MOLECULAR VARIATION AND GENETIC DIVERGENCE IN T. AMAN RICE GENOTYPES USING SSR MARKERS

Rice is staple food in many countries of Africa and a major part of the diet in many others. However, Africa’s demand for rice exceeds production with the deficit of 40% being imported. One way to improve Africa’s rice production is through breeding high yielding varieties suitable for the different environment conditions. This study was conducted to assess the genetic variability and stability performance of 48 lowland rice genotypes including 37 interspecific (Oryza glaberrima × Oryza sativa ssp. indica) and 11 intraspecific (O. sativa ssp. indica × O. sativa ssp. indica) in 12 environments in Nigeria, Benin Republic and Togo using Additive Main Effect and Multiplicative Interaction (AMMI) and Genotype+ Genotype x Environment (GGE) biplot models. The combined analysis of variance revealed significant differences (P<0.01) among the genotypes, environments, and genotypes x environment interaction. Both the AMMI and GGE models identified NERICA-L8 and NERICA-LI2 as the best genotypes for cultivation across environments. Ouedeme environments in Benin Republic were the most stable and ideal for rice cultivation, while Ibadan sites were the most unstable. TOG 5681 had the least yield and was the most unstable across seasons. Genetic diversity was analyzed using 22 important morpho-agronomic traits and 50 simple sequence repeat (SSR) markers and the results were subjected to principal components analysis (PCA). The results revealed that the first eight PC axes (PC1–8) accounted for 75.13% of the total variation, while PC1–4 accounted for 50.39% of the total variation among rice genotypes. However, 10 of the 50 SSR markers were polymorphic and generated 49 alleles (average = 4.9 alleles per locus), suggesting moderate to substantial genetic diversity among the rice genotypes. The polymorphic information content (PIC) ranged from 0.24 to 0.65, with an average PIC value of 0.45. Two structured populations were observed which clustered into five heterotic groups and an outgroup, respectively. This suggests that heterosis could be exploited in the next hybridization program by crossing one of the genotypes in any SSR marker-defined cluster, with the rice accession TOG 5681 in cluster I. The results of this study suggest that morpho-agronomic traits should be used to compliment SSR data in rice diversity studies, especially if a few polymorphic SSR markers are to be used.

Cassava, family Euphorbiaceae, is the fifth most important staple food crop. The study of variability and diversity present in diverse cassava accessions maintained at the field genebank of TCRS, TNAU is essential to design the breeding program of cassava in TNAU. Hence, a study was carried out to assess the genetic diversity based on morphological traits and Simple Sequence Repeat (SSR) markers, identifying the SSR markers linked to a cassava mosaic disease-resistant QTL region. Genetic diversity among cassava germplasm accessions was estimated using 28 morphological traits and three SSR markers. The polymorphic information content (PIC) result gives high information for all the SSR markers. Morphological traits revealed 10 clusters and the SSR markers revealed 13 clusters at a similarity coefficient of 2.514 and 0.75, respectively. The SSR marker diversity revealed that the most promising clusters (II and XII) possessing accessions with yield attributing traits and cluster IX were grouped together due to low yield and CMD susceptibility. Morphological descriptors revealed variance for yield contributing traits. The first three principle components accounted for 10.8, 9.1, and 8.4%, respectively. Correlation studies showed significant correlation among yield and yield-attributing traits, which in turn influences yield. The morphological traits diversity reveals that cluster VI was the most promising cluster in which genotypes ME209 and ME460 possess higher yield and starch content with cassava mosaic disease (CMD) resistance. Most of the accessions grouped under cluster X belong to selection from International Centre for Tropical Agriculture (CIAT) collections and the cultivars under clusters I and II were true cassava seed (TCS) selections of India. The CMD-linked SSR marker which is reported in the previous studies have generated eight alleles and each were regressed with CMD resistance and resulted in non-significant linkage to CMD resistance.

