SSR Primers Research Articles

Evaluation of Allelic Variation in Cedrus deodara (Roxb.) G. Don to identify Region Specific Nuclear Microsatellite Markers (nSSRs) in Uttarakhand, India

Cedrus deodara is an excellent timber species extensively prone to illicit felling and smuggling of timber. Knowledge of molecular marker techniques can help in tracing the origin of the stolen timber back to the forest area for legal purposes. The present study demonstrates the use of nSSR markers for timber tracking in Cedrus deodara. In this research twentyfive individual trees were randomly selected from two geographically distinct populations Kathiyan and Jageshwar forest of Uttarakhand. Out of 47 nuclear SSR primers, only nine showed positive amplification out of which five (pdms009, pm01, pm05, SSRPtctg4698 and RPTest9) were finally selected to reveal allelic variation. Total of 10 alleles of individuals were detected at all five loci for both populations. For Kathiyan, population effective number of alleles ranged from 1.26 to 2 while for Jageshwar, it ranged from 1.14 to 1.98. Expected heterozygosity (HE) for Chakrata population ranged from 0.21 to 0.51 while for Jageshwar it was between 0.13 to 0.5. Analysis of molecular variance (AMOVA) shows that most of the variations were found within a population (97.81 %) as compared to among the population (2.19 %) with an FST value of 0.02. In this research, we found that the same primer pairs gave a different product length for both geographically distinct populations of C. deodara, for Kathiyan population locus pdms009, pm01, pm05, SSRPtctg4698 and RPTest9 product lengths for both alleles a and b were 197 and 125, 304 and 221,402 and 317,261 and 208, 332 and 246 base pair respectively, while for Jageshwar population, it were 339 and 238, 319 and 203, 456 and 283, 245 and 197, 309 and 235 base pair respectively.

Genetic Diversity of Turkish Cultivated Emmer (Triticum dicoccum Schrank) Revealed by Microsatellite Markers

The aim of this study is to evaluate the genetic diversity of twelve populations for cultivated emmer (Triticum dicoccum Schrank ex Schübl) and bread wheat (Triticum aestivum L.) species, which are important wheat gene resources and grown in different villages in Kars province (Turkey), using eleven microsatellite markers. SSR primers produced a total of 41 alleles and the average polymorphism percentage was 86.2%. The average number of alleles obtained from primers was 3.72. Polymorphic Information Content (PIC) values varied between 0.14 and 0.37 with the means of 0.26 value. The primers of Xgwm-46 (0.287), Xgwm-154 (0.304) and Xgwmn-361 (0.325) were identified as the most effective primers in understanding the genetic diversity of emmer genotypes. Local emmer wheat had a little higher allelic richness and gene diversity than cultivated wheat. Due to geographic and climatic variations, genetic differentiation was detected in these wheat populations.

A novel twisted bud mutant from Ziziphus jujubaMill. ‘Dongzao’

The twisted growth of organs is a distinctive and attractive trait for woody plants. A bud mutant with twisted branches was discovered from ‘Dongzao’, the most popular and tasty table cultivar of Chinese jujube (Ziziphus jujuba Mill.). This study explored the morphological and nutritional characteristics and SSR marker changes of the novel variant. Compared to its mother plant and an external control ‘Dongzao’, the variant had shorter fruit (oblate rather than round) and shorter bearing shoots with smaller internode length. Although the sugar/acid ratio increased in the variant fruit, the soluble sugar, titrated acid and ascorbic acid contents of the fruit were not significantly different between the variant and its mother. No differences in the DNA fingerprint were observed between the variant and its mother using 50 pairs of SSR primers. The novel twisted variant of ‘Dongzao’ could be a valuable germplasm for jujube breeding because it has high-quality fruit and good yield. It also has great potential for direct economic utilization and could be used as a desirable material for research on the mechanism of twisted growth.

