SSR Loci Research Articles

Molecular identification of Bambusa changningensis is the natural bamboo hybrid of B. rigida × Dendrocalamus farinosus.

Bamboo is one of the fastest-growing plants commonly used in food, fibre, paper, biofuel, ornamental and medicinal industries. Natural hybridization in bamboo is rare due to its long vegetative period followed by gregarious flowering and death of the entire population. In the current study, a new bamboo species, Bambusa changningensis, shows intermediate characteristics of Dendrocalamus farinosus and B. rigida morphologically, but it is unknown whether B. changningensis is a natural hybrid. Moreover, B. changningensis has been identified as a superior variety of Sichuan Province with high pulping yield, fibre length and width. Therefore, we analyzed the morphological characteristics, DNA markers, DNA barcoding and chloroplast genomes to identify the hybrid origin of B. changningensis and possible maternal parent. We have developed the transcriptomic data for B. changningensis and mined the SSR loci. The putative parental lines and hybrid were screened for 64 SSR makers and identified that SSR14, SSR28, SSR31 and SSR34 markers showed both alleles of the parental species in B. changningensis, proving heterozygosity. Sequencing nuclear gene GBSSI partial regions and phylogenetic analysis also confirm the hybrid nature of B. changningensis. Further, we have generated the complete chloroplast genome sequence (139505 bp) of B. changningensis. By analyzing the cp genomes of both parents and B. changningensis, we identified that B. rigida might be the female parent. In conclusion, our study identified that B. changningensis is a natural hybrid, providing evidence for bamboo's natural hybridization. This is the first report on confirming a natural bamboo hybrid and its parents through SSR and chloroplast genome sequence.

Genetic analysis of heat tolerance in hot pepper: insights from comprehensive phenotyping and QTL mapping.

High temperatures present a formidable challenge to the cultivation of hot pepper, profoundly impacting not only vegetative growth but also leading to flower and fruit abscission, thereby causing a significant reduction in yield. To unravel the intricate genetic mechanisms governing heat tolerance in hot pepper, an F2 population was developed through the crossing of two distinct genotypes exhibiting contrasting heat tolerance characteristics: DLS-161-1 (heat tolerant) and DChBL-240 (heat susceptible). The F2 population, along with the parental lines, was subjected to comprehensive phenotyping encompassing diverse morphological, physiological, and biochemical heat-related traits under high temperature conditions (with maximum temperature ranging from 31 to 46.5°C and minimum temperature from 15.4 to 30.5°C). Leveraging the Illumina Nova Seq-6000 platform, Double digest restriction-site associated DNA sequencing (ddRAD-seq) was employed to generate 67.215 Gb data, with subsequent alignment of 218.93 million processed reads against the reference genome of Capsicum annuum. Subsequent variant calling and ordering resulted in 5806 polymorphic SNP markers grouped into 12 LGs. Further QTL analysis identified 64 QTLs with LOD values ranging from 2.517 to 11.170 and explained phenotypic variance ranging from 4.05 to 19.39%. Among them, 21 QTLs, explaining more than 10% phenotypic variance, were identified as major QTLs controlling 9 morphological, 3 physiological, and 2 biochemical traits. Interestingly, several QTLs governing distinct parameters were found to be colocalized, suggesting either a profound correlation between the QTLs regulating these traits or their significant genomic proximity. In addition to the QTLs, we also identified 368380 SSR loci within the identified QTL regions, dinucleotides being the most abundant type (211,381). These findings provide valuable insights into the genetics of heat tolerance in hot peppers. The identified QTLs and SSR markers offer opportunities to develop heat-tolerant varieties, ensuring better crop performance under high-temperature conditions.

Genome-wide identification and molecular evolution of NAC gene family in Dendrobium nobile.

