Simple Sequence Repeat Primers Research Articles

Molecular characterization of some currants (Ribes species) from Türkiye using inter-primer binding site (iPBS) and simple sequence repeat (SSR) markers

AbstractThis study was carried out to determine the genetic relationships and variation levels among currant genotypes and cultivars in Türkiye. Ten currant cultivars and one hybrid Jostaberry cultivar, which are breeding products of Europe but are best known and have high commercial value in Türkiye, were used. One black-fruited currant cultivar from Türkiye was included. In addition, 14 currant genotypes naturally grown in Türkiye were used. In this study, 14 retrotransposon-based inter-primer binding site (iPBS) and 10 microsatellite-based simple sequence repeat (SSR) markers were used for a total of 26 currant samples. By evaluating the allele profiles obtained from PCR and capillary electrophoresis with iPBS and SSR primers, the size range, average number of alleles, total number of alleles, number of polymorphic alleles, polymorphism rate, heterozygosity, polymorphism information content, marker index and discriminating power were determined successfully. In addition, unweighted pair group method with arithmetic mean (UPGMA) dendrograms and principal coordinate analysis (PCoA) were constructed and performed. The amounts of amplification product produced using iPBS and SSR markers for currant species with different fruit colours were also compared. In this study, it was proven that the level of genetic relatedness among currant cultivars and genotypes can be determined with high accuracy by iPBS markers. Moreover, the use of iPBS markers in currants was performed for the first time in this study. On the other hand, with the preferred SSR markers, the level of genetic relatedness among currants was successfully determined.

Development of SSRs Based on the Whole Genome and Screening of Bolting-Resistant SSR Marker in Brassica oleracea L.

Simple sequence repeats (SSRs), also known as microsatellites, stand out as the most crucial molecular markers in both animals and plants owing to their high polymorphism, extensive information content, ease of detection through polymerase chain reaction (PCR) assays, and widespread distribution across the genome. In this study, a total of 125,443 SSR loci were identified from the whole-genome sequence of B. oleracea, and 82,948 primer pairs for SSR have been designed. Furthermore, each primer pair is designated with a unique identifier (ranging from BolSSR00001 to BolSSR82984). Our findings indicated that certain markers within them could be transferred to other cruciferous crops. In addition, a total of 336 pairs of SSR primers have been used to screen the polymorphism between the bolting-resistant and bolting-easy gene pools. After the test of verification with F2 generation individual plants, we obtained an SSR dominant marker, BolSSR040196, linked with bolting-resistant locus in cabbage, and the genetic distance between this SSR marker and the bolting-resistant locus was 10.69 cM. Moreover, BolSSR040196 is located on the C05 chromosome with a CT motif, characterized by a repeat of 9 in bolting-easy plants and 11 in bolting-resistant plants. Haplotype analysis showed that the correct prediction rate reached 82.35%. The BolSSR040196 marker can be used in marker-assisted selection (MAS) breeding, offering a straightforward and efficient approach for bolting-resistant cabbage breeding in the future.

[Genetic background of lily germplasm resources based on SSR markers].

To study the genetic background of lily (Lilium spp.) germplasm resources, and accurately evaluate and select excellent germplasm for genetic improvement of lily, we analyzed the genetic background of 62 lily germplasm accessions from 11 provinces of China by using simple sequence repeat (SSR) molecular markers. The results showed that 15 out of 83 pairs of lily SSR primers were polymorphic. A total of 157 allelic loci were amplified, with the number of alleles per locus ranging from 5 to 19 and the average number of effective alleles per locus being 4.162 8. The average observed heterozygosity and expected heterozygosity were 0.228 2 and 0.694 1, respectively. The average polymorphic information content was 0.678 8. The average Nei's diversity index and Shannon's information index were 0.694 1 and 1.594 9, respectively, indicating that the tested lily germplasm had high genetic diversity. The 62 germplasm accessions were classified into 5 groups by the unweighted pair group method with arithmetic mean (UPGMA) and into 3 groups by the principal component analysis. The two analyses revealed a geographic correlation among different groups. The majority of lily germplasm accessions from the same source tended to cluster together. The population structure analysis classified the lily accessions into 4 populations and 1 mixed population. The above results provide a theoretical basis and genetic resources for the precise identification and breeding of lily germplasm resources.