Molecular characterization and genetic diversity studies of Indian soybean (Glycine max (L.) Merr.) cultivars using SSR markers

BackgroundThe genetic base of soybean cultivars in India has been reported to be extremely narrow, due to repeated use of few selected and elite genotypes as parents in the breeding programmes. This ultimately led to the reduction of genetic variability among existing soybean cultivars and stagnation in crop yield. Thus in order to enhance production and productivity of soybean, broadening of genetic base and exploring untapped valuable genetic diversity has become quite indispensable. This could be successfully accomplished through molecular characterization of soybean genotypes using various DNA based markers. Hence, an attempt was made to study the molecular divergence and relatedness among 29 genotypes of soybean using SSR markers.Methods and resultsA total of 35 SSR primers were deployed to study the genetic divergence among 29 genotypes of soybean. Among them, 14 primer pairs were found to be polymorphic producing a total of 34 polymorphic alleles; and the allele number for each locus ranged from two to four with an average of 2.43 alleles per primer pair. Polymorphic information content (PIC) values of SSRs ranged from 0.064 to 0.689 with an average of 0.331. The dendrogram constructed based on dissimilarity indices clustered the 29 genotypes into two major groups and four sub-groups. Similarly, principal coordinate analysis grouped the genotypes into four major groups that exactly corresponded to the clustering of genotypes among four sub-groups of dendrogram. Besides, the study has reported eight unique and two rare alleles that could be potentially utilized for genetic purity analysis and cultivar identification in soybean.ConclusionIn the present investigation, two major clusters were reported and grouping of large number of genotypes in each cluster indicated high degree of genetic resemblance and narrow genetic base among the genotypes used in the study. With respect to the primers used in the study, the values of PIC and other related parameters revealed that the selected SSR markers are moderately informative and could be potentially utilized for diversity analysis of soybean. The clustering pattern of dendrogram constructed based on SSR loci profile displayed good agreement with the cultivar’s pedigree information. High level of genetic similarity observed among the genotypes from the present study necessitates the inclusion of wild relatives, land races and traditional cultivars in future soybean breeding programmes to widen the crop gene pool. Thus, hybridization among diverse gene pool could result in more heterotic combinations ultimately enhancing genetic gain, crop yield and resistance to various stress factors.

Genetic diversity and population structure of apple germplasm from Eastern Black Sea region of Turkey by SSRs

Turkey has a diverse apple germplasm and Northeastern Anatolia had great contributions to domestication of the Malus genus. Despite quite high rainfall (900–2300 mm) and humidity (70–85%) levels, several local apple cultivars have been successfully grown for centuries at the coastal zone of Eastern Black Sea region of Northeastern Anatolia without any pesticide applications. In this study, a set of 206 local genotypes, including 6 international cultivars as references was analyzed using 13 SSR primers with the aim of assessing the genetic diversity and population structure among the genotypes. Bayesian genetic structure analysis was used to reveal differentiation of the groups and the results were confirmed by factorial correspondence analysis (FCA). A total of 234 alleles (average 18) were generated by 13 SSR markers. CH02c06 was the most informative (PI: 0.017) and CH03g07 was the least informative (PI: 0.07) locus. The average He was 0.859 with a range of 0.775–0.929 and the average Ho was 0.760 with a range of 0.556–0.872. Considerable genetic variation was detected among the genotypes and genetic similarity varied between 11 and 96%. A Bayesian genetic structure analysis indicated three subpopulations (K) and admixture among the accessions. Several loci yielded three alleles in 39 accessions. No synonyms or identical cultivars were detected but several genotypes known by the same names formed 22 homonym groups. The present SSR data will have great contributions to future germplasm management efforts as well as to further comparative studies that investigate genetic relationships among the local apples of Northeastern Anatolia.

Molecular Characterization of Drought Tolerant Genotypes of Pearl Millet [Pennisetum glaucum (L.) R. Br.] for A1 Zone

Pearl millet is a widely grown, climate resilient rainfed cereal crop cultivated on 29 million ha in the arid and semi-arid tropical regions of Asia and Africa accounting for almost half of global millet production. It is useful for minimizing the adverse effect of climate change, hence facilitating income and food security among farming communities. It has deep root system and exhibit climate-resilient features including adaptation to a wide range of ecological conditions, less irrigational requirements, better growth and productivity in low nutrient input conditions, less dependent on synthetic fertilizers and minimum vulnerability to environmental stresses and thus can survive in harsh climatic conditions, less fertile soil under water scarcity. Breeding of drought tolerant varieties and selecting genotypes for better water use efficiency is important in pearl millet to mitigate the changing climatic scenario. In this study, 24 genotypes of pearl millet which are drought tolerant and specific for A1 zone were characterized using 15 drought specific SSR primers. All the 15 SSRs amplified products of varying sizes ranging between 90-550 bp. A total of 40 alleles were obtained in this study and the number of alleles per locus varied between 2 to 5 with an average of 2.67 alleles. Polymorphic Information Content (PIC) varied from 0.34 to 0.76 with an average of 0.53 PIC value. This study will be useful for developing high yielding, dual purpose cultivars for low rainfall areas i.e. A1 zone and increasing pearl millet productivity.