Rice is staple food in many countries of Africa and a major part of the diet in many others. However, Africa’s demand for rice exceeds production with the deficit of 40% being imported. One way to improve Africa’s rice production is through breeding high yielding varieties suitable for the different environment conditions. This study was conducted to assess the genetic variability and stability performance of 48 lowland rice genotypes including 37 interspecific (Oryza glaberrima × Oryza sativa ssp. indica) and 11 intraspecific (O. sativa ssp. indica × O. sativa ssp. indica) in 12 environments in Nigeria, Benin Republic and Togo using Additive Main Effect and Multiplicative Interaction (AMMI) and Genotype+ Genotype x Environment (GGE) biplot models. The combined analysis of variance revealed significant differences (P<0.01) among the genotypes, environments, and genotypes x environment interaction. Both the AMMI and GGE models identified NERICA-L8 and NERICA-LI2 as the best genotypes for cultivation across environments. Ouedeme environments in Benin Republic were the most stable and ideal for rice cultivation, while Ibadan sites were the most unstable. TOG 5681 had the least yield and was the most unstable across seasons. Genetic diversity was analyzed using 22 important morpho-agronomic traits and 50 simple sequence repeat (SSR) markers and the results were subjected to principal components analysis (PCA). The results revealed that the first eight PC axes (PC1–8) accounted for 75.13% of the total variation, while PC1–4 accounted for 50.39% of the total variation among rice genotypes. However, 10 of the 50 SSR markers were polymorphic and generated 49 alleles (average = 4.9 alleles per locus), suggesting moderate to substantial genetic diversity among the rice genotypes. The polymorphic information content (PIC) ranged from 0.24 to 0.65, with an average PIC value of 0.45. Two structured populations were observed which clustered into five heterotic groups and an outgroup, respectively. This suggests that heterosis could be exploited in the next hybridization program by crossing one of the genotypes in any SSR marker-defined cluster, with the rice accession TOG 5681 in cluster I. The results of this study suggest that morpho-agronomic traits should be used to compliment SSR data in rice diversity studies, especially if a few polymorphic SSR markers are to be used.

BackgroundThis study aimed to develop a set of perfect simple sequence repeat (SSR) markers with a single copy in the cotton genome, to construct a DNA fingerprint database suitable for authentication of cotton cultivars. We optimized the polymerase chain reaction (PCR) system for multi-platform compatibility and improving detection efficiency. Based on the reference genome of upland cotton and 10× resequencing data of 48 basic cotton germplasm lines, single-copy polymorphic SSR sites were identified and developed as diploidization SSR markers. The SSR markers were detected by denaturing polyacrylamide gel electrophoresis (PAGE) for initial screening, then fluorescence capillary electrophoresis for secondary screening. The final perfect SSR markers were evaluated and verified using 210 lines from different sources among Chinese cotton regional trials.ResultsUsing bioinformatics techniques, 1 246 SSR markers were designed from 26 626 single-copy SSR loci. Adopting a stepwise (primary and secondary) screening strategy, a set of 60 perfect SSR markers was selected with high amplification efficiency and stability, easy interpretation of peak type, multiple allelic variations, high polymorphism information content (PIC) value, uniform chromosome distribution, and single-copy characteristics. A multiplex PCR system was established with ten SSR markers using capillary electrophoresis detection.ConclusionsA set of perfect SSR markers of cotton was developed and a high-throughput SSR marker detection system was established. This study lays a foundation for large-scale and standardized construction of a cotton DNA fingerprint database for authentication of cotton varieties.