Identification and characterization of functionally relevant SSR markers in natural Dalbergia odorifera populations

BackgroundDalbergia odorifera is a rare and precious rosewood specie, which is valued for its amber tones, abstract figural patterns, and impermeability to water and insects. However, the information on genetic diversity and marker-assisted selection breeding of D. odorifera is still limited. Simple sequence repeat (SSR) markers are an ideal tool for genetic diversity analysis and marker-assisted molecular breeding for complex traits.ResultsHere, we have developed SSR markers within candidate genes and used them to explore the genetic diversity among D. odorifera germplasm resources. A total of 635 SSR loci were identified. The proportions of mono-, di- and tri-nucleotide repeat motifs were 52.28%, 22.99% and 21.42%, respectively. From these, a total of 114 SSR primers were synthesized, of which 24 SSR markers displayed polymorphism (polymorphic information content (PIC) > 0.25). Subsequently, these polymorphic markers were used for the genetic diversity analysis of 106 D. odorifera individuals from 11 natural populations. According to the genetic diversity analysis of D. odorifera natural populations, the average observed heterozygosity (Ho) was 0.500, the average expected heterozygosity (He) was 0.524, and the average Shannon’s information index (I) was 0.946. These indicated that the natural populations had moderate genetic diversity. AMOVA analysis showed that 5% of the total variation was within the individuals of a population, whereas 95% of the variation was among the individuals of the populations, indicating a high degree of genetic variation between populations. On the basis of their genetic structures, these populations could be divided into four groups.ConclusionsOur study provides important experimental resources for genetic studies and assists in the program of molecular breeding of D. odorifera wood formation.

Molecular delineation and genetic diversity of Fusarium species complex causing tea dieback in India and their sensitivity to fungicides

Fusarium dieback is a prevalent disease of tea (Camellia sinensis (L.) Kuntze) in India and China. The pathogen affects plucking shoots and causes complete crop loss under favourable conditions. Although, other fungal diseases that infect tea were widely explored for pathogen identification and characterization, little is known about Fusarium dieback in major tea growing regions of India. Therefore, in this study 24 isolates of Fusarium species causing dieback on tea in 24 tea gardens of Assam and North Bengal were isolated and characterized through micro-morphological and molecular analysis using multi-locus DNA sequences to determine whether multiple species of Fusarium are responsible for dieback disease. Molecular analysis revealed the association of four species of Fusarium, namely F. concentricum, F. solani, F. fujikuroi, and F. oxysporum. The most dominant species was F. concentricum (62.5%), whereas the occurrence frequencies of F. solani, F. fujikuroi, and F. oxysporum were 20.8%, 12.5%, and 0.04%, respectively. All Fusarium isolates were pathogenic to tea leaves when individually and co-inoculated, with F. concentricum HPE0F10 and NHT0F17 exhibiting 19.3 mm lesion length being the most virulent isolates. The growth rate of the pathogen was positively correlated with lesion lengths (P < 0.001). Co-infection of two or multiple Fusarium species enhanced the disease severity and quicker disease development compared to single inoculation. All the isolates showed diverse genetic backgrounds based on Inter Simple Sequence Repeat (ISSR) primers and principal coordinate analysis. In fungicide screening test, all isolates except HPE0F10, NDS0F18, and SNY0F22 were insensitive to M1 fungicide (copper oxychloride). Isolates GNP0F8 and LKW0F16 were insensitive to MBC + M3 (Carbendazim + Mancozeb), whereas HPE0F10, NLG0F11, IND0F13, and NLW0F19 were insensitive to DMI (respective each two to Hexaconazole and Valextra) fungicides. Notably, all the isolates were susceptible to propiconazole. Further, ISSR segregated isolates insensitive to MBC + M3 and M1 fungicides into two separate clusters, representing their distinctiveness. The fungicide screening results indicate that fungicide resistance can be managed by rotating different modes of action of fungicides against multiple Fusarium species causing tea dieback.

Directional Breeding Generates Distinct Genetic Diversity in Hybrid Turf Bermudagrass as Probed with Simple Sequence Repeat Markers

Bermudagrass (Cynodon spp.) is a drought-resistant warm-season turfgrass adapted to the southern and transitional zones in the United States. Multiple hybrid cultivars have been developed and released for use as turfgrass, and others are still undergoing development. Increasing genetic diversity of commercial cultivars is vital to stress tolerance. A DNA profiling study of 21 experimental selections from the Oklahoma State University turfgrass breeding program and 11 cultivars was conducted using 51 simple sequence repeat primer pairs across the bermudagrass genome. A pairwise genetic relationship analysis of the genotypes using 352 polymorphic bands showed genetic similarity coefficients ranging from 0.59 to 0.89. The average pairwise population differentiation values were 0.012 for the 11 cultivars and 0.169 for the 21 selections. A cluster analysis using the unweighted paired group with the arithmetic average method grouped the entries into six clusters. A correlation analysis identified different levels of pairwise genetic relationships among the entries that largely reflected parental relationship. Directional breeding and selection for cold hardiness or drought resistance created progeny that had distinct genetic diversity in the tested bermudagrasses. It is evident that an increase in genetic diversity of the existing cultivar pool with the release of one or more experimental selections for commercial use will strengthen and improve bermudagrass systems.

