SSR Molecular Markers Research Articles

Assessment of genetic relationships among common buckwheat (Fagopyrum esculentum Moench) varieties from Western Balkans using morphological and SSR molecular markers

Assessment of genetic relationships among common buckwheat (Fagopyrum esculentum Moench) varieties from Western Balkans using morphological and SSR molecular markers

Identification of the male parent of superior half-sib Populus tomentosa individuals based on SSR markers

When hundreds of cross-pollinated trees planted in The National Gene Bank of Populus tomentosa blossom and bear fruit, in theory, half-sib progeny should originate from hundreds of combinations. Paternity testing was performed on the superior half-sib progeny with outstanding performance using microsatellite markers, a process that can be used to rapidly screen for superior cross combinations and significantly improve the efficiency and effectiveness of genetic improvements. Based on the comprehensive evaluation of the eight main growth traits of 2292 plants of 30 half-sib families, 77 plants consisting of 43 triploid and 34 diploid plants were selected as superior. We used 14 pairs of highly polymorphic SSR primers to identify the male parents of the 77 superior plants. Our results identified 29 male trees as the pollen parents of 59 superior plants from 49 cross combinations. The probabilities of 2-14, 3-48-1, 1903, 2-1, 4234, 5099, and 4114 as male parents were significantly higher than those of other males, suggesting that these male parents have potential for applications in breeding strategies of P. tomentosa. Thus, this study used SSR molecular markers to identify the male parents of superior individuals in a P. tomentosa population, laying a foundation for the application of maternal efficiency and construction of optimal hybrid parent groups.

Morphological and genetic diversity of bamboos in Arboretum Universitas Padjadjaran, Sumedang, Jawa Barat

Annisa, Mardliyyah A, Kusmoro J. 2017. Morphological and genetic diversity of bamboos in Arboretum Universitas Padjadjaran, Sumedang, Jawa Barat. Pros Sem Nas Masy Biodiv Indon 3: 351-360. Bamboos are versatile plant that have been used for a long time by Indonesian people in everyday life. The utilization of bamboo, continuously, could possibly reduced species and population of bamboos that available in nature. The study of morphological and genetic diversity can assist in gathering data for the purpose of conservation and sustainability use of bamboo’s germplasm. Research aimed to identify morphological and genetic diversity of bamboos in Arboretum Universitas Padjadjaran. The research of morphological diversity was done by identification based on the result of morphological observation and vegetative organ morphometric, while the research of genetic diversity was done by using SSR molecular marker. This research used observation method which included morphological identification and determination of location for collecting plant materilas, collecting plant materials, DNA isolation, visualization of DNA products, and amplification by using SSR marker. Morphology and amplification data were analysed descriptively. Clustering analysis based on genetic was done by UPGMA on Numerical Taxonomy and Multivariate Analysis System (NTSYS) 2.02. Result of morphological diversity research showed there were ten species of bamboos in Arboretum Universitas Padjadjaran that consisted of four genus such as Bambusa, Gigantochloa, Phyllostachys, and Schizostachyum. Result of genetic diversity research showed that ten loci of SSR markers designed from Phyllostachys in China and Bambusa in India can be applied on Arboretum’s bamboos . The total alleles were 117 alleles with range size of 83-893bp. PIC value of the primers were ranged between 0.84-0.94, which categorized as very informative. Based on similarity coefficient values it can be concluded that bamboo Arboretum Universitas Padjadjaran has high level genetic diversity.

Assessment of Genetic Variation within Commercial Iranian Pomegranate (Punica granatum L.) Cultivars, Using ISSR and SSR Markers

Pomegranate is one of the most important horticultural crops in Iran, and has been cultivated for thousands of years in this country. At this period due to selection of superior cultivars from nature or mutation emerged in these cultivars, and their vegetative propagation, substantial genetic variation has occurred within and among the cultivars. Thus, each cultivar may consist of different clones. According to this issue, diversity within four commercial cultivars of pomegranate was analyzed. Two molecular marker systems including ISSR and SSR were used to evaluate variability between 36 samples of four commercial cultivars. ISSR markers produced 114 amplification products, out of which 97 were polymorphic (83.23%). Mean resolving power was 2.96 for ISSR markers. 19 SSR molecular markers were used, 15 of which amplified polymorphic products, while the remaining ones monomorphic., The number of polymorphic alleles per locus ranged from two to four (average 3.6). The observed and expected heterozygosities ranged from 0.04 to 0.92 and 0.14 to 0.62, respectively. In addition, mean polymorphic information content was 0.45 for SSR loci. Our results showed that commercial Iranian pomegranate have different clones. Therefore, ISSR and SSR markers can be a useful tools for detecting clones of each cultivar.