The molecular characterization and genetic variability between two of commercial and polyemberyonic Egyptian mango cultivars, namely: Zebda and Ewais, with thirty seedlings of their offspring were analyzed using 8 Simple Sequences Repeat (SSR) markers. LMMA _15 marker was discarded in data analysis because of producing only one band (monomorphic locus). Other seven markers produced total of 22 alleles with a high level of Polymorphism (~100 percent). The effective number of alleles ranged from 1.7, to 3.4 with average value of 1.47. Heterozygosity per locus varied from 0.00 to 0.75 with an average of 0.36. Polymorphic Information content (PIC) value scored from 0.41 to 0.70 with average of 0.57. The discrimination power (Dp) ranged between 0.11 and 0.72 with an average of 0.50 per locus. Generally, the genetic similarity values varied between 0.12 and 100% over 32 genotypes. A cluster analysis was used to determine genetic similarities. The dendrogram can be grouped into two major clusters (I and II). Cluster I consists of Ewais seedlings exhibiting 94-100% genetic similarity among them. Cluster II consists of all seedlings of Zebda cultivar exhibiting 52-100% genetic similarity and divided to two sub clusters. Seedling (Z_C_S2) was the most divergent in first sub cluster and second sub cluster exhibited less distance and consists of all other Zebda seedlings. This study additionally indicates that SSR markers are useful for distinguishing and characterizing mango genotypes. The genetic relatedness among these genotypes could provide useful information for conservation and selection of cross parents in breeding.

<p>Brown planthopper is one of the most destructive insect pest of rice in Indonesia and other Asian countries. Pyramiding some brown planthopper resistance genes is a valuable approach to create more durable resistance against the pest. The objective of this study was to identify polymorphisms of Brown Planthopper Resistance genes (Bph) on 20 genotypes of rice, and to obtain genetic relationship among genotypes tested. The experiment was conducted from June to September 2012 at Green House and Laboratory of Plant Analysis and Biotechnology, Faculty of Agriculture, Universitas Padjadjaran, Jatinangor. Twenty genotypes were analyzed, and two of them were used as check varieties. Simple Sequence Repeat (SSR) markers were applied to detect Bph3, Bph4, Qbph3, and Qbph4 genes. Polymorphic levels were analyzed by calculating PIC (Polymorphic Information Content). The grouping of rice genotypes were done based on principal components analysis (PCA) of SSR data, and the genetic relationship based on the presence of Bph genes was estimated using UPGMA (Unweighted Pair Group With Arithmetic mean). Results showed that RM313, RM8072, RM8213, RM5953, RM586, and RM589 markers were polymorphic. Rice genotypes PTB 33, Diah Suci, Cibogo, Cisantana, Digul, Ciherang, Inpari 13, Inpari 10, and Memberamo had Bph3, Qbph3, Bph4, and Qbph4. Meanwhile Bph3, Qbph3, and Bph4 were supposed to be belonged by IR 64, Aek Sibundong, Batang Gadis, IR 66, and Mekongga. Kalimas and Tukat Penatu had Bph3, Qbph3, dan Qbph4. IR 74 had Bph3 and Qbph3, and Fatmawati had Bph3 and Bph4. UPGMA clustering resulted in two main clusters, in which the first cluster consisted of 2 subclusters. PTB-33 was closely related with Memberamo, Tukat Penatu, Digul, Diah Suci, and Kalimas. The SSR markers used in this study were proven to be valuable in molecular detection of Bph genes and in estimating genetic relationsips of rice genotypes. PTB-33 was a good donor of resistance genes, as well as Memberamo, Tukat Penatu, Digul, Diah Suci, and Kalimas which were identified as promising donors in rice breeding resistance to brown planthopper. </p><p><strong>Keywords :</strong> Bph gene, Brown Planthopper, Genetic relationship, SSR markers.</p>

The objective of the present study was to find the genetic diversity of eight improved varieties and eighty-four traditional accessions of rice using nine polymorphic simple sequence repeat (SSR) markers. The SSR markers detected 32 alleles ranging from 2 to 5, with an average of 3.55 alleles per locus, indicating a high genetic diversity. The number of effective alleles (ne) ranged from 1.85 (RM208) to 3.84 (RM493), with an average of 2.64 alleles per locus, which reconfirms an existing broad genetic diversity. Nei’s genetic diversity index (Nei, 1973) was very high (0.5955), indicating a high mean expected heterozygosity (HE). RM493 recorded the maximum polymorphism information content (PIC) value (0.8814). The mean PIC value was 0.5955 for the used SSR markers. Out of nine SSR markers, seven scored more than 0.5 PIC values, proving their potential to be used as genetic markers. Shannon’s information index (I) ranged from 0.65 (RM208) to 1.36 (RM493), with a mean value of 1.00. All genetic diversity indicators, na, ne, HE, PIC and I reflect the high genetic differentiation in representative sample of rice genotypes. According to the unweighted pair group method with arithmetic mean dendrogram and Nei’s genetic distance, the ninety-two rice genotypes were classified into seven groups at an ~85 level of similarity with additional sub-clusters within each group. Rice genotypes with significant genetic divergence can be chosen for upcoming breeding programmes by assessing their positions within the dendrogram. In the dendrogram, genotypes sharing the same name were not clustered together, indicating their distinct genetic backgrounds despite them sharing the same name. The traditional rice accessions clustered together in the dendrogram can be further analysed using more polymorphic SSR markers.