NAC transcription factors are an important genes that regulate plant growth and development, and can regulate functions such as fruit ripening in plants. Based on genome data of Dendrobium nobile, the NAC gene family was identified and analyzed by bioinformatics methods. In this study, we identified 85 NAC genes in Dendrobium nobile genome, and systematically analyzed the NAC gene family. We found that they were distributed unevenly in the nineteen chromosomes. The amino acid length of D. nobile NAC gene family (DnoNACs) ranged from 80 to 1065, molecular weight ranged from 22.17 to 119.02 kD, and isoelectric point ranged from 4.61~9.26. Its promoter region contains multiple stress responsive elements, including light responsive, gibberellin-responsive, abscisic acid responsiveness, MeJA-responsiveness and drought-inducibility elements. Phylogenetic analysis indicates that the D. nobile NAC gene family is most closely related to Dendrobium catenatum and Dendrobium chrysotoxum. Analysis of SSR loci indicates that the fraction of mononucleotide repeats was the largest, as was the frequency of A/T. Non-coding RNA analysis showed that these 85 NAC genes contain 397 miRNAs. The collinearity analysis shows that 9 collinear locis were found on the chromosomes of D. nobile with Arabidopsis thaliana, and 75 collinear locis with D.chrysotoxum. QRT-PCR experiment under different salt concentration and temperature conditions verified the response mechanism of DnoNAC gene family under stress conditions. Most DnoNAC genes are sensitive to salt stress and temperature stress. The results of this study provide a reference for further understanding the function of NAC gene in D. nobile.

Genome-wide microsatellite characterization and marker development in Diospyros oleifera

Diospyros oleifera, a diploid Diospyros, have a wide range of applications in biomedical science and the food, wood, and chemical industries. In addition, it is an excellent model plant to study Diospyros. While, to date, the genome-wide mining of SSRs (or Microsatellite) in D. oleifera is still absent. In this study, we constituted the first deep characterization of genome-wide microsatellites for D. oleifera and developed polymorphic SSR markers across Diospyros lotus and Diospyros kaki. A total of 330,507 SSRs were identified from D. oleifera genome with an average density of 385.95 SSRs per Mb, representing a higher SSR density. D. oleifera SSR motifs are not randomly distributed across genomic regions and motif types. Although, the distribution of SSRs differed slightly among D. oleifera chromosomes, only chromosomes 1 and 5 had considerably high densities. There are significantly lower frequencies of SSRs in the coding regions than that in the whole genomic region, which was only about 3.49% of the density of the D. oleifera genome (13.48/Mb vs. 385.95/Mb). Of the different SSR motif types, mononucleotide repeats (47.63%) were the most abundant in genome, while trinucleotide repeats (68.57%; 1924) were the most abundant in coding regions. Therefore, unlike in the D. oleifera genome, the AT-rich motif lost its numerical dominance in the coding regions. In addition, 231,809 primer pairs were identified, and 87.65% were high-resolution markers in the D. oleifera genome, which generated no more than three in silico products. 25,387 potential for interspecific polymorphic SSR markers between two Diospyros species were also identified. An amplification rate of 92% from testing Diospyros species were clarified through with a subset of candidate polymorphic markers (100 markers) test. Then, 29 novel polymorphism SSR markers were employed to distinguish among 12 Diospyros accessions with high mean PIC values of 0.6009. Thus, these SSR locus we identified in this study had the potential to reveal genotypic diversity in Diospyros.

Analysis of the Conservation Status, Genetic Diversity and Population Structure of Endangered Ostrya rehderiana Resources Using SSR Markers

Climate change and anthropogenic habitat destruction have led to the extinction of many species. Ostrya rehderiana (Betulaceae) is a typical rare and endangered species, and only five wild individuals have survived. In the 1980s, the offspring of the five wild trees were planted for ex situ conservation and have grown into stable cultivated populations. To protect O. rehderiana resources, the genetic diversity and genetic structure of this species were analysed using SSR markers. A total of 167 alleles were detected among 116 individuals from the only wild population and five cultivated populations at 12 SSR loci. The genetic diversity level of O. rehderiana was He = 0.88. Genetic differentiations occurred among populations (Fst = 0.17), which was also validated via an analysis of molecular variance (AMOVA). The cultivated populations TM1, TM2 and WC showed considerable genetic differences from the wild population WP based on Bayesian clustering analysis, phylogenetic tree reconstruction and principal coordinate analysis (PCoA). The cultivated populations had more genetic diversity than the wild one. It is speculated that novel alleles may have emerged out of natural processes of evolution and adaptation. The cultivated population QY with the most unique alleles has begun to propagate seedlings naturally, and the small population size and geographical isolation may negatively influence the founding of this population. To weaken the effects of bottlenecks and genetic drift, anthropogenic gene flow among populations is necessary. In addition to the wild population, the cultivated population QY and six individuals from populations QY and WC were also found to be important for the conservation of O. rehderiana. The results of this study may guide the development of conservation policies for endangered O. rehderiana.