Analysis of genetic diversity among medicinal therapist Trigonella foenum-graecum L. genotypes through RAPD and SSR markers

Trigonella is recognized as a medicinal therapist throughout the globe due to its multifaceted rare medicinal properties. It is indigenous from Iran to Northern India but has gained global acceptance towards cultivation and consumption for its yellow-to-amber colored seed which substantially contributes to food, pharmaceutical, nutraceutical and cosmetic industry. Genetic diversity serves as an excellent tool for developing improved crop varieties with breeder preferred traits. Unfortunately, very little information available on variability existing in commercial Trigonella genotypes considerably impedes the crop improvement. In this study, ninety Trigonella genotypes belonging to most productive North Indian states were subjected to multilocus genotyping using RAPD (49) and SSR (13) primers and detected an average of 55.60 and 50.16% polymorphism, respectively. The percentage polymorphism range (RAPD, 16.7–90.90; SSR, 33.30–66.66) average band informativeness (RAPD, 0.182–0.85; SSR, 0.21–0.91) and resolving power (RAPD, 0.95–9.984; SSR, 1.68–7.28) obtained revealed the wide range of diversity prevailing among these genotypes. Hierarchical clustering of genotypes in nine different clusters showed Trigonella’s genetic variability has wide genetic distribution across different agro-climatic zones. No consistency was observed while grouping Trigonella varieties based on eco-geographical region. Eventually, knowledge of these genetic differences significantly contributes in designing intra-specific crosses with potential interest to spice breeding programs. To the best of our knowledge, this is the first report of genetic diversity using SSR molecular markers in Trigonella foenum-graecum L.

Genetic variation of european maize genotypes (Zea mays L.) Detected using ssr markers

The SSR molecular markers were used to assess genetic diversity in 40 old European maize genotypes. Ten SSR primers revealed a total of 65 alleles ranging from 4 (UMC1060) to 8 (UMC2002 and UMC1155) alleles per locus with a mean value of 6.50 alleles per locus. The PIC values ranged from 0.713 (UMC1060) to 0.842 (UMC2002) with an average value of 0.810 and the DI value ranged from 0.734 (UMC1060) to 0.848 (UMC2002) with an average value of 0.819. 100% of used SSR markers had PIC and DI values higher than 0.7 that means high polymorphism of chosen markers used for analysis. Probability of identity (PI) was low ranged from 0.004 (UMC1072) to 0.022 (UMC1060) with an average of 0.008. A dendrogram was constructed from a genetic distance matrix based on profiles of the 10 maize SSR loci using the unweighted pair-group method with the arithmetic average (UPGMA). According to analysis, the collection of 40 diverse accessions of maize was clustered into four clusters. The first cluster contained nine genotypes of maize, while the second cluster contained the four genotypes of maize. The third cluster contained 5 maize genotypes. Cluster 4 contained five genotypes from Hungary (22.73%), two genotypes from Poland (9.10%), seven genotypes of maize from Union of Soviet Socialist Republics (31.81%), six genotypes from Czechoslovakia (27.27%), one genotype from Slovak Republic (4.55%) and one genotype of maize is from Yugoslavia (4.55%). We could not distinguish 4 maize genotypes grouped in cluster 4, (Voroneskaja and Kocovska Skora) and 2 Hungarian maize genotypes - Feheres Sarga Filleres and Mindszentpusztai Feher, which are genetically the closest.