Only a limited number of simple sequence repeat (SSR) markers is available for the genome of garlic (Allium sativum L.) despite the fact that SSR markers have become one of the most preferred DNA marker systems. To develop new SSR markers for the garlic genome, garlic expressed sequence tags (ESTs) at the publicly available GarlicEST database were screened for SSR motifs and a total of 132 SSR motifs were identified. Primer pairs were designed for 50 SSR motifs and 24 of these primer pairs were selected as SSR markers based on their consistent amplification patterns and polymorphisms. In addition, two SSR markers were developed from the sequences of garlic cDNA-AFLP fragments. The use of 26 EST-SSR markers for the assessment of genetic relationship was tested using 31 garlic genotypes. Twenty six EST-SSR markers amplified 130 polymorphic DNA fragments and the number of polymorphic alleles per SSR marker ranged from 2 to 13 with an average of 5 alleles. Observed heterozygosity and polymorphism information content (PIC) of the SSR markers were between 0.23 and 0.88, and 0.20 and 0.87, respectively. Twenty one out of the 31 garlic genotypes were analyzed in a previous study using AFLP markers and the garlic genotypes clustered together with AFLP markers were also grouped together with EST-SSR markers demonstrating high concordance between AFLP and EST-SSR marker systems and possible immediate application of EST-SSR markers for fingerprinting of garlic clones. EST-SSR markers could be used in genetic studies such as genetic mapping, association mapping, genetic diversity and comparison of the genomes of Allium species.

The Genetic diversity was assessed among 96 pigeonpea accessions including 15 male sterile, 13 maintainer and 68 germplasm lines using 44 Simple Sequence Repeats (SSR) markers distributed over all the 11 chromosomes. Out of 44 SSR markers, 33 were polymorphic which showed 75% polymorphism among the used markers. For an individual primer, the alleles amplified varied from 2 to 4 with an average of 2.54. The Polymorphic Information Content (PIC) values ranged from 0.26 (CCM 0183) to 0.78 (CCM 0402 and CCM 0721). Based on 112 alleles amplified by SSR markers, the 96 genotypes were alienated into eight clusters. Cluster I and cluster VII were the largest with 22 genotypes each, cluster III and cluster IV were the smallest with two genotypes each, while cluster II, cluster V, cluster VI and cluster VIII consisted of 10, 15, 14 and 9 genotypes, respectively. Genotypes Pusa 991 and ULA 11 were found to be the most distant genotypes with highest dissimilarity coefficient (32%) where as AL 112A and AL 113A were the least distant genotypes with lowest dissimilarity coefficient (2%). Thus, highly distant genotypes can be used in pigeonpea improvement programs for getting desirable segregants. The selected panel of polymorphic SSR markers performed well in detection of genetic diversity patterns and can be used for future germplasm characterization studies in pigeonpea.

