that 106 allele bands were detected among the sesame accessions, and the Shannon’s information index (I) was 0.4092. Analysis results of UPGMA clustering, two-dimension principal components and population structure were basically consistent; 545 varieties were divided into three groups in UPGMA dendrogram or three subgroups in population structure. There were the lower genetic differentiation coefficient and the closer sibship in worldwide sesame. The similarity coefficient of seven ecological groups in China ranged from 0.9811 to 0.5462, and there was no close genetic relationship between varieties from northern regions and other regions in China. The similarity coefficients for foreign varieties were from 0.9726 to 0.7442, and the genetic relationships of varieties from Africa zone was close to those from Japan zone, but not from China. It showed that the genetic basis of varieties from China was relatively narrow, and the genetic diversities of varieties were not completely related to geographic distribution, but the genetic diversities of varieties from abroad presented abundance. Therefore, the introduction of foreign resources should be reinforced, and the domestic resources from different regions should be utilized in the future sesame breeding.

Identification of true hybrid progenies in cassava using simple sequence repeat (SSR) markers

For the maping of Cassava mosaic disease (CMD) resistance gene, CO2 and MNga-1 parents were used for hybridization to develop F1 hybrid seeds and 153 progenies were planted in the field for genotyping. Out of the 75 SSR primers used for parental polymorphism, 57 produced polymorphic bands between parents and these were used to screen the 153 progenies of the cross for genotyping. Out of these 57 SSR primer pairs SSRY32, SSRY36, SSRY83, SSRY339, NS149 and NS890 loci produced single marker which clearly distinguished the parents. Based on the said six primers, 12 progenies out of 153 were identified as self pollinated seeds of CO2 female parent. So these types of SSR markers were very much useful for identification of true hybrid seedlings in the early stage of growth and genotyping of the progenies more accurately for gene mapping. DOI: http://dx.doi.org/10.3329/bjb.v42i1.15906 Bangladesh J. Bot. 42(1): 155-159, 2013 (June)

Identification of Rose (Rosa x hybrida) Varieties Using Simple Sequence Repeat (SSR) Markers

The objective of this study was to evaluate the suitability of simple sequence repeat markers for variety identification in 69 rose (Rosa x hybrida) varieties. A set of 112 SSR primer pairs was evaluated and 43 primer pairs showed polymorphism in 12 varieties. Twenty-two primer pairs out of 43 primer pairs showed high levels of polymorphism and reproducibility. The genetic relationship of 69 varieties was analyzed based on the marker genotypes of 22 SSRs. A total of 114 polymorphic amplified fragments were obtained by using 22 SSR markers. Two to ten SSR alleles were detected for each locus with an average of 5.18 alleles per locus. Average polymorphism information content (PIC) was 0.621, ranging from 0.211 to 0.813. A total of 114 marker loci were used to calculate Jaccard’s distance coefficients for cluster analysis using unweighted pair-group method with arithmetical average (UPGMA). Cluster analysis of genetic diversity revealed that these SSR marker sets identified each genotypes of 69 rose varieties. These SSR markers may be used for wide range of practical application in variety identification of rose.

Genetic Relationships Among Chinese Maize OPVs Based on SSR Markers

Bulk-SSR method was used to analyze the genetic diversity of 44 open-pollinated varieties collected from Henan, Shandong, Shanxi, and Jilin provinces and Guangxi Zhuang Autonomous Region, China using 70 pairs of SSR primers. The purposes of this study were to (1) compare the genetic diversity among 44 Chinese maize open-pollinated varieties; (2) estimate the minimum number of alleles for construction of a stable dendrogram; and (3) trace the genetic relationships among local germplasm from different regions of China. In total, these 70 SSR primers yielded 292 alleles in 176 samples (4×44) analyzed. The number of alleles per locus was 4.17 on average and ranged from 2 to 8. The highest number of alleles per open-pollinated variety (55.25) was detected in Shanxi germplasm, which indicated that open-pollinated varieties from Shanxi possessed the largest genetic diversity among those from the five locations. The correlation coefficients between different genetic similarity matrices suggested that 200 alleles were sufficient for analysis of the genetic diversity of these 44 open-pollinated varieties. The cluster analysis showed that 44 open-pollinated varieties collected from three growing regions in China were accurately classified into three groups that were highly consistent with their geographic origins, and there is no correlation between GS and geographic distance in this study.