Evaluation of the genetic structure of Bromus inermis populations from chemically and radioactively polluted areas using microsatellite markers from closely related species

Hypothesis: The anthropogenic effects can be manifested in a decrease in genetic diversity and the population differentiation, and increase in the frequencies of rare and/or private alleles. Materials and methods: We have selected a collection of primers for B. inermis, consisting of 21 microsatellite loci from B. sterilis, B. tectorum and Triticum aestivum. Results: Only 38% of SSR primers showed good transferability and were used for B. inermis population studies from technogenic pollution areas. We revealed 42 alleles at eight loci, and the number of alleles per locus varied from one to 13 per populations. The percentage of polymorphic loci in B. inermis populations was 48.44%. A total of 22 rare, 14 private and 9 both rare and private alleles were reported. There were no correlations between geographic and genetic distances. Only 6.8% of the genetic variability was distributed among B. inermis populations. Conclusion: There was no decrease in genetic diversity found in B. inermis populations growing under anthropogenic stress. No significant differences in the number of rare and private alleles in the background and impact populations of B. inermis were found. The smooth brome is characterized by low differentiation of the populations. Possible reasons for this phenomenon are discussed.

Genetic and Morphological Variability among the Isolates of Fusarium oxysporum f. sp. lentis causing Wilt of Lentil

Background: Lentil wilt is one of the most important diseases of lentil directly contributing to the yield losses of the crop. It is caused by Fusarium oxysporum f. sp. lentis which shows the great genetic and morphological diversity. Methods: Present investigation was conducted during 2017-2019 with 22 isolates of the pathogen, collected from lentil grown areas of Uttarakhand and Uttar Pradesh to explore genetic and morphological variability employing eight ISSR and five SSR primers. Result: The isolates showed huge morphological variability based on the color, pigmentation and shape of conidia. Study on molecular variability revealed the versatility in the genome of different isolates of pathogen. The diversity among the isolates of pathogens collected from Uttarakhand and Uttar Pradesh was also evident. Generated work on genetic and morphological variability and the population diversity among the different isolates of the pathogen impact on developing ideal disease management strategies.

Tissue-specific transcriptome for Rheum tanguticum reveals candidate genes related to the anthraquinones biosynthesis.

The online version contains supplementary material available at 10.1007/s12298-021-01099-8.

Estimation of Genetic Diversity among Canola Accessions using Simple Sequence Repeat Markers

Genetic studies through molecular markers proved important to find out the genetic diversity of canola. In this study, 50 lines of canola were used to find the polymorphism using 15 SSR primers and investigated the genetic diversity, PIC values, frequency-based genetic distance, and allelic frequencies. Mean gene diversity, frequency-based genetic distance, and PIC values were 0.8777, 0.233 and 0.8666, respectively for the canola lines. A good range of genetic diversity was found among studied canola lines with value 85.91% polymorphism. Maximum and minimum genetic distances among 50 lines were 1 and 0.26, respectively. Accessions ACC-26068, ACC-24241, ACC-24244, ACC-24233, ACC-24423 and ACC-24224 have maximum genetic distance. Accessions ACC-24879 and ACC-24169 had minimum genetic distance i.e., 0.26. Dendrogram based on genetic distances showed four main clusters that were further dividing into several sub-clusters. The primers utilized in the present study, were valuable to identify different accessions of canola to find the variability present. This variability will be helpful to initiate the breeding program with their molecular genetic basis.