Genome-wide identification of microsatellite and development of polymorphic SSR markers for spotted sea bass (Lateolabrax maculatus)

The pea (Pisum sativum L.) is one of the more important legume crops produced globally. We studied the structure and genetic diversity in a collection of 50 pea accessions with 16 simple sequence repeat (SSR) markers, whose average polymorphic information content (PIC) was 0.62. The SSR markers amplified a total of 28 alleles with an average of 4 alleles per locus, with locus AB71 and D21 amplifying the largest number of alleles (6). The observed heterozygosity (Ho) was 0.09±0.08 and the expected heterozygosity (He) was 0.42, indicating an elevated level of inbreeding (Fis = 0.60). The genetic relationships were inferred with a similarity index (DICE) and a bayesian analysis (STRUCTURE), detecting 2 clusters for the genotypes, with a high similarity of the morphological characteristics of each genotype. The results of this study will be useful for the creation of future breeding programs.The pea (Pisum sativum L.) is one of the more important legume crops produced globally. We studied the structure and genetic diversity in a collection of 50 pea accessions with 16 simple sequence repeat (SSR) markers, whose average polymorphic information content (PIC) was 0.62. The SSR markers amplified a total of 28 alleles with an average of 4 alleles per locus, with locus AB71 and D21 amplifying the largest number of alleles (6). The observed heterozygosity (Ho) was 0.09±0.08 and the expected heterozygosity (He) was 0.42, indicating an elevated level of inbreeding (Fis = 0.60). The genetic relationships were inferred with a similarity index (DICE) and a bayesian analysis (STRUCTURE), detecting 2 clusters for the genotypes, with a high similarity of the morphological characteristics of each genotype. The results of this study will be useful for the creation of future breeding programs.

Fig (Ficus carica L.) is a traditional crop in Turkey and widely cultivated around the Mediterranean areas. The gynodioecious fig species is present in two sexual forms, i.e. the domesticated fig (female tree) and the caprifig (male tree). Caprifigs are crucial for high quality fig production and breeding while, the studies on assessment of genetic relationship among caprifigs is limited. The aim of this study was to determine genetic diversity among 45 caprifigs and 2 female figs collected from four provinces in Marmara and Aegean Sea Regions of Turkey using simple sequence repeat (SSR) markers. In this work, 24 SSR markers were tested, one was monomorphic and the remaining markers amplified 82 alleles. The number of polymorphic alleles per SSR marker ranged from 2 to 7. The observed heterozygosity (Ho) differed from 0.18 to 0.76 and expected heterozygosity (He) ranged between 0.24 and 0.81. The polymorphism information content (PIC) varied from 0.42 to 0.98. A UPGMA analysis based on Dice similarity matrix clustered fig genotypes into two main groups and similarly, STRUCTURE analysis placed fig genotypes into two different gene pools (K=2). Fig genotypes collected from the same region were not clustered together in a group indicating that the fig genotypes did not cluster on the basis of their collection sites. Our results demonstrated that caprifigs and female figs are not genetically distinct and they clustered together in a group. All fig genotypes had distinct SSR marker profiles suggesting that there were no synonyms or homonyms. These results revealed a high genetic variation among fig genotypes and 23 SSR markers were enough to discriminate all fig genotypes analysed in this study demonstrating that SSR marker system is suitable for genetic analysis in figs.

Tomato landraces are less sensitive to environmental stresses and grown mainly under rain fed conditions. They are still grown in small farms due to quality and special demand of consumers. These landraces are valuable sources of genetic traits, and plant breeders can use breeding programs for crop improvement. One of the primary needs of the crop improvement is the estimation of genetic diversity. Development of microsatellite simple sequence repeat (SSR) markers from map-referenced bacterial artificial chromosomes (BAC) clones is a very effective means of targeting markers to marker scarce positions in the genome. This study was aimed at developing a set of functional SSR markers via in silico analysis of publicly available tomato DNA sequences. As a result, 17 SSR markers were developed and tested on one tomato commercial cultivar and eight local landraces. 12 loci (27 alleles) were scored and showed 100% polymorphic patterns. The calculated polymorphism information content (PIC) values for the SSR markers developed ranged from 0.62 to 0.97 (mean 0.89). The SSR motifs CT(26) AT(27) and TTC(6) TTA(4) had the highest PIC value (0.97), while CAA(5)A(8) had the lowest PIC value (0.62). According to tomato expressed sequence tag (EST) analysis, some of these developed SSR markers, such as mono and di-nucleotide are related to some genes. The T(16) motif is related to hydroxyproline-rich glycoprotein, which is a family protein from Arabidopsis thaliana . On the other hand, the SSR with tri-nucleotide repeat motif AAC(4)A(11) was related to a putative homologous protein to A7Q2S4 from Vitis vinifera . Keywords: Tomato landraces, in silico simple sequence repeat (SSR) markers, DNA markers, genetic diversity