Tetraploid citrus progenies arising from FDR and SDR unreduced pollen in 4x X 2x hybridizations

Polyploid citrus arise by somatic and sexual polyploidization, and both have been used in triploid breeding programs. Sexual polyploidization is mainly achieved by first-division restitution (FDR) or second-division restitution (SDR) meiotic restitution mechanisms. In citrus, mostly SDR producing 2n ovule has been described. However, we obtained 72 tetraploid hybrids from 4x X 2x sexual hybridizations using two doubled-diploid mandarins as female parents (‘Moncada’ mandarin and ‘Fina’ clementine) and a diploid hybrid tangor as male parent (clementine X sweet orange—‘CSO’) suggesting 2n pollen formation. This material was used to confirm the existence of 2n pollen in Citrus and to analyze its origin. SSR and SNP molecular marker analyses revealed that 64 out of the 72 recovered tetraploid plants resulted from the fertilization of a reduced diploid female gamete by unreduced (diploid) pollen from ‘CSO’, whereas eight tetraploid plants arose from self-pollination of the tetraploid parent. The maximum-likelihood method based on parental heterozygosity restitution (PHR) of centromeric loci identified both FDR and SDR as the mechanisms leading to unreduced male gamete formation. From the 64 unreduced gametes produced by diploid ‘CSO’ tangor, 41 (64.1%) were originated by FDR, whereas 12 (18.8%) were significant for SDR. Non-conclusive results were obtained for 11gametes (17.2%). The pattern of PHR variation of markers located along the linkage group 2 confirmed our results at population level. To our knowledge, this is the first report of tetraploid citrus progenies arising from unreduced pollen and the first description of the coexistence of two meiotic restitution mechanisms (SDR and FDR) producing unreduced pollen in citrus.

Genetic linkage analysis using DNA markers in sweetpotato.

Sweetpotato is one of the most important food crop species in the world, with more than 104,000,000 tons produced each year, and the breeding of superior cultivars with agronomically important traits, such as improved disease resistance, yield, and nutrient richness, is necessary, especially in developing countries. However, as a result of the crop’s complex genomic architecture, which results from its hexaploidy (2n = 6× = 90), high heterozygosity, huge genome, and outcrossing nature, the analysis of genetic linkage in sweetpotato has been challenging. In addition, the lack of whole genome sequences or gene annotations for cultivated hexaploids has interrupted the validation of mapped QTL regions and gene cloning. In spite of these technical difficulties, linkage map construction and QTL mapping analysis have been reported. This review summarizes the results of these linkage analyses, which used SSR, AFLP, and retrotransposon-based molecular markers, and describes future directions for the genetic analysis and marker-assisted breeding of this important but genetically complex crop species.

Relationships between genetic distance, combining ability and heterosis in rapeseed (Brassica napus L.)

Present study was conducted to evaluate the relationship between parental genetic distances, combining ability and heterosis in rapeseed (Brassica napus L.). Nine elite inbreds widely used in Chinese rapeseed breeding programs, were crossed in a diallel design to develop 36 hybrids and evaluated together with their parents across four environments. Genetic distances among lines were measured from 11 morphological traits, and SSR and SRAP molecular markers. Results showed that genetic distance evaluated by total molecular markers (GDtotal) had no correlation with heterosis. In order to make a better prediction, significant markers and favoring markers were selected from the total molecular markers, and results revealed that genetic distance measured by favoring markers (GDfavor) significantly and positively correlated with number of seeds per silique, thousand seed weight, seed yield per plant and seed yield per plot for high-check heterosis (HCK) and sum of parental general combining ability (GCA(f+m)). The selected favoring markers could be used for the further study on prediction of heterosis for yield and yield related traits. The magnitudes of correlation coefficients of morphological distances with mid-parent heterosis, high-parent heterosis and HCK for yield and other traits were mostly not significant. Interestingly, more higher levels of correlations were found between GCA(f+m) and heterosis, which indicated that using the information of GCA(f+m) to predict heterosis appeared an important method in rapeseed. Three out yielding crosses, 8D129 × 8E001 (HCK, 23.56%), 8C189 × 8C272 (23.31%), and 8C343 × 8D129 (20.08%), were identified as ideal hybrids evaluated across four environments and may be promoted for adoption and commercialization in China.