Genome-Wide Datasets of Chicories (Cichorium intybus L.) for Marker-Assisted Crop Breeding Applications: A Systematic Review and Meta-Analysis.

Cichorium intybus L. is the most economically important species of its genus and among the most important of the Asteraceae family. In chicory, many linkage maps have been produced, several sets of mapped and unmapped markers have been developed, and dozens of genes linked to traits of agronomic interest have been investigated. This treasure trove of information, properly cataloged and organized, is of pivotal importance for the development of superior commercial products with valuable agronomic potential in terms of yield and quality, including reduced bitter taste and increased inulin production, as well as resistance or tolerance to pathogens and resilience to environmental stresses. For this reason, a systematic review was conducted based on the scientific literature published in chicory during 1980-2023. Based on the results obtained from the meta-analysis, we created two consensus maps capable of supporting marker-assisted breeding (MAB) and marker-assisted selection (MAS) programs. By taking advantage of the recently released genome of C. intybus, we built a 639 molecular marker-based consensus map collecting all the available mapped and unmapped SNP and SSR loci available for this species. In the following section, after summarizing and discussing all the genes investigated in chicory and related to traits of interest such as reproductive barriers, sesquiterpene lactone biosynthesis, inulin metabolism and stress response, we produced a second map encompassing 64 loci that could be useful for MAS purposes. With the advent of omics technologies, molecular data chaos (namely, the situation where the amount of molecular data is so complex and unmanageable that their use becomes challenging) is becoming far from a negligible issue. In this review, we have therefore tried to contribute by standardizing and organizing the molecular data produced thus far in chicory to facilitate the work of breeders.

Comparative analysis of nine Tilletia indica genomes for the development of novel microsatellite markers for genetic diversity and population structure analysis.

Karnal bunt (KB; Tilletia indica) is the prime quarantine concern for quality wheat production throughout the world. The most effective approach to dealing with this biotic stress is to breed KB-resistant wheat varieties, which warrants a better understanding of T. indica genome architecture. In India, the North Western Plain Zone is the prime hot spot for KB disease, but only limited efforts have been made to decipher T. indica diversity at the genomic level. Microsatellites offer a powerful and robust typing system for the characterization and genetic diversity assessment of plant pathogens. At present, inadequate information is available with respect to the development of genome-derived markers for revealing genetic variability in T. indica populations. In current research, nine complete genome sequences of T. indica (PSWKBGH_1, PSWKBGH_2, PSWKBGD_1_3, RAKB_UP_1, TiK_1, Tik, DAOMC236408, DAOMC236414, and DAOMC236416) that exist in the public domain were explored to know the dynamic distribution of microsatellites. Comparative genome analysis revealed a high level of relative abundance and relative density of microsatellites in the PSWKBGH_1 genome in contrast to other genomes. No significant correlation between microsatellite distribution for GC content and genome size was established. All the genomes showed the dominance of tri-nucleotide motifs, followed by mono-, di-, tetra-, hexa-, and penta-nucleotide motifs. Out of 50 tested markers, 36 showed successful amplification in T. indica isolates and produced 52 different alleles. A PCR assay along with analysis of the polymorphic information content (PIC) revealed 10 markers as neutral and polymorphic loci (PIC 0.37). The identified polymorphic SSR loci grouped a geographically distinct T. indica population of 50 isolates representing seven Indian regions (Jammu, Himachal Pradesh, Punjab, Haryana, Uttarakhand, Uttar Pradesh, and Rajasthan) into four distinct clusters. The results of the analysis of molecular variance identified 94% genetic variation within the population and 6% among the population. Structure analysis also confirmed the existence of four genetically diverse groups containing admixtures of T. indica isolates across populations. In nutshell, the current study was successful in identifying novel, neutral and polymorphic microsatellite markers that will be valuable in offering deep insight into the evolutionary relationship and dynamics of the T. indica population for devising effective KB management strategies in wheat.