Zanthoxylum is a versatile economic tree species utilized for its spice, seasoning, oil, medicinal, and industrial raw material applications, and it has a lengthy history of cultivation and domestication in China. This has led to the development of numerous cultivars. However, the phenomenon of mixed cultivars and confusing names has significantly obstructed the effective utilization of Zanthoxylum resources and industrial development. Consequently, conducting genetic diversity studies and cultivar identification on Zanthoxylum are crucial. This research analyzed the genetic traits of 80 Zanthoxylum cultivars using simple sequence repeat (SSR) and inter-Primer Binding Site (iPBS) molecular markers, leading to the creation of a DNA fingerprint. This study identified 206 and 127 alleles with 32 SSR markers and 10 iPBS markers, respectively, yielding an average of 6.4 and 12.7 alleles (Na) per marker. The average polymorphism information content (PIC) for the SSR and iPBS markers was 0.710 and 0.281, respectively. The genetic similarity coefficients for the 80 Zanthoxylum accessions ranged from 0.0947 to 0.9868 and from 0.2206 to 1.0000, with mean values of 0.3864 and 0.5215, respectively, indicating substantial genetic diversity. Cluster analysis, corroborated by principal coordinate analysis (PCoA), categorized these accessions into three primary groups. Analysis of the genetic differentiation among the three Zanthoxylum (Z. bungeanum, Z. armatum, and Z. piperitum) populations using SSR markers revealed a mean genetic differentiation coefficient (Fst) of 0.335 and a gene flow (Nm) of 0.629, suggesting significant genetic divergence among the populations. Molecular variance analysis (AMOVA) indicated that 65% of the genetic variation occurred within individuals, while 35% occurred among populations. Bayesian model-based analysis of population genetic structure divided all materials into two groups. The combined PI and PIsibs value of the 32 SSR markers were 4.265 × 10− 27 and 1.282 × 10− 11, respectively, showing strong fingerprinting power. DNA fingerprints of the 80 cultivars were established using eight pairs of SSR primers, each assigned a unique numerical code. In summary, while both markers were effective at assessing the genetic diversity and relationships of Zanthoxylum species, SSR markers demonstrated superior polymorphism and cultivar discrimination compared to iPBS markers. These findings offer a scientific foundation for the conservation and sustainable use of Zanthoxylum species.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12870-024-05373-1.

To evaluate the genetic diversity among 48 genotypes of chickpea comprising cultivars, landraces and internationally developed improved lines genetic distances were evaluated using three different molecular marker techniques: Simple Sequence Repeat (SSR); Start Codon Targeted (SCoT) and Conserved DNA-derived Polymorphism (CDDP). Average polymorphism information content (PIC) for SSR, SCoT and CDDP markers was 0.47, 0.45 and 0.45, respectively, and this revealed that three different marker types were equal for the assessment of diversity amongst genotypes. Cluster analysis for SSR and SCoT divided the genotypes in to three distinct clusters and using CDDP markers data, genotypes grouped in to five clusters. There were positive significant correlation (r = 0.43, P < 0.01) between similarity matrix obtained by SCoT and CDDP. Three different marker techniques showed relatively same pattern of diversity across genotypes and using each marker technique it's obvious that diversity pattern and polymorphism for varieties were higher than that of genotypes, and CDDP had superiority over SCoT and SSR markers. These results suggest that efficiency of SSR, SCOT and CDDP markers was relatively the same in fingerprinting of chickpea genotypes. To our knowledge, this is the first detailed report of using targeted DNA region molecular marker (CDDP) for genetic diversity analysis in chickpea in comparison with SCoT and SSR markers. Overall, our results are able to prove the suitability of SCoT and CDDP markers for genetic diversity analysis in chickpea for their high rates of polymorphism and their potential for genome diversity and germplasm conservation.