SSR analysis on stress effect of transgenic hybrid poplar 741 on Clostera anachoreta (Fabricius) (Lepidoptera: Notodontidae)

The genetic differentiation of the experimental population of Clostera anachoreta fed on different resistant transgenic 741 poplar leaves was analyzed by SSR molecular marker technique to investigate stress effect of transgenic poplar Bt gene as food on target insect. The experimental population of C. anachoreta fed on transgenic 741 poplar high resistant strains 'Pb29', medium resis-tant strains 'Pb17' and non-transgenic poplar (CK), and the screened ten pairs of SSR primers were used. The results showed that 76 alleles were observed in ten pairs of primers. The average allele was 7.6, the average effective number of alleles was 2.2, the average observed heterozygosity was 0.5167, the average expected heterozygosity was 0.5167, and the average percentage of polymorphic loci was 96.7%. The genetic diversity level of C. anachoreta experimental population fed on transgenic poplar 741 was significantly higher than that fed on non-transgenic populations, and C. anachoreta fed on high resistance had the lowest genetic similarity with CK samples, which showed an increasing trend of the genetic diversity of the experimental population fed on transgenic Bt poplar. It was thus clear that transgenic hybrid poplar 741 had stress effects on genetic differentiation of C. anachoreta experimental population by SSR.

The Current Status of Germplum Database: a Tool for Characterization of Plum Genetic Resources in Romania

In Romania, Prunus genetic resources are kept in collections of varieties, populations and biotypes, mainly located in research and development institutes or fruit growing stations and, in the last years, by some private enterprises. Creating the experimental model for the Germplum database based on phenotypic descriptors and SSR molecular markers analysis is an important and topical objective for the efficient characterization of genetic resources and also for establishing a public-private partnership for the effective management of plum germplasm resources in Romania. The technical development of the Germplum database was completed and data will be added continuously after characterizing each new accession.

Archaeobotanical reconstructions of vegetation and report of mummified apple seeds found in the cellar of a first-century Roman villa on Elba Island

In the late Roman Republic period (2nd–1st century BC), in the area of San Giovanni on Elba Island, previously subject to intense extraction of iron ore, a rustic villa was established by Marco Valerio Messalla, a supreme Roman magistrate. The foundations of the walls were discovered and excavated by an archaeological mission. Palaeobotanical analysis of a set of stratigraphic layers was performed. Palynological slides showed remains of palynomorphic and non-pollen objects, while data combined with anthracological investigations confirmed the hypothesis that in the 1st century AD the villa was destroyed by a fire that created a compact crust under which were discovered four broken Roman amphorae containing about five hundred apple seeds. Comparisons of archaeological and fresh seeds from reference collections showed discontinuous morphology except for one group of archaeological samples. DNA was isolated from seeds that had well-preserved embryos in all groups. DNA extracts from archaeological, wild and modern domestic seeds (controls) were amplified by PCR and tested with SSR molecular markers, followed by genome analysis.

Morphological characterization and genetic parameter estimation in backcrossed progenies of Passiflora L. for ornamental use

Morphological characterization and genetic parameter estimation were performed in four progenies obtained by backcrossing the donor genitor Passiflora sublanceolata and four recurrent genitor F1 HD13 hybrids (P. sublanceolata vs. P. foetida). The use of two different germination methodologies showed the existence of significant differences between the germination potential of two-way crossing, indicating time differences to overcome seed dormancy. SSR and ISSR molecular markers allowed the backcrossed character to be confirmed in most plants within progenies, discarding the hypothesis of self-pollination in the origin of backcrossed germplasm. Broad qualitative variation was observed, reflecting 30 different floral patterns in the backcrossed population. ANOVA revealed a significant effect between both characterization periods and for most descriptors analyzed separately in the first and second characterization periods. Scott-Knott clustering revealed that most of the features of the progenies showed intermediate values for the mean of the genitors; however, they have similar mean values between them. Generally higher heritability was observed for floral traits, suggesting high genetic control in phenotypic expression. Data indicates the success of the backcrossing for the recovery of ornamental characteristics with broad variability among progenies. Genetic parameters indicate that the selection of progenies should be made based on floral characteristics, yet all progenies showed higher mean values of the recurrent parents for the most important floral descriptor. However, the selection of plants within progeny should be performed after the characterization of individual plants, either for direct use as an ornamental plant, or for use in other breeding programs.