In vitro mutagenesis and directed screening for salt-tolerant mutants in peanut

To expand the salt-tolerant gene resources of peanut, we conducted in vitro mutagenesis with pingyangmycin (PYM) as the mutagen and directed screening with medium containing NaCl. After embryonic leaflets from mature peanut seeds (variety Huayu 22) were cultured on somatic embryogenesis-induction medium containing 4 mg/L PYM for 4 weeks, the surviving somatic embryos were sequentially transferred to a germination medium containing 15 and then 20 g/L NaCl. The 30 NaCl-tolerant plantlets obtained were grafted and transplanted in the field in 2011, and the mature seeds of 26 regenerated plants were harvested. In 2012, all seeds from each plant were sown in the field. The offspring (M2 generation) of 23 of 26 NaCl-tolerant, regenerated plants differed from their mutagenic parent in vigor, growth habit, flowering habit, pod shape, and seed coat color, and they also exhibited trait segregation from the same NaCl-tolerant, regenerated plant. In a germination test with a 0.7 % NaCl solution and the M3-generation seeds from 18 of the NaCl-tolerant, regenerated plants, the germination rate was substantially higher for the seeds from 6 plants than for seeds from the mutagenic parent (Huayu 22). To determine whether the changes in plant traits might be associated with gene mutations, DNA polymorphisms between the mutagenic parent and 19 M3 generation individuals from different NaCl-tolerant, regenerated plants were analyzed with 39 pairs of SSR primers, and all mutants differed from the mutagenic parent in >2 loci. The results indicate that the use of PYM-based mutagenesis in combination with directed in vitro screening with NaCl is effective for creating and identifying salt-tolerant mutants of peanut.

Molecular Genetic Diversity of Major Indian Rice Cultivars over Decadal Periods

Genetic diversity in representative sets of high yielding varieties of rice released in India between 1970 and 2010 was studied at molecular level employing hypervariable microsatellite markers. Of 64 rice SSR primer pairs studied, 52 showed polymorphism, when screened in 100 rice genotypes. A total of 184 alleles was identified averaging 3.63 alleles per locus. Cluster analysis clearly grouped the 100 genotypes into their respective decadal periods i.e., 1970s, 1980s, 1990s and 2000s. The trend of diversity over the decadal periods estimated based on the number of alleles (Na), allelic richness (Rs), Nei’s genetic diversity index (He), observed heterozygosity (Ho) and polymorphism information content (PIC) revealed increase of diversity over the periods in year of releasewise and longevitywise classification of rice varieties. Analysis of molecular variance (AMOVA) suggested more variation in within the decadal periods than among the decades. Pairwise comparison of population differentiation (Fst) among decadal periods showed significant difference between all the pairs except a few. Analysis of trends of appearing and disappearing alleles over decadal periods showed an increase in the appearance of alleles and decrease in disappearance in both the categories of varieties. It was obvious from the present findings, that genetic diversity was progressively on the rise in the varieties released during the decadal periods, between 1970s and 2000s.

Development of an expressed gene catalogue and molecular markers from the de novo assembly of short sequence reads of the lentil (Lens culinaris Medik.) transcriptome

Genomic resources such as ESTs, molecular markers and linkage maps are essential for crop improvement. However, these resources are still limited in important legumes such as lentil (Lens culinaris Medik.), which is valued world wide as a rich source of dietary protein. In this study, the de novo transcriptome assembly of 119,855,798 short reads, generated by Illumina paired-end sequencing, was performed using various assembly programs. This resulted in 42,196 nonredundant high-quality transcripts of average length 810 bases, N50 value of 1,432 and an average expression per transcript of 26.21 rpkm reads per kilobase per million(RPKM). Similarity search with the unigenes and protein sequences of other plants resulted in maximum similarity with soybean. A total of 20,009 nonredundant transcripts showed similarity with the UniProtKB database and of these, 18,064 transcripts were grouped into three main GO categories, that is, biological process (15,126), molecular function (15,505) and cellular component (9,434). Annotated transcripts were mapped to 289 predicted Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and 8,893 transcripts were classified into 24 functional categories based on Cluster of Orthologous Groups (COG) of proteins. Mining the data set for the presence of SSRs resulted in 8,722 SSRs with a frequency occurrence of one SSR per 3.92 kb. From these, 5,673 SSR primer pairs were designed, and a subset of these were utilized for diversity analysis. This study, which provides a large data set of annotated transcripts and gene-based SSR markers, would serve as a foundation for various applications in lentil breeding and genetics.