Analysis of Genetic Variability of Potato Induced Mutants Using Simple Sequence Repeat (SSR) Markers

Aims: Potato (Solanum tuberosum L.) is ranked fourth amongst the world’s major stable food crop and second in Kenya after maize and thus plays a vital role in food and nutrition security and sustainable development. Despite its importance, potato production in Kenya is still low due to abiotic and biotic constraints. Traditional breeding and improvement have been difficult due to the narrow genetic diversity of the crop owed to the complex tetrasomic inheritance patterns. Induced mutation has been used to generate genetic variations in potato from which desired putative mutants are selected. In most cases the level of genetic variability is not known. The objective of this study was to determine the genetic variability of potato mutants using SSR markers. Study Design: The study involved three potato varieties; Asante, Kenya Mpya and Kenya Sherekea which are high yielding and commercially grown varieties in Kenya. Place and Duration of Study: Sample: They were irradiated at different dose rates of gamma rays at Co60 source at FAO/IAEA laboratories, Seiberdorf Austria in 2014. A total of 163 mutants were advanced to M1V4 generation at the University of Eldoret between 2015 and 2018. Methodology: Genomic DNA was extracted to assess the diversity with 20 SSRs markers. Results: All the 20 SSR primers were polymorphic with 6-19 bands amplified per primer and marker STM5127 showed the highest allele number (19) using PowerMarker software. The STRUCTURE analysis suggested that the potato mutants were clustered into six sub-populations based on the unweighted pair-group method of arithmetic averages (UPGMA) thus, the accessions were divided into three major clusters. Analysis of molecular variance (AMOVA) indicated that 8.6% of total molecular variance was attributed to diversity among sub-populations, while 91.4% of variance was associated with differences within sub-populations. Conclusion: This study highlights the most comprehensive investigation of the genetic diversity and population structure of potato mutants, and provides valuable information for genetic improvement, and systematic utilization.

Genetic variability and population divergence of Rhododendron platypodum Diels in China in the context of conservation

Genetic diversity in endangered species is of special significance in the face of escalating global climate change and alarming biodiversity declines. Rhododendron platypodum Diels, an endangered species endemic to China, is distinguished by its restricted geographical range. This study aimed to explore genetic diversity and differentiation among its populations, gathering samples from all four distribution sites: Jinfo Mountain (JFM), Zhaoyun Mountain (ZYM), Baima Mountain (BMM), and Mao’er Mountain (MEM). We employed 18 pairs of Simple Sequence Repeat (SSR) primers to ascertain the genetic diversity and structural characteristics of these samples and further utilized 19 phenotypic data points to corroborate the differentiation observed among the populations. These primers detected 52 alleles, with the average number of observed alleles (Na) being 2.89, the average number of effective alleles (Ne) being 2.12, the average observed heterozygosity (Ho) being 0.57, and the expected heterozygosity (He) being 0.50. This array of data demonstrates the efficacy of the primers in reflecting R. platypodum’s genetic diversity. SSR-based genetic analysis of the populations yielded Ho, He, and Shannon index (I) values ranging from 0.47 to 0.65, 0.36 to 0.46, and 0.53 to 0.69, respectively. Notably, the ZYM population emerged as the most genetically diverse. Further analysis, incorporating molecular variance, principal component analysis, UPGMA cluster analysis, and structure analysis, highlighted significant genetic differentiation between the Chongqing (BMM, JFM, ZYM) and Guangxi (MEM) populations. Morphological data analysis corroborated these findings. Additionally, marked genetic and morphological distinctions were evident among the three Chongqing populations (BMM, JFM, and ZYM). This suggests that, despite the observed regional differentiation, R. platypodum’s overall genetic diversity is relatively constrained compared to other species within the Rhododendron genus. Consequently, R. platypodum conservation hinges critically on preserving its genetic diversity and protecting its distinct populations.