Patterns of genetic structure and evidence of gene flow among Tunisian Citrus species based on informative nSSR markers

This study investigates the extent of genetic diversity, phylogenetic relationships and the amount of gene flow among Tunisian Citrus species based on a set of 15 informative nuclear SSR molecular markers. Genotyping data highlighted an allelic richness among Tunisian Citrus species and has allowed the detection of 168 alleles among them 104.19 were effective. The partition of the total genetic diversity (HT=0.832) showed that the highest amount of variation within the Citrus species is HS=0.550, while the relative amount of the between-species genetic diversity GST does not exceed 0.338. This pattern of genetic structure was supported by low-to-moderate FST pairwise values and the presence of a gene flow (Nm) among the eight Citrus species. The lowest genetic differentiation was revealed between the species C. sinensis and C. insitorum (FST=0.111, Nm=1.99), while the highest genetic differentiation was recorded between the species C. aurantifolia and C. paradisi (FST=0.367, Nm=0.43). The established Neighbor Joining analysis showed that all genotypes were widely discriminated and clearly pooled according to their species of origin, with minor exceptions.

Population Structure and Genetic Diversity in Sweet Cassava Cultivars from Paraná, Brazil

The objectives of the present study were to assess the population structure and genetic diversity in traditional sweet cassava cultivars collected in “backyard” cultivations in the municipalities of Maringa, Cianorte, and Toledo, State of Parana, Southern Brazil, using 13 SSR molecular markers. All the loci analyzed were considered polymorphic with a mean of 3.15 alleles per locus; the mean polymorphism information content (PIC) value found was 0.4598, indicating that the primers were reasonably informative and the heterozygosity amplitude observed ranged from 0.0270 (GA134) to 0.8718 (SSRY45), with a mean of 0.4762, while the mean genetic diversity obtained was 0.5407, ranging from 0.3138 (GA21) to 0.6502 (GA140). The most divergent combinations were BGM434T-BGM20M, BGM35C-BGM20M, BGM430T-BGM232M, BGM430T-BGM164M, BGM430T-BGM322M, and BGM35C-BGM164M. The analysis of population structure distributed the traditional cultivars assessed in four different groups. The analysis of molecular variance (AMOVA) estimated that 11, 15, and 74 % of the variance were between groups, between individuals within groups, and between individuals within the population as a whole, respectively. The F is (0.170) and F it (0.259) values indicated a number of heterozygotes present in the population under study lower than that necessary to reach the Hardy-Weinberg equilibrium. The genetic variability found among the traditional sweet cassava cultivars assessed was considered wide, and the groups that were most distant were mostly cultivars from Toledo and Maringa.

Traditional Banana Diversity in Oceania: An Endangered Heritage.

This study aims to understand the genetic diversity of traditional Oceanian starchy bananas in order to propose an efficient conservation strategy for these endangered varieties. SSR and DArT molecular markers are used to characterize a large sample of Pacific accessions, from New Guinea to Tahiti and Hawaii. All Pacific starchy bananas are shown of New Guinea origin, by interspecific hybridization between Musa acuminata (AA genome), more precisely its local subspecies M. acuminata ssp. banksii, and M. balbisiana (BB genome) generating triploid AAB Pacific starchy bananas. These AAB genotypes do not form a subgroup sensu stricto and genetic markers differentiate two subgroups across the three morphotypes usually identified: Iholena versus Popoulu and Maoli. The Popoulu/Maoli accessions, even if morphologically diverse throughout the Pacific, cluster in the same genetic subgroup. However, the subgroup is not strictly monophyletic and several close, but different genotypes are linked to the dominant genotype. One of the related genotypes is specific to New Caledonia (NC), with morphotypes close to Maoli, but with some primitive characters. It is concluded that the diffusion of Pacific starchy AAB bananas results from a series of introductions of triploids originating in New Guinea area from several sexual recombination events implying different genotypes of M. acuminata ssp. banksii. This scheme of multiple waves from the New Guinea zone is consistent with the archaeological data for peopling of the Pacific. The present geographic distribution suggests that a greater diversity must have existed in the past. Its erosion finds parallels with the erosion of cultural traditions, inexorably declining in most of the Polynesian or Melanesian Islands. Symmetrically, diversity hot spots appear linked to the local persistence of traditions: Maoli in New Caledonian Kanak traditions or Iholena in a few Polynesian islands. These results will contribute to optimizing the conservation strategy for the ex-situ Pacific Banana Collection supported collectively by the Pacific countries.