Marker-Aided Selection of Polyploid Poplars

Polyploid breeding is an important means for creating elite varieties for the development of poplar plantations. However, polyploid poplars are rare in natural stands. In this study, we established an analytical toolkit to perform marker-aided selection of polyploid poplars. This toolkit contains 12 SSR primer pairs with sites located in the exonic DNA regions and resulting amplified microsatellites in the intronic/intergenic regions. Highly conserved primer pairs were selected by testing in eight species from four poplar sections. The amplified loci’s variability was examined using trees from a germplasm collection of Populus deltoides. Subsequently, copy numbers amplified by the highly variable primers were experimentally determined using progeny of a full-sib diploid pedigree. Based on the above tests, a subset of primers were finally selected and used for marker-aided selection of polyploid poplars from a set of natural Populus tomentosa stands. The reliability of the established analytical toolkit was further verified using a flow cytometer. We established a fast and reliable technique to screen polyploid poplars from natural stands.

Marker assisted selection for developing high yielding submergence tolerant rice (Oryza sativaL.) genotypes with slender grain

Submergence was a major abiotic stress in rice growing areas, particularly in the rainfed low land ecosystem of eastern India. Tolerance of complete submergence was recognized in some landraces and can be conferred by the Sub1 locus in chromosome 9. Farmers’ adaptation to these landraces and previously developed submergence tolerance genotypes was poor due to either their low yield potential and/or poor grain quality. The purpose of this study was introgressing Sub1 locus from tolerant genotype to high yielding susceptible cultivars with an aim of developing high yielding submergence tolerant genotypes with slender grain preferred by local consumer. During this study fifty lines were selected on the basis of higher yield potential from an advance generation (F4) of a cross between a submergence tolerant, natural mutant (P-1) and submergence susceptible rice cultivar (IR-36). Three already established SSR Primer pairs (RM-265, RM-219 and RM-464A), linked with submergence tolerance were employed to detect p...

Molecular Mapping of Powdery Mildew Resistance Gene pmCH83 in a Putative?Wheat- Thinopyrum intermedium Cryptic Introgression Line

Wheat introgression line CH09W83 is immune or highly resistant to wheat powdery mildew caused by Blumeria graminis f. sp. tritici(Bgt) pathotypes E09, E20, E21, E23, E26, Bg1, and Bg2 at seedling stage in China. This line was derived from the cross between wheat–Thinopyrum intermedium line TAI7047(resistant) and wheat cultivar Jintai 170(susceptible). To identify the Bgt resistance gene(s) in CH09W83, we developed segregating populations(F2, F2:3, and BC1) by crossing/backcrossing CH09W83 with susceptible wheat parents. The result of genetic analysis showed that the adult resistance to Bgt E09 in CH09W83 was controlled by a single recessive gene, which was tentatively designated as pmCH83. Using the F2population of Taichang 29(susceptible) × CH09W83 and 658 pairs of SSR primers, four codominant SSR markers, i.e., Xgwm193,Xgwm251, Xwmc652, and Xgpw7272, were identified to be linked with pmCH83 according to bulked segregant analysis, and the genetic distances between the target gene and flanking markers were 4.3 cM to Xgwm251 and 3.8 cM to Xwmc652. Using Chinese Spring nullitetrasomic and ditelosomic lines, pmCH83 was located on chromosomal arm 4BL of wheat. In combination with evidence of bivalent pairing at metaphase I and GISH, CH09W83 is probably a cryptic wheat–Th. intermedium translocation. Based on pedigree analysis and linkage map, we deduced that pmCH83 is a novel Bgt resistance gene originating from Th. intermedium.