Short Communication: Analysis of simple sequence repeats variations for the assessment of genotypic diversity in Centella asiatica (L.) Urb.

Abstract. Maruzy A, Susandarini R. 2024. Short Communication: Analysis of simple sequence repeats variations for the assessment of genotypic diversity in Centella asiatica (L.) Urb. Biodiversitas 25: 734-740. Centella asiatica (L.) Urb. is a perennial plant widely known as herbal ingredient in traditional and modern medicine, as well as in the cosmetics industry. This herbaceous species grows in various types of habitat, and is known for its morphological variations among populations, indicating the existence of genotypic variation. In this study, the assessment of genotypic diversity using simple sequence repeats (SSR) was carried out on C. asiatica accessions from 32 populations collected from eight mountains in Central Java. Representative individuals from four populations grow at different altitudes were sampled in each mountain. Amplification of genomic DNA using the SSR primer mCaCIR002 produced a single amplicon of 180 bp. The results of SSR analysis showed that there is high genotypic diversity in C. asiatica which is characterized by variations in the number of microsatellite repeats of (CT)10 to (CT)19. Variations in the number of repeats are mainly caused by insertions and deletions in DNA sequences at the positions 102-145. Haplotype analysis also showed high genetic diversity within and between populations of C. asiatica, indicated by the haplotype diversity of 1.00 in all populations. The haplotype network analysis indicated that there was no gene flow between the four populations at each mountain where C. asiatica populations grow. The results of this study provide evidence for the role of SSR as a suitable molecular marker for studying genotypic diversity at the intraspecific level, and confirming the morphological variability in this species.

Association mapping analysis for cultivated and weedy types of Perilla crop collected from South Korea using morphological characteristics and SSR markers

There are two cultivated and weedy types of Perilla crop (TCWTPC), and they are widely distributed and cultivated in East Asia, especially in South Korea and Japan. The objective of this study is to create simple sequence repeat (SSR) markers linked to morphological traits that show differences between accessions of the TCWTPC using recently designed SSR primer sets in Perilla crop. Genetic diversity within 52 accessions of the TCWTPC, gathered from South Korea, was assessed using 28 novel Perilla SSR primer sets. Based on the assessment, a collection of 28 Perilla SSR primer sets were shown to exhibit polymorphism and yielded a total of 142 alleles across the 52 accessions of the TCWTPC. Through inspection of a phylogenetic tree and population structure, the 52 accessions of the TCWTPC were classified into three major groups. Although most accessions of the TCWTPC were relatively clearly distinguished, SSR markers failed to distinguish several accessions belonging to the two weedy types of the Perilla crop. By using an association mapping analysis (AMA) of the 28 Perilla SSR markers and seven morphological characteristics in the 52 TCWTPC accessions, we detected that three of the Perilla SSR markers (KNUPF134, KNUPF137, KNUPF149) were associated with plant and seed characteristics. The novel SSR primer sets developed in Perilla crop should be useful in AMA for assessing genetic diversity and relationships between and within TCWTPC accessions, and this information will be helpful for genetic mapping in breeding programs for Perilla crop.

Genetic diversity among genotypes of Parkia platycephala (Benth.), a typical tree of northeastern Brazil

Parkia platycephala (Fabaceae) is a useful tree. The leaves are rich in protein, fiber and minerals and are good quality, low-cost fodder for animals, making it a great option for animal feed during the dry season in Northeast Brazil. In addition, wood is used in small buildings and as fuel. With the aim of exploring genetic variation within the species, we have assessed the molecular and morpho-agronomic characteristics of 10 accessions (F1 - F10) of the species originating from the same location and maintained in the Forage Collection at Embrapa Meio Norte. Clustering analyses based on the amplicons generated by 12 inter simple sequence repeat (ISSR) primers and on 36 qualitative/quantitative markers separated the accessions into two groups, the constituents of which were dependent on the characteristics considered. The most divergent genotypes according to ISSR analysis were F3 and F8. Morpho-agronomic analysis identified F2 and F7 as the most divergent, while the traits that contributed most (36.5%) to total diversity were, in order of importance, mature inflorescence length, stem length, immature inflorescence width, seed weight and pedicel length. The results revealed that there is sufficient genetic variability among the studied accessions. These accessions with greater diversity are candidates for actions that promote the conservation, domestication and genetic improvement of the species.