Genetic Analysis of the Cultivars of Ping’ou Hybrid Hazelnut (C. heterophylla Fisch. × C. avellana L.) in China Based on SSR Markers

Ping’ou hybrid hazelnut is one of the most profitable tree nuts in China, but economically important cultivars must first be genetically validated to meet industrial demand. Traditional approaches used for cultivar identification are mainly trait-based and unreliable. Previous approaches at the DNA level, focusing on the identification of species or/and varieties that originated in China, were not used widely in hybrid hazelnut because there was no proper standard sample. In this research, a multiplexed fingerprinting test was conducted to allow for hazelnut cultivar identification using SSR markers derived from European hazelnut. Twenty-seven SSR markers were used to fingerprint 57 genetically unique Ping’ou hybrid hazelnut and related wild species. All markers showed a high level of polymorphism, as indicated by mean values for observed heterozygosity (Ho = 0.84), expected heterozygosity (He = 0.80), and polymorphism information content (PIC = 0.78). A total of 301 alleles were detected, and the number of effective alleles varied from 6 for KG817 and GB818 to 18 for B654, with an average of 11.2 alleles per locus. Moreover, the Shannon’s information index (I) ranged from 1.293 for BR215 to 2.385 for B654, with an average of 1.908. The neighbor-joining tree, principal coordinate analysis, and Bayesian analysis revealed clear separation between hybrid cultivars and wild forms (Cluster/group I), as well as the differentiation within hybrid genotypes (Clusters/groups II and III). Additionally, the NJ dendrogram demonstrated a further split within Clusters/group III (III a and III b). Altogether, with the comparable SSR information of the European hazelnut cultivar ‘Barcelona’, the newly developed marker sets can assist in the germplasm identification of hazelnut cultivars and reproductive materials. Importantly, these combined SSR loci can be applied to characterize the genetic relationships and population structures among wild genotypes and hybrid cultivars, which will then provide information to guide hazelnut breeding based on their genetic background.

Microsatellite marker-based genetic diversity assessment among exotic and native maize inbred lines of Bangladesh

Hybrid development is basically dependent on the variability among available genetic resources. Polymorphism among the maize inbreds is essentially needed for maize hybridization. This study aimed at the assessment of diversity among 22 maize inbreds by 18 microsatellite markers. The study identified 187 alleles at 18 SSR loci. The amplified allele frequency per microsatellite locus was 10.4 and the highest allele per locus was 17 in SSR primer pair phi026. SSR primer set p-umc1292, phi074 and phi090 showed the lowest 6 alleles per genotype per locus. The locus phi026 showed the highest degree of gene diversity (0.92), and the locus p-umc1292 had the lowest of gene diversity (0.77) with a mean value of 0.862 among the microsatellites. At each site, the most prevalent allele varied between 0.14 (bnlg371) and 0.36. (p-umc1292). At any given locus, an average of 0.22 out of the 22 selected maize inbred lines had a common major allele. The average value of the polymorphic information content (PIC) was 0.85, within the range of 0.74 at the lowest to 0.92 at the highest. The higher PIC values of phi026 and nc013 established them to be the best markers for maize inbred lines. The UPGMA clustering generated seven distinct groups having 12.5% of similarity coefficient. The results revealed that inbred lines E10, E27, E19, E34, E35, E4, E43, E28, E11, E21, E17, E38, E25, E34, E14, E16, E39 and E3 were more diversified. These lines are promising to be used as parent materials for hybrid maize development in the future.

Chloroplast Genome Structure and Phylogenetic Analysis of 13 Lamiaceae Plants in Tibet.