SSR Molecular Marker and Analysis Report of Variety of Chicken for their Varying Weight

SSR Molecular Marker and Analysis Report of Variety of Chicken for their Varying Weight

The characterisation of Vitis vinifera ’Refošk’ with AFLP and SSR molecular markers and ampelographic traits

The genetic diversity and ampelographic variability of autochthonous red wine cultivar ‘Refošk’ (Vitis vinifera L.) grown in Slovenia were evaluated with AFLP molecular markers and OIV descriptors, respectively. SSR molecular markers were employed to confirm cultivar identity of analysed samples. Eight AFLP primer combinations, one was monomorphic, produced 16 polymorphic markers in 41 out of 113 samples, what classified samples into monomorphic and polymorphic group. Dendrogram constructed with simple matching coefficient and unweighted pair-group method analysis presented genetic diversity within polymorphic group. Refošk biotypes from monomorphic and polymorphic groups were evaluated with 22 OIV descriptors related to bunch, berry and must, but on the basis of ampelographic characterization samples were not differentiated among two major groups obtained with AFLP analysis. Results of genetic analysis indicated that ‘Refošk’ originated from closely related plants that are phenotypically very similar. With regard to low observed genetic diversity more attention should be dedicated to the selection in order to conserve remaining genetic diversity.

Molecular Categorization of Some Water Yam (Dioscorea alata L.) Germplasm in Ghana Using Microsatellite (SSR) Markers

The yam species, Dioscorea alata has an advantage for sustainable cultivation due to its comparatively good agronomic characteristics. Breeders are therefore keen to improve the food quality of the species. Nevertheless, published data on molecular classification and genetic diversity of this crop are scanty. This research therefore investigated genetic variability and relationships among some collected Ghanaian D. alata accessions (35) together with 14 introductions from IITA in Nigeria. The true genetic identity and population structure of the accessions were determined using 14 Simple Sequence Repeats primer pairs available for yams. Dimensional scatter diagram of the principal coordinates showed a wide dispersion among the accessions. Cluster analysis using unweighted neighbour-joining method clearly separated the 49 accessions into five main groupings. From the allele frequency analysis, the size of amplified alleles ranged from 100 to 510 bp. The mean polymorphic information content (PIC) values for all markers used was 0.91 and ranged between 0.86 and 0.94 in loci YM13 and YM30 respectively. Gene diversity was high and the average observed heterozygosity was 0.77. Gene diversity was high ranging from 0.87 in YM13 to 0.94 in YM30 with a mean of 0.92. Generally, the allele frequency of all the primers was below 0.95 indicating that they were all polymorphic in character. The findings of this study confirm that SSR molecular markers are able to identify closely related materials within species.

Interspecific potato somatic hybrids between Solanum tuberosum and S. cardiophyllum, potential sources of late blight resistance breeding

Solanum cardiophyllum Lindl. (PI 341233) (2n = 2x = 24, 1EBN) is a diploid wild potato species highly resistant to late blight, the most serious disease of potato. However, it poses a problem of sexual incompatibility with common potato. So to circumvent this problem, in this study, we developed interspecific potato somatic hybrids between cultivated S. tuberosum dihaploid C-13 and wild species S. cardiophyllum via protoplast fusion. Out of 26 regenerants, only 4 were confirmed as true somatic hybrids containing both parental genomes based on molecular markers and phenotypes. Somatic hybrids were identified by RAPD, ISSR, SSR, AFLP and cytoplasm (chloroplast and mitochondrial genomes) type molecular markers. Intermediate phenotypes of somatic hybrids were confirmed by leaf, flower and tuber traits. Late blight resistance of the hybrids was assessed by challenge inoculation of P. infestans under controlled conditions. Somatic hybrids were found tetraploid by flow cytometry, exhibited high pollen stainability by acetocarmine staining and formed berries and viable seeds after crossing with a common potato variety. Somatic hybrids possessed diverse cytoplasm types (W/α, W/γ and T/β) as assessed by chloroplast and mitochondrial genome-specific markers. Further, cluster analysis based on the Jaccard’s coefficient of molecular profiles generated by all above markers showed genetic distinctness in somatic hybrids and parents. Taken together, these interspecific somatic hybrids with diverse cytoplasm background have potential to be employed in potato breeding programmes to widen the cultivated gene pool through conventional and molecular methods.