Genetic diversity and population structure of wild and cultivated apricots collected from Kyrgyzstan

AbstractCentral Asia contains rich genetic resources for apricots. With its mountainous geography, Kyrgyzstan is the country where wild and cultivated apricot forms are preserved. The present study revealed genetic diversity and population structures in 91 apricot accessions from different regions of Kyrgyzstan. Fifteen simple sequence repeat (SSR) primers were used, capillary electrophoresis was performed, and the number of alleles per primer ranged from 4 to 11. A significant level of variation was determined among apricots originating from Kyrgyzstan. In the resulting dendrogram, all apricot accessions were divided into five groups, and the materials belonging to the northern and southern regions were grouped separately. At the same time, the population structure of apricots was analyzed. Accordingly, individuals have 0.80 or more membership coefficients; therefore, they are likely pure and non-admixed. There were 34 apricot accessions that have been determined as pure. The remaining 53 individuals had varying membership coefficients and were likely to be admixed by at least two subpopulations. This study is the most comprehensive research on Kyrgyzstan-origin apricots, providing vital information on their identification, preservation, and use in future research.

[Development and application of SSR markers of Saposhnikovia divaricata based on transcriptome].

Transcriptome sequencing was employed to mine the simple sequence repeat(SSR) locus information of Saposhnikovia divaricata and design specific primers, which aimed to provide a basis for the research on the genetic diversity of S. divaricata germplasm resources. The seed purity, 1 000-seed weight, germination rate, and seed vigor were determined. MISA was used to obtain the SSR locus information from 12 606 unigene longer than 1 kb in the transcriptome database. Forty-three pairs of SSR primers designed in Primer 3 were used to analyze the polymorphism of 28 S. divaricata samples of different sources. The results showed that there were differences in the seed purity, 1 000-seed weight, germination rate, vigor, and seed length and width among S. divaricata samples of different sources. Particularly, the germination rate and seed vigor had significant differences, and HB-ZJK1, NMG-CF4, NMG-BT, NMG-HLE1, and NMG-CF2 had significantly higher 1 000-seed weight, germination rate, and seed vigor than the samples of other sources. Among the 86 233 unigene, 12 606(14.62%) unigene contained 15 958 SSR loci, with one SSR locus every 5 009 bp on average. The SSR loci were mainly single nucleotide and dinucleotide repeats, which were dominated by G/C and TC/AG, respectively. All the primers were screened by using 28 S. divaricata sample from different habitats, and the primers corresponding to the amplification products with clear bands and stable polymorphism were obtained. The clustering results of the biological characteristics and genetic diversity of the 28 S. divaricata samples were basically consistent, and the samples of the same origin(HB-AG1, HB-AG2, HB-ZJK1, and HB-ZJK2) generally gathered together and had close genetic relationship. The SSRs in S. divaricata transcriptome has high frequency, rich types, and high polymorphism, which provides candidate molecular markers for the germplasm identification, genetic map construction, and molecular-assisted breeding.

Comprehensive Tools of Alkaloid/Volatile Compounds-Metabolomics and DNA Profiles: Bioassay-Role-Guided Differentiation Process of Six Annona sp. Grown in Egypt as Anticancer Therapy.

Trees of the Annona species that grow in the tropics and subtropics contain compounds that are highly valuable for pharmacological research and medication development and have anticancer, antioxidant, and migratory properties. Metabolomics was used to functionally characterize natural products and to distinguish differences between varieties. Natural products are therefore bioactive-marked and highly respected in the field of drug innovation. Our study aimed to evaluate the interrelationships among six Annona species. By utilizing six Start Codon Targeted (SCoT) and six Inter Simple Sequence Repeat (ISSR) primers for DNA fingerprinting, we discovered polymorphism percentages of 45.16 and 35.29%, respectively. The comparison of the profiles of 78 distinct volatile oil compounds in six Annona species was accomplished through the utilization of GC-MS-based plant metabolomics. Additionally, the differentiation process of 74 characterized alkaloid compound metabolomics was conducted through a structural analysis using HPLC-ESI-MSn and UPLC-HESI-MS/MS, and antiproliferative activities were assessed on five in vitro cell lines. High-throughput, low-sensitivity LC/MS-based metabolomics has facilitated comprehensive examinations of alterations in secondary metabolites through the utilization of bioassay-guided differentiation processes. This has been accomplished by employing twenty-four extracts derived from six distinct Annona species, which were subjected to in vitro evaluation. The primary objective of this evaluation was to investigate the IC50 profile as well as the antioxidant and migration activities. It should be noted, however, that these investigations were exclusively conducted utilizing the most potent extracts. These extracts were thoroughly examined on both the HepG2 and Caco cell lines to elucidate their potential anticancer effects. In vitro tests on cell cultures showed a significant concentration cytotoxic effect on all cell lines (HepG2, HCT, Caco, Mcf-7, and T47D) treated with six essential oil samples at the exposure time (48 h). Therefore, they showed remarkable antioxidant activity with simultaneous cytotoxic effects. In total, 50% and 80% of the A. muricata extract, the extract with the highest migratory activity, demonstrated a dose-dependent inhibition of migration. It was strong on highly metastatic Caco cells 48 h after treatment and scraping the Caco cell sheet, with the best reduction in the migration of HepG2 cells caused by the 50% A. reticulata extract. Also, the samples showing a significant IC50 value showed a significant effect in stopping metastasis and invasion of various cancer cell lines, making them an interesting topic for further research.