Genetic diversity in maize dent landraces assessed by morphological and molecular markers

Maize Research Institute ?Zemun Polje? genebank maintains a collection of landraces grouped into 18 agro-ecological collected from ex-Yugoslavia territories. The application and comparison of different marker systems are important for the characterization and use of maize landraces in breeding program, as potential sources of desirable traits. In this study, 15 morphological traits, 7 RAPD primers and 10 SSR primer pairs were applied to i) to determine genetic distance between 21 maize dent landraces and ii) compare results obtained on morphological and molecular markers. Phenotypic analysis showed high level of heterogeneity between landraces. Higher level of genetic diversity was obtained with SSR than with RAPD. Genetic distance mean value for RAPD data was 0.35 i.e. for SSR 0.48. Based on the morphological traits and molecular markers, unweighted pairgroup method (UPGMA) analysis was applied for cluster analysis, using statistical NTSYSpc program package. Cluster analysis of morphological and molecular markers distances did not show the same population grouping. Better agreement with agro-ecological data was obtained with RAPD markers. Correlations between dissimilarity matrices for different types of markers were low. Data obtained in this work could be useful for further study of a larger number of landraces, and conservation of genetic resources and their genetic diversity.

Development of EST-SSRs in Brassica napus

EST-SSRs were developed in Brassica napus by database mining. We isolated 7,802 EST-SSRs from the B. napus 643,946 ESTs deposited in the NCBI. With the cut-off value of >10 repeats in di-nucleotide repeats and >7 tri-nucleotide repeats, 303 ESTs were suitable for primer designing for PCR amplification. Of the sixteen possible di-nucleotide combinations, only three types of repeats (AC/GT, AG/CT, and AT/TA) were present among the di-nucleotide EST-SSRs. Whereas, 27 tri-nucleotide repeat motifs from the 64 possible combinations were present. The repeat numbers ranged from 10-15 in di-nucleotide repeats and 7-9 in tri-nucleotide repeat motifs, respectively. By checking PCR amplification in 10 Korean rapeseed breeding lines or cultivars, 234 primer pairs showed successful PCR amplification and 142 of the 234 primer pairs revealed polymorphism among the control cultivars or breeding lines. While the repeat length does not related with the SSR polymorphisms, the repeat motif number showed positive relation with the polymorphism generation by showing higher repeat numbers with higher polymorphisms. We are here presenting the PCR amplifiable primer sequences of the 234 SSR primer pairs to aid in the germplasm management and breeding programs of the B. napus in Korea.

Assessment of inter- and intra-cultivar variations in olive using SSR markers

Olive (Olea europaea L.) production in the world has been made by using many cultivars, and the genetic uniformity of commercial cultivars is important for standard olive oil and table olive production. The genetic variation among and within commonly cultivated olive cultivars in Turkey was analyzed using SSR markers. A total of 135 leaf samples were collected from 11 commonly cultivated olive cultivars from 11 provinces in four geographical regions of Turkey. Seven SSR primer pairs generated 46 SSR markers, and the number of SSR markers per primer pair ranged from 4 (UDO-14) to 9 (GAPU-89) with an average of 6.57. This high level of SSR polymorphism suggests that olive production in Turkey has been made using genetically diverse olive cultivars and this high level of genetic variation is probably due to the location of Turkey in the center of the origin of olive. The UPGMA dendrogram, developed to visualize the estimated genetic relationships among the 135 samples, demonstrated that the clustering of olive cultivars was not based on geographical regions of cultivation. Presence of genetic variation was detected within a nationwide grown Turkish olive cultivar, called 'Gemlik'. Olive growers successfully discriminated olive cultivars with distinct morphological and pomological characters. However, there was some confusion about the identification of cultivars with similar phenotypic traits. To prevent misidentification of olive cultivars and to minimize intra-cultivar variation, certified propagation materials which were characterized using DNA based molecular markers should be used during the establishment of new olive orchards.