Genetic Analysis of Yield Attributes in Rice for Water Deficit Under Egyptian Conditions التحليل الوراثى لصفات المحصول فى الارز للاجهاد المائى تحت الظروف المصرية

Rice parental genotypes with good combining abilities provide an efficient tool to enhance rice yield under water deficit conditions. Using 15 F1 hybrids derived from a half diallel mating involving six parental genotypes, two experiments were carried out during 2019 and 2020 growing seasons to investigate the genetic analysis and combining ability for yield and its components under irrigated (E I) and water stress (E II) environments. Eight agronomic traits were studied over a period of two seasons, and cluster analysis based on 16 SSR primers found that the genotypes under study differed greatly in terms of variation. Overall, all yield attributes have been significantly affected by water deficit and was governed by both additive and non-additive gene actions. Based on the mean values and GCA effects, Gaori and IET1444 are suitable for incorporation of earliness and drought tolerance traits. Obviously that Gaori, IET1444, IRAT 170 and WAB 450 would serve as good general combiners for grain yield attributes under (E II) conditions. Due importance should be given for six combinations viz, Giza 177 x IRAT 170, Giza 177x Gaori, Sakha 101x IRAT 170, Sakha 101x IET 1444, Gaori x WAB 450 and IET 1444x WAB 450 to identify as new genotypes with high grain yield under water deficit condition. There might be a great possibility to get the best new combinations through crossing genotypes with highest genetic distance. Therefore, these findings should be considered when selecting elite genotypes for developing superior new rice crosses under water-stress conditions.

Allelic and bio-chemical characterization of Indian wheat (Triticum aestivum) varieties and breeding lines for quality traits

Thirteen released and two elite genotypes of wheat (Triticum aestivum L.) from North-West Plain Zone of India were subjected to biochemical and molecular characterization to study the genetic variability, their grouping and identification using a combination of biochemical and molecular markers during 2014-16 at Department of Genetics and Plant Breeding, COA, CCSHAU, Hisar. HD 2967, WH 1142 and WH 1080 were overall better performing for protein content, sedimentation value, hectolitre weight and grain appearance. A total of 99 and 117 polymorphic bands were detected using ISSR and SSR primers, respectively. The similarity indices for SSR ranged from 0.64 to 0.77 while ISSR showed the range of 0.63-0.92. Present study could be effectively utilized in DNA fingerprinting, identification of wheat varieties and elite lines for bio-chemical traits and determination of seed purity.

Effect of culture media, auxins and genotypes on plantlet regeneration from oil palm (Elaeis guineensis Jacq.) zygotic embryos through somatic embryogenesis

Aim: This study evaluated efficient culture media for the regeneration of elite material through somatic embryogenesis from oil palm zygotic embryos. Methodology: For callus induction, zygotic embryos of four elite genotypes (G1-264T, G2-238DX17P, G3-37DX17P and G4-237T) were cultured on three basal media (Y3, MS and N6) with different auxin 2 mg l-1 (Picloram, 2,4-D and Dicamba) combinations. Subculture was made every month for three passages. It evaluated various callus characters. The embryogenic calli from callus induction media were transferred to the embryo maturation medium and subcultured until the polyembryoids formed. For shoot and root formation, somatic embryo clumps were transferred into regeneration media. In-vitro plantlets with well-grown roots were hardened in pots for six weeks and assessed clonal fidelity using polymorphic SSR primers. Results: Among the treatments, calli from N6+2,4-D, Y3+2,4-D and N6+Picloram showed the highest embryogenic callus potential. G4-237T induced more embryogenic calli (32.982) among genotypes, which was on par with G1-264T (24.196). Embryogenic calli grown on N6 media with Dicamba showed the highest proliferation rate (1.141). After 60 days of culture on regeneration media, the highest number of plantlets per somatic embryogenic clump was obtained from G1-264T on N6 media supplemented with Dicamba. Interpretation: Culture media salt concentration showed a significant difference among media by causing perturbations of auxin flow during somatic embryogenesis affecting callus induction, proliferation and plantlet regeneration. This may be useful for standardizing the genotype-specific regeneration media in oil palm.

Virulence and SSR Diversity of Brown Planthopper (Nilaparvata lugens) Adapted on Differential Rice Host Varieties

Brown planthopper biotype 1, 2, 3 and a representative field population are required for resistance screening of promising rice lines in Indonesia, but the current biotype stocks has shown deviation in virulence patterns. The objectives of this study were to develop a set of brown planthopper populations with differential virulence and to investigate their genetic variability using SSR marker. Females originated from two field populations were selected on variety Mudgo (carries Bph1 gene) or ASD7 (bph2 gene) using honeydew excretion as the virulence parameter. Selection cycles resulted in population T, M, A, and R, which was raised and adapted on variety TN1 (carries no Bph gene), Mudgo, ASD7, and Rathu Heenathi (Bph3, Bph17), respectively. Population R was the most virulent as expected and can be used to represent a field population, but the remaining populations still showed high virulence level. AMOVA and PCoA results based on analysis with 38 SSR primer pairs revealed partial genetic separation among populations, with population R was the most genetically distant from the remaining populations. The desired virulence character of the remaining populations is expected could be achieved after further selection and prolonged adaptation on their respective hosts.