Development and utility of SSR markers based on Brassica sp. whole-genome in triangle of U.

Simple sequence repeats (SSR), also known as microsatellites, are crucial molecular markers in both animals and plants. Despite extensive previous research on SSRs, the development of microsatellite markers in Brassica crops remains limited and inefficient. Krait software was used to identify microsatellites by genome-wide and marker development based on three recently sequenced basic species of Brassica crops in the triangle of U (Brassica rapa, B. nigra and B. oleracea), as well as three allotetraploids (B. juncea, B. napus and B. carinata) using public databases. Subsequently, the primers and the characteristics of microsatellites for most of them were accordingly designed on each chromosome of each of the six Brassica species, and their physical locations were identified,and the cross-transferability of primers have been carried out. In addition, a B-genome specific SSR marker was screened out. A total of 79341, 92089, 125443, 173964, 173604, and 222160 SSR loci have been identified from the whole genome sequences of Brassica crops within the triangle of U crops, B. rapa (AA), B. nigra (BB), B. oleracea (CC), B. napus (AACC), B. juncea (AABB) and B. carinata (BBCC), respectively. Comparing the number distribution of the three allotetraploid SSR loci in the three subgenomes AA, BB and CC, results indicate that the allotetraploid species have significant reduction in the number of SSR loci in the genome compared with their basic diploid counterparts. Moreover, we compared the basic species with their corresponding varieties, and found that the microsatellite characters between the allotetraploids and their corresponding basic species were very similar or almost identical. Subsequently, each of the 40 SSR primers was employed to investigate the polymorphism potential of B. rapa (85.27%), B. nigra (81.33%) and B. oleracea (73.45%), and B. rapa was found to have a higher cross-transfer rate among the basic species in the triangle of U. Meanwhile, a B-genome specific SSR marker, BniSSR23228 possessing the (AAGGA)3 sequence characteristics was obtained, and it located in chromosome B3 with a total length of 97 bp. In this study, results suggest that the pattern of distribution may be highly conserved during the differentiation of basic Brassica species and their allotetraploid counterparts. Our data indicated that the allotetraploidization process resulted in a significant reduction in SSR loci in the three subgenomes AA, BB and CC. The reasons may be partial gene dominated chromosomal homologous recombination and rearrangement during the evolution of basic diploid species into allotetraploids. This study provides a basis for future genomics and genetic research on the relatedness of Brassica species.

SSR-marker Assisted Evaluation of Genetic Diversity in Greengram [Vigna radiata (L.) Wilcezk]

Background: Assessing genetic diversity in greengram is a prerequisite for its genetic improvement for yield and quality. DNA markers such as simple sequence repeats (SSRs) have been preferred in this crop for the analysis of genetic diversity because SSR markers are locus-specific, widely dispersed throughout the genome, highly polymorphic due to variation in repeat units, highly informative because of co-dominant nature, high reproducibility and ease of assay by PCR (Polymerase chain reaction). The current study aimed to study the genetic diversity among the mutants of greengram at the molecular level using SSR markers. Methods: In this study, twenty-five SSR markers were used to analyze the genetic diversity amongst twenty mutant genotypes of greengram along with their parents. Genomic DNA was isolated from the leaves by using the standard CTAB DNA extraction method. Then DNA purification and PCR amplifications were carried out. The genetic variability and diversity among genotypes was examined by assessing the scoring of the amplified bands by SSR -PCR amplification. Result: Seventeen SSR primers generated 102 polymorphic bands with an average of the six polymorphic bands per primer. The number of alleles per locus ranged from four to nine. The size of amplification product varied for each primer and the range found to be 100bp to 2000bp. The mean polymorphic information content (PIC) value for SSR markers was found to be 0.6335. The value of Jaccard’s similarity coefficient had ranged from 0.07-0.70 with an average value of 0.38. The dendrogram constructed on SSR molecular markers data through the unweighted pair group method with arithmetic averages (UPGMA) method had enabled grouping of the genotypes into thirteen clusters. The results indicate the usefulness of SSR markers in the assessment of genetic variability and diversity among the mutant genotypes of greengram.