The chloroplast (cp) genome has unique and highly conserved characteristics and is therefore widely used in species identification and classification, as well as to improve the in-depth understanding of plant evolution. In this study, the cp genomes of 13 Lamiaceae plants in the Tibet Autonomous Region of China were sequenced, assembled and annotated using bioinformatics methods. Phylogenetic trees were constructed to reveal the phylogenetic relationship of related species in the Lamiaceae. The results showed that all 13 cp genomes had a typical four-segment structure, including one large single-copy (LSC) region, one pair of inverted repeat (IR) regions and one small single-copy (SSC) region. The sequence lengths of the 13 cp genomes were between 149,081 bp and 152,312 bp, and the average GC content was 37.6%. These genomes contained 131-133 annotated genes, including 86-88 protein-coding genes, 37-38 tRNA genes, and 8 rRNA genes. A total of 542 SSR loci were detected using MISA software. The repeat types were mostly single-nucleotide repeats, accounting for 61% of simple repeats. A total of 26,328-26,887 codons were detected in 13 cp genomes. According to the RSCU value analysis, the codons mostly ended with A/T. Analysis of IR boundaries showed that the other species were relatively conserved, except for Nepeta laevigata (D. Don) Hand.-Mazz., which differed in gene type and location on both sides of the boundary. By analysing nucleotide diversity, two highly mutated regions located in the LSC and SSC regions were identified in the 13 cp genomes. Using the cp genome of Lycium ruthenicum Murray as the outgroup, 97 cp genomes of the Lamiaceae were used to construct an Maximum Likehood (ML) phylogenetic tree, in which these species were divided into eight major clades, corresponding to eight subfamilies based on morphological classification. The phylogenetic results based on monophyletic relationships were consistent with the morphological classification status at the tribe level.

Structure, sequence and phylogenetic analysis of the complete chloroplast genome of the brown algae Saccharina sp. ye-E (Laminariaceae: Phaeophyceae) from Sakhalin Oblast, Russia

For this investigation, the chloroplast genome of Saccharina sp. ye-E from Russia was uncovered and annotated with Illumina sequencing data to examine the phylogenetic relationship of Saccharina in Laminariaceae from a molecular biology perspective. Analysis of the structural characteristics, simple repeat sequence (SSR) loci, relative species differences, codon preference, and phylogenetic relationships was conducted. The results revealed that the chloroplast genome of Saccharina sp. ye-E had a length of 130,624 bp, containing 139 protein-coding genes (PCGs), 6 ribosomal RNAs (rRNAs) and 57 transfer RNAs (tRNAs) genes, and a total GC content of 31.1%. There were 15 SSR loci in the genome. Effective codon number (ENC) and codon adaptation index (CAI) both indicated the strong codon randomness and codon preference. The phylogenetic tree, based on the complete chloroplast genomes of 10 brown algae, showed that four species of Saccharina genus formed a clade, with Saccharina sp. ye-E and Saccharina latissimi having the most related genetic affinity. It is believed that the determination of the chloroplast genome of Saccharina sp. ye-E will be beneficial for future algal genetics evolution and systematic studies in the Laminariaceae.

Development of EST-SSR primers and genetic diversity analysis of the southern blight pathogen Sclerotium rolfsii using transcriptome data.

Sclerotium rolfsii Sacc. is a globally dispersed pathogenic fungus that causes southern blight disease in many crops and Chinese herbal medicine. The high degree of variation and diversity in the fungi altered population genetic structure. Therefore, the important factors of variation within the pathogen population should be considered during the development of management strategies for the disease. In this study, S. rolfsii isolates from 13 hosts in 7 provinces of China were collected and analyzed to identify their morphological features and perform molecular characterization. To develop EST-SSR primers, transcriptome sequencing was performed on isolated CB1, and its SSR loci were comprehensively analyzed. In addition, we analyzed the polymorphisms among different populations based on screened EST-SSR primers. The results showed that all of these clean reads with total 36,165,475 assembled bases were clustered into 28,158 unigenes, ranged from 201 bp to 16,402 bp on the length, of which the average length was 1,284 bp. Of these, the SSR sequence appeared at an average interval of 15.43 kB, and the frequency of SSR was 0.0648 SSR/kB. Polymorphism of 9 primers was observed among 22 populations, and was verified by the Shannon's index (average = 1.414) and polymorphic information index (> 0.50). The genetic diversity analysis revealed diversity in all host populations and geographical populations. Further, molecular variance analysis (AMOVA) showed that the differences between groups were mainly related to geographical location. Based on cluster analysis, the 7 populations were roughly divided into 3 groups, and the results were highly consistent with those based on the geographical location, ultimately aligning with the results of STRUCTURE analysis. The findings build on current knowledge of the distribution of S. rolfsii in the southwest area of China, adding value to current knowledge base on the population structure and genetic diversity of S. rolfsii, specifically in the context of Chinese herbal medicine cultivation in China. Overall, our findings may provide valuable information for breeding of crops with enhanced resistance toward S. rolfsii.

Screening and validation of salt-stress responsive cg-SSR markers in wheat (Triticum aestivum L.) germplasm of Pakistan.