Screening of Mango Landraces for Polyembryony and Confirmation of Seedling Origin using Microsatellite Markers

Background: The introduction of polyembryonic rootstocks in the area of propagation is of great importance since they produce one zygotic and several nucellar plantlets. Proper identification of sexual embryo from each hybrid seed is necessary in order to preserve only the nucellar seedlings, which would help to maintain the rootstock’s genetic characteristics as well as to overcome the major constraints in the area of fruit breeding especially in hybridization programme by eliminating the nucellar ones to advanced generations. Contrasting reports exists regarding the vigour of zygotic seedlings of polyembryonic mango genotypes. It is necessary to identify/ distinguish the zygotic seedling from the nucellar population at an early stage, for which, microsatellite analysis could be a reliable tool. Methods: The experiment was laid out in completely randomized design (CRD) with 20 treatments replicated thrice. The twenty local mango landraces from Thiruvananthapuram (Kerala) were screened for polyembryony and were geo-referenced. Germination studies were conducted. Microsatellite analysis of all the plantlets from two varieties which exhibited the highest polyembryony were done using SSR primers and their banding patterns were compared with those of their respective mother plants. Result: Out of twenty mango varieties screened, seventeen were polyembryonic. Kappa Manga recorded the highest germination, germination index and seedling vigour index-I. Kotookonam Varikka recorded the highest polyembryony and followed by Kochu Kilichundan. Microsatellite analysis revealed that all the seedlings obtained from the respective stones of Kotookonam Varikka and Kochu Kilichundan had identical SSR profile to the mother plant, which indicated nucellar origin of seedlings having similar genetic composition to the mother plant.

Analysis of Genetic Diversity and Population Structure in Bitter Gourd (Momordica charantia L.) Using Morphological and SSR Markers.

The present investigation was carried out using 51 diverse bitter gourd accessions as material for studying genetic diversity and relatedness using morphological and SSR markers. A wide variation was observed for morphological traits like the number of days to the first female flower anthesis (37.33–60.67), the number of days to the first fruit harvest (47.67–72.00), the number of fruits/plant (12.00–46.67), fruit length (5.00–22.23 cm), fruit diameter (1.05–6.38 cm), average fruit weight (20.71–77.67 g) and yield per plant (513.3–1976 g). Cluster analysis for 10 quantitative traits grouped the 51 accessions into 6 clusters. Out of 61 SSR primers screened, 30 were polymorphic and highly informative as a means to differentiate these accessions. Based on genotyping, a high level of genetic diversity was observed, with a total of 99 alleles. The polymorphic information content (PIC) values ranged from 0.038 for marker BG_SSR-8 to 0.721 for S-24, with an average of 0.429. The numbers of alleles ranged from 2 to 5, with an average of 3.3 alleles per locus. Gene diversity ranged from 0.04 for BG_SSR-8 to 0.76 for S-24, showing a wide variation among 51 accessions. The UPGMA cluster analysis grouped these accessions into 3 major clusters. Cluster I comprised 4 small, fruited accessions that are commercially cultivated in central and eastern India. Cluster II comprised 35 medium- to long-sized fruited accessions, which made up an abundant and diverse group. Cluster III comprised 11 long and extra-long fruited accessions. The polymorphic SSR markers of the study will be highly useful in genetic fingerprinting and mapping, and for association analysis in Momordica regarding several economic traits.

Development of genome-wide SSR markers in rapeseed by next generation sequencing.