Evaluation for Parental Polymorphism and Identification of Microsatellites Linked to Drought Tolerance in Rice (Oryza sativa L.)

Aims: To identify the ready-to-use polymorphic microsatellite markers associated with drought tolerance for marker-assisted backcross breeding through a polymorphism survey between Jyothi and Chuvannamodan rice varieties.&#x0D; Place and Duration of Study: Centre for plant biotechnology and molecular biology, Thrissur, Kerala, India, during January to May 2023.&#x0D; Methodology: The genomic DNA of Jyothi and Chuvannamodan was isolated by following CTAB (Cetyltrimethyl Ammonium Bromide) method with modification. Isolated DNA from both varieties was subjected to PCR amplification using 208 Simple Sequence Repeats (SSRs) primer pairs distributed in 12 chromosomes. The amplified PCR products were electrophoresed in 3% agarose gel and separated fragments were visualized and documented in the Gel Documentation System. Different allele sizes produced by the same SSRs primers between two varieties are identified as polymorphic markers. Polymorphism per-cent was calculated, and frequency distribution and chromosome distribution of polymorphic markers were analyzed.&#x0D; Results: A total of 208 SSR primers were surveyed for the parental polymorphism. Out of which, 85 SSR primers exhibited clear polymorphism between Jyothi and Chuvannamodan and the remaining 123 were monomorphic primers. The amplicon size ranged from 83bp (RM430) to 495bp (RM18919) among the different primers. The survey revealed maximum parental polymorphism on chromosome 4 (69.23%), followed by chromosome 5 (64.28%), and minimum polymorphism on chromosome 8 (21.73%). The average per cent of polymorphism between the parents was 40.86%. Among the 85 polymorphic markers, 66 had dinucleotide repeats, 17 had trinucleotide repeats, and 1 had tetranucleotide repeats.&#x0D; Markers with dinucleotide repeats showed higher level of polymorphism. A group of 28 polymorphic markers were identified to be linked with traits including root-related traits, grain yield, leaf rolling and leaf drying under drought conditions.&#x0D; Conclusion: The polymorphic markers identified in the present study form the basis for tagging drought-tolerant QTLs/genes, fine mapping of those genes, and subsequently in marker-assisted breeding programs. The polymorphic markers linked with the QTLs for grain yield under drought can be pyramided in Jyothi through marker-assisted backcross breeding.

Genetic Diversity of Durian (Durio zibethinus Murr.) from North and South Nias using Simple Sequence Repeat (SSR)

Durian is a tropical fruit that has economic value for Nias Island especially in North and South Nias. Anthropogenic activity already occured in Nias and caused disturbance resulting in destruction and extinction of various durian. The aim of the research was to analyzed the genetic diversity of Durian from North and South Nias using SSR primers namely: Dz844, DzGCCG01, DzGCAG01, and DzMTb021. Total DNA was successfully isolated from 20 accessions. The DNA was successfully amplified resulting in DNA alleles sizing from 170-1100 bp. All primers showed 33 alleles with 28 polymorphic bands. The DNA profile were further analyzed with Ntsys which showed that 20 accession has coefficient of similarity 0.66-0.94 and clustered into nine groups at the similarity coefficient of 0.80. The research showed that the ninth accession of durian from North and South Nias has a high genetic diversity.