Soil salinity has been affecting wheat production worldwide over past few decades. Evaluation of wheat genotypes for salinity tolerance at germination and vegetative growth level is crucial. Marker assisted selection is a technique used extensively for choosing salt-tolerant genotypes from breeding populations to introduce novel genes. The current study's main goal was to discover salt-stress resistant genes; genetic divergence and genome-wide connection by using recently designed candidate gene-based simple-sequence-repeat markers (cg-SSRs). The phenotypic connection of morphological features during the germination growth stage i.e., germination period, root length/weight and shoot length/weight, and vegetative growth stages i.e., root length/weight and shoot length/weight were tested in a group of 50 wheat genotypes. Significant difference was observed in germination rate, root length and weight among control and saline treatments. Total 30 SSR markers were utilized to test salinity resistance genes in wheat genotypes. Three (10%) of which were monomorphic, one (3.34%) showed no result, and the other 26 (86%) were polymorphic. Using 30 polymorphic markers discovered total 37 alleles. The polymorphic information content (PIC), quantifies each SSR locus capacity to discriminate between wheat, varied from 0.00 to 0.38 with an average of 0.19. Association analysis revealed that 26 primers were associated with morphological features, 03 with root length and the remaining 23 with germination. Utilizing morphological data, stress tolerance index (STI) was designed concluding that Auqab-2000, Margala-99 and Ufaq showed better resistance against salinity among other wheat genotypes. Cluster analysis demonstrated that wheat genotypes have vast genetic variability.

Microsatellite markers reveal genetic diversity and population structure of Portunus trituberculatus in the Bohai Sea, China

The swimming crab, Portunus trituberculatus, is one of the main aquaculture species in Chinese coastal regions due to its palatability and high economic value. To obtain a better understanding of the genetic diversity of P. trituberculatus in the Bohai Sea, the present study used 40 SSR loci to investigate the genetic diversity and population structure of 420 P. trituberculatus individuals collected from seven populations in the Bohai Sea. Genetic parameters revealed a low level of genetic diversity in the cultured population (SI = 1.374, He = 0.687, and PIC = 0.643) in comparison with wild populations (SI ≥ 1.399, He ≥ 0.692, and PIC ≥ 0.651). The genetic differentiation index (Fst) and gene flow (Nm) ranged from 0.001 to 0.060 (mean: 0.022) and 3.917 to 249.750 (mean: 31.289) respectively, showing a low differentiation among the seven populations of P. trituberculatus. Population structure analysis, phylogenetic tree, and principal component analysis (PCA) demonstrated that the seven groups of P. trituberculatus were divided into four subpopulations (K = 4), but the correlation between genetic structure and geographical distribution was not obvious. These results are expected to provide useful information for the fishery management of wild swimming crabs.

Evaluation of the Genetic Structure of Some Accessions Belonging to Onobrychis spp. Using Microsatellite DNA Markers

Turkey is in a very convenient position for animal husbandry in terms of both natural resources and ecological conditions. Forage crops, which has a very important place in agricultural activities, is the insurance of plant and animal production. Sainfoin is a perennial forage legume species that grown in the northern temperate regions of the world from the Mediterranean region and the Caucasus, and to Central Asia. In this study the genetic diversity of 100 genotypes representing 44 accessions from 18 different Onobrychis species (O. arenaria subsp. arenaria, O. inermis, O. petraea, O. cyri, O. iberica, O. altissima, O. vassilczenkoi, O. conferta subsp. argentea, O. alba subsp. laconica, O. biebersteinii, O. grandis, O. kachetica, O. kemulariae, O. oxyodonta, O. megataphros, O. pallasii, Onobrychis spp., and O. viciifolia) were evaluated using 8 simple sequence repeat (microsatellite) markers. Based on the results, OVK036, OVK094, OVK125, OVM033, OVK161, OVK046, OVM061, and OVK174 loci were polymorphic. The observed number of alleles per SSR locus ranged from 6 to 21 alleles (mean of 11.625). Maximum allele frequency ranged from 0.51 to 0.93 with a mean value of 0.73. The PIC value ranged from 0.124 to 0.244. The mean polymorphism information content of loci was 0.188. Genetic diversity coefficients according to the UPGMA ranged from 0.000 to 0.9375. Cluster analysis divided the 100 sainfoin genotypes into two main groups (Cluster-I and Cluster-II). All diploid genotypes (except for 1 diploid genotype) used in the study formed a separate group within Cluster-I. The results revealed that SSR markers used in this study are useful for molecular characterization and assessing genetic diversity of sainfoin accessions. The obtained SSR alleles and genetic variability in a studied certain loci provided significant information about the genetic structure of sainfoin accessions that could be used as parental lines in sainfoin breeding programs.