Rapeseed (Brassica napus L.) is an important oil crop with a huge genome. This study used next generation sequencing technology to develop SSR markers in rapeseed. A total of 213,876 sequence reads were obtained in 58.8Mb. For these reads, 21,523 SSRs were recovered from 18,575 microsatellites sequences and 8,964 SSR primer pairs were identified. Di- and mono-nucleotides were the most abundant, accounting for 47.5% and 30.7% of all SSRs, respectively. A total of 8,776 SSRs were designed from contigs and 100 SSR primers were tested for validation of SSR locus amplification. Nearly all (94%) of the markers were found to produce clear amplicons and to be reproducible. For these markers, forty-three SSRs showed polymorphic bands in eight rapeseed accessions. Thirty-four SSRs were then applied to 78 rapeseed accessions from China to evaluate the genetic diversity. Result showed that the allele number varied from two to seven, with a mean value of 3.59. The effective allele number of ranged from 1.14 to 3.25, with an average of 2.09. The average values of observed heterozygosity and expected heterozygosity were 0.54 and 0.49, respectively. The Nei's gene diversity varied from 0.12 to 0.69, with a mean value of 0.48. Resulting of the markers testing showed that the identified genome-wide SSRs were useful in rapeseed genetic studies, including genetic diversity, QTL mapping and marker-assisted selection for breeding.

De novo transcriptome assembly and EST-SSR markers development for Zelkova schneideriana Hand.-Mazz. (Ulmaceae).

The online version contains supplementary material available at 10.1007/s13205-021-02968-5.

Genetic diversity of sugar palm (Arenga pinnata) derived from nine regions in Indonesia based on SSR markers

Abstract. Rinawati DY, Reflinur, Dinarti D, Sudarsono. 2021. Genetic diversity of sugar palm (Arenga pinnata) derived from nine regions in Indonesia based on SSR markers. Biodiversitas 22: 3749-3755. Sugar palm (Arenga pinnata (Wurmb) Merr.) has an important economic and conservation value. Indonesia has genetic diversity potential of sugar palm, considering the widespread distribution of sugar palm in Indonesia which has variations in geographical type. This study aims to determine the diversity and relationship of sugar palm from nine regions in Indonesia based on SSR markers. The genetic material consists of 141 sugar palm accessions derived from Bangka, Lampung, Lebak, Bogor, Tasikmalaya, Brebes, Gowa, Bombana, and Muna. Nine pairs of SSR primers were used for genotyping. The highest and lowest genetic diversity was found in the Bangka and Muna populations, respectively. The genetic diversity within a population (79%) was higher than the genetic diversity between populations (21%). The genetic distance between Bangka and Lebak is the closest (0.033), while between Lampung and Muna is the farthest (0.283). The accession relationship is divided into three major clusters. Clusters 1 consisted of Bangka, Lampung, Bogor, Tasikmalaya, Brebes, Gowa, Bombana and Muna accessions. Cluster 2 consisted of Bangka, Lampung, Lebak, Bogor, Tasikmalaya, Brebes, and Gowa accessions. Cluster 3 consisted of Bangka, Lebak, Brebes, Tasikmalaya, and Gowa accessions. Accession clustering does not show a typical relationship pattern based on geographic location.

Inheritance analysis and identification of SSR markers associated with fusarium wilt resistance in melon

ABSTRACT Fusarium wilt (FW), caused by Fusarium oxysporum Schlechtend f. sp. melonis (Leach & Currence) Snyd. & Hans (Fom) is a substantial threat to muskmelon (Cucumis meloL.) cultivation around the world. Identification of new sources of resistance and transfer of the resistance genes to elite cultivars is the effective breeding strategy to reduce the FW-associated crop losses in muskmelon. In the present study, inheritance studies and molecular mapping for FW resistance were conducted in a muskmelon inbred line KP4HM-15 with FW resistance introgressed from snapmelon. Inheritance studies in the F2 population derived from the cross Punjab Sunehri//KP4HM-15 indicated that FW resistance in KP4HM-15 is governed by a single dominant gene. Bulked segregant analysis was conducted to map the Fom-5(t) gene in KP4HM-15 using 527 SSR primers. Four primer pairs, CMCTN35, DM0096, CSWCTT02, and ECM181, were found to show differential polymorphism in the resistant and susceptible bulks and were analysed in the whole of the population. Two SSR markers, DM0096 and CSWCTT02, mapped close to the Fom-5(t) gene at a genetic distance of 1.4 and 2.5 cM, respectively. These markers can potentially be used to transfer Fom-5(t) in elite muskmelon genotypes through marker-assisted backcross breeding until more tightly linked markers are available.