Evaluation of Biological Control of Sorghum Strains Using Bacillus Thuringiensis and Pseudomonas Aeruginosa Under Drought Stress

Background: Sorghum is an economically significant staple food crop for more than half a billion people in developing nations, especially in arid and semi-arid locations where drought stress is a significant limiting factor. Despite usually being regarded as tolerant, sorghum suffers severely from drought stress, which lowers its productivity and nutritional quality throughout its principal cultivation areas. Objective: Improvements in DNA fingerprinting by ISSRs, SSRs, and RAPD markers have also been employed in sorghum genetic modification (GMOs) to enhance the economic characteristics of this crop. Materials and methods: To provide a natural defence against pests, the most tolerant plants among the seven varieties of sorghum bicolour were selected and planted in the second season of 2020–2021 under treatment with two microorganisms, B. thuringiensis and P. aeruginosa. This study considered seven varieties of sorghum bicolour planted under 50% water deficiency in 2019–2020. Genetic variability analysis of sorghum genotypes was performed using seven Inter-Simple Sequence Repeat (ISSR) primers, six Simple Sequence Repeat (SSR) primers, and five Random Amplified Polymorphic DNA (RAPD) primers. Seven Sorghum bicolour accessions were collected from various regions of Egypt and their phylogenetic relationships were evaluated. Additionally, DNA fingerprinting and analyses of the genetic diversity and evolutionary linkages in the sorghum germplasm employed the (ISSR) molecular marker technique. Results and conclusion: The Fisher Least Significant difference test (LSD) at P &lt; 0.05, based on RAPD, ISSR, and SSR markers demonstrated a significant connection.  The findings demonstrated that 51 bands with a size range of 100–1500 bp and polymorphism percentage of 72.5% were created using five RAPD primers. Seven ISSR primers generated 45 bands With a 57.8(%) polymorphism percentage, ranging in size from 100 to 3000 bp. six SSR primers generated 28 bands with (67.86%) polymorphism percentage of 67.86 %, ranging in size from 100 to 1500 bp. Morphological characteristics and ISSR, SSR, and RAPD analyses were used to group the UPGMA Dendrogram into groups. Jaccard's coefficient was used to analyse the genetic similarity matrix. The maximum similarity was observed for ISSR between Hybrid Sh1 and Hybrid Sh306 (0.984%), SSR between Hybrid Sh306 and Sudan grass (0.964%), and RAPD between Giza 15 and Indian Millet (0.706%). The classification of sorghum germplasm, breeding initiatives, and conservation efforts rely heavily on the determination of the genetic diversity among sorghum species. Identification of genetic variants, morphological features, and genetic analysis of ISSR, SSR, and RAPD are useful techniques. These findings demonstrate a large ratio of variation in sorghum. This work could serve as a guide for future research on sorghum and aid in the understanding of species and breeding initiatives.

Agro-morphological, biochemical, and molecular markers of barley genotypes grown under salinity stress conditions

The aim of this study was to evaluate the impact of salt stress on morphological, yield, biochemical, and molecular attributes of different barley genotypes. Ten genotypes were cultivated at Fayoum Research Station, El-Fayoum Governorate, Egypt, during two seasons (2020–2021 and 2021–2022), and they were exposed to two different salt concentrations (tap water as a control and 8000 ppm). The results showed that genotypes and salt stress had a significant impact on all morphological and physiological parameters. The morphological parameters (plant height) and yield attributes (spike length, number of grains per spike, and grain yield per plant) of all barley genotypes were significantly decreased under salt stress as compared to control plants. Under salt stress, the total soluble sugars, proline, total phenol, total flavonoid, ascorbic acid, malondialdehyde, hydrogen peroxide, and sodium contents of the shoots of all barley genotypes significantly increased while the potassium content decreased. L1, which is considered a sensitive genotype was more affected by salinity stress than the tolerance genotypes L4, L6, L9, and Giza 138. SDS-PAGE of seed proteins demonstrated high levels of genetic variety with a polymorphism rate of 42.11%. All genotypes evaluated revealed significant variations in the seed protein biochemical markers, with new protein bands appearing and other protein bands disappearing in the protein patterns of genotypes cultivated under various conditions. Two molecular marker techniques (SCoT and ISSR primers) were used in this study. Ten Start Codon Targeted (SCoT) primers exhibited a total of 94 fragments with sizes ranging from 1800 base pairs to 100 base pairs; 29 of them were monomorphic, and 65 bands, with a polymorphism of 62.18%, were polymorphic. These bands contained 21 unique bands (9 positive specific markers and 12 negative specific markers). A total of 54 amplified bands with molecular sizes ranging from 2200 to 200 bp were produced using seven Inter Simple Sequence Repeat (ISSR) primers; 31 of them were monomorphic bands and 23 polymorphic bands had a 40.9% polymorphism. The techniques identified molecular genetic markers associated with salt tolerance in barley crop and successfully marked each genotype with distinct bands. The ten genotypes were sorted into two main groups by the unweighted pair group method of arithmetic averages (UPGMA) cluster analysis based on molecular markers and data at a genetic similarity coefficient level of 0.71.