Genome-wide analysis of fatty acid desaturase genes in chia (Salvia hispanica) reveals their crucial roles in cold response and seed oil formation

Chia (Salvia hispanica) is a functional food crop with high α-linolenic acid (ALA), the omega-3 essential fatty acid, but its worldwide plantation is limited by cold-intolerance and strict short-photoperiod flowering feature. Fatty acid desaturases (FADs) are responsible for seed oil accumulation, and play important roles in cold stress tolerance of plants. To date, there is no report on systemically genome-wide analysis of FAD genes in chia (ShiFADs). In this study, 31 ShiFAD genes were identified, 3 of which contained 2 alternative splicing transcripts, and they were located in 6 chromosomes of chia. Phylogenetic analysis classified the ShiFAD proteins into 7 groups, with conserved gene structure and MEME motifs within each group. Tandem and segmental duplications coursed the expansion of ShiFAD genes. Numerous cis-regulatory elements, including hormone response elements, growth and development elements, biotic/abiotic stress response elements, and transcription factor binding sites, were predicted in ShiFAD promoters. 24 miRNAs targeting ShiFAD genes were identified at whole-genome level. In total, 15 SSR loci were predicted in ShiFAD genes/promoters. RNA-seq data showed that ShiFAD genes were expressed in various organs with different levels. qRT-PCR detection revealed the inducibility of ShiSAD2 and ShiSAD7 in response to cold stress, and validated the seed-specific expression of ShiSAD11a. Yeast expression of ShiSAD11a confirmed the catalytic activity of its encoded protein, and its heterologous expression in Arabidopsis thaliana significantly increased seed oleic acid content. This work lays a foundation for molecular dissection of chia high-ALA trait and functional study of ShiFAD genes in cold tolerance.

DNA marker identification of downy mildew resistance locus Rpv10 in grapevine genotypes.

One of the most common and harmful diseases of grapevine is downy mildew, caused by Plasmopara viticola. Cultivars of Vitis vinifera, the basis of high-quality viticulture, are mainly not resistant to downy mildew. Varieties with natural resistance to downy mildew belong to the vine species of North America and Asia (V. aestivalis, V. berlandieri, V. cinerea, V. labrusca, V. amurensis, etc.), as well as Muscadinia rotundifolia. The breeding of resistant cultivars is based on interspecific crossing. Currently, molecular genetic methods are increasingly used in pre-selection work and directly in breeding. One of the major loci of downy mildew resistance, Rpv10, was first identified in the variety Solaris and was originally inherited from wild V. amurensis. DNA markers that allow detecting Rpv10 in grapevine genotypes are known. We used PCR analysis to search for donors of resistance locus among 30 grape cultivars that, according to their pedigrees, could carry Rpv10. The work was performed using an automatic genetic analyzer, which allows obtaining high-precision data. Rpv10 locus allele, which determines resistance to the downy mildew pathogen, has been detected in 10 genotypes. Fingerprinting of grape cultivars with detected Rpv10 was performed at 6 reference SSR loci. DNA marker analysis revealed the presence of a resistance allele in the cultivar Korinka russkaya, which, according to publicly available data, is the offspring of the cultivar Zarya Severa and cannot carry Rpv10. Using the microsatellite loci polymorphism analysis and the data from VIVC database, it was found that Korinka russkaya is the progeny of the cultivar Severnyi, which is the donor of the resistance locus Rpv10. The pedigree of the grapevine cultivar Korinka russkaya was also clarified.

Genome survey sequencing-based SSR marker development and their validation in Dendrocalamus longispathus.

Bamboo is an important genetic resource in India, supporting rural livelihood and industries. Unfortunately, most Indian bamboo taxa are devoid of basic genomic or marker information required to comprehend the genetic processes for further conservation and management. In this study, we perform genome survey sequencing for development of de novo genomic SSRs in Dendrocalamus longispathus, a socioeconomically important bamboo species of northeast India. Using Illumina platform, 69.49 million raw reads were generated and assembled into 1,145,321 contig with GC content 43% and N50 1228bp. In total, 46,984 microsatellite repeats were mined-out wherein di-nucleotide repeats were most abundant (54.71%) followed by mono- (31.91%) and tri-repeats (9.85%). Overall, AT-rich repeats were predominant in the genome, but GC-rich motifs were more frequent in tri-repeats. Afterwards, 21,596 SSR loci were successfully tagged with the primer pairs, and a subset of 50 were validated through polymerase chain reaction amplification. Of these, 36 SSR loci were successfully amplified, and 16 demonstrated polymorphism. Using 13 polymorphic SSRs, a moderate level of gene diversity (He = 0.480; Ar = 3.52) was recorded in the analysed populations of D. longispathus. Despite the high gene flow (Nm = 4.928) and low genetic differentiation (FST = 0.119), severe inbreeding (FIS = 0.407) was detected. Further, genetic clustering and STRUCTURE analysis revealed that the entire genetic variability is captured under two major gene pools. Conclusively, we present a comprehensive set of novel SSR markers in D. longispathus as well as other taxa of tropical woody bamboos.

Production and verification of lemon × acid lime hybrid populations via embryo rescue

Interspecific hybridization between canker-tolerant Konkan Seedless lemon (C. limon Burm) andcommercially grown Kagzi lime (C. aurantifolia Swingle) is significantly impeded by the occurrence of nucellarembryony. Hence, the objective of our work was to enhance hybrid seedlings’ recovery and to confirm theirhybridity through polymorphic SSR markers. Results revealed quite a satisfactory germination (< 58%) from allthe stages of embryos and media used, but younger and oldest embryos produced more than 87% germinationon all three media tested. MS medium supplemented with 500 mg l-1 malt extract plus 1.5 mg l-1 GA3 and 20μll-1NAA proved better for the survival of plantlets, obtained from the culture of either aged or younger embryos,though it was highest in plantlets, obtained from the culture of 80-90 days old embryos. The seedlings obtainedfrom culture 80-90 old embryos (DAP or days after pollination) on MS medium supplemented with 500 mg l-1malt extract plus 1.5 mg l-1 GA3 and 20μl l-1 NAA had the highest root and shoot length at 30 days after culture(DAC). SSR loci CCSM4 and CAC-33 expressed the highest polymorphism, showing their ability to distinguishthe hybrids of Konkan Seedless (lemon) × Kagzi lime (acid lime) .

Molecular genotyping of sweet potato (Ipomoea batatas L. Lam) accessions using microsatellites

The experiment was conducted to ascertain the level of genetic diversity in sweet potato accessions using microsatellites. Thirty sweet potato accessions obtained from the International Potato Center (CIP), Kumasi, Ghana, Mozambique, and local germplasm of the National Root Crops Research Institute (NRCRI), Umudike, Abia State, Nigeria, as well as sweet potato vines from local farmers' fields in Jos, Plateau State, and Bauchi State, Nigeria, were analyzed for genetic diversity using five microsatellite markers. The results showed that the polymorphic SSR loci revealed diverse relationship among the sweet potato cultivars, which was grouped into four major clusters by unweighted pair group method analysis (UPGMA) method. Cluster analysis showed a Jaccard co-efficient ranging from 0.0 to 3.0 indicating high genetic diversity. The primers detected a total of 18 alleles and the number of alleles per locus was 4 for IBR-19, IBR-286, IBR-297 and 3 for IBR-16 and IBR-242 with an average of 3.67 alleles per locus. The polymorphic information content (PIC) of the markers varied from 0.35 to 0.72 with an average of 0.497. Marker IBR-19 revealed the highest PIC of 0.72, while marker IBR-297 had the lowest PIC of 0.35. Observed heterozygosity ranged from 0.32 to 0.89 with a mean of 0.675 across the five SSR loci. The results from the Analysis of molecular variance (AMOVA) which was used to quantify the diversity level and genetic relationship among the thirty sweet potato accessions indicated that a high diversity was mostly distributed within the populations for sweet potato accessions (75.12%) and (15.67%) among the populations.