Simple Sequence Repeat Molecular Markers Research Articles

Seed genetic purity testing of F1 Benincasa hispida var. chieh-qua hybrids using SSR molecular marker analysis

To ensure that farmers can access high-quality seeds, it is essential to find a simple, rapid and accurate method to assess seed purity. In recent years, heterosis in chieh-qua has been widely applied to production. Using the whole genome sequence of chieh-qua, we designed simple sequence repeat (SSR) primers specific for chieh-qua. The parental lines of nine hybrids were screened using 200 SSR primers, seven of which exhibited polymorphisms. The bands were clear, stable and reproducible. We found dominant and co-dominant bands between the parents of the nine hybrids. The seven pairs of SSR primers were successfully used to assess genetic purity of the nine chieh-qua hybrids. The SSR molecular marker purity assessment results were consistent with the results obtained from a field grow-out test (GOT). However, the use of SSR markers provided a more accurate, reliable and faster method for seed purity testing than the GOT. We propose using SSR molecular marker technology to assess the genetic purity of chieh-qua hybrid seeds. With this method, the seed quality can be determined faster, which may help to accelerate the chieh-qua breeding process.

Indigenous Mota-named coarse rice germplasm is distinct from fine-grained rice collected from south-central coastal Bangladesh as compared with the morphological descriptors and molecular markers

While crop enhancement can be performed using the agronomical adaptive traits of local crop varieties, human actions and climatic conditions alter their diversity. An assessment and characterization of 20 germplasm accessions of Mota-named coarse rice diversity alongside eight fine-grained rice from the south-central region of Bangladesh is our primary objective. In this original study, we perform the evaluation of genetic variability of Mota-named coarse rice in comparison with the fine rice from south-central coastal Bangladesh using morphological characteristics and SSR molecular markers. Nineteen morphological descriptors showed distinct variations. Three prominent groups surfaced from simple sequence repeat (SSR). Eighty-seven percent and 13% of genetic variation within and among populations were observed, respectively, using analysis of molecular variance. Five subpopulations emerged from model-based structure study. The studied SSR loci showed 0.642 (for mean polymorphism content value per primer), 0.862 (as highest value), and 0.683 (as average value) for gene diversity of every locus. Geographical information-based molecular analysis revealed high allelic richness (18–21) in the upazilas (counties) of Bauphal, Galachipa, and Dumki, all in the district of Patuakhali, division of Barisal. Morphological–molecular analysis of Mota-named coarse rice germplasm revealed a considerable amount of genetic diversity and a prominent level of population structure. Our results provide information useful for future breeding and conservation programs.

English

The assessment of genetic diversity among improved crop germplasm can facilitate the expansion of the genetic base for crop improvement in breeding program. However, little effort has been made to assess the level of genetic relatedness among released varieties and elite soybean [Glycine max (L.) Merr.] genotypes. The objective of this study was to determine the degree of genetic diversity that exists among released and elite soybean genotypes in Uganda. In this study, 21 polymorphic simple sequence repeat (SSR) molecular markers were used to determine the degree of genetic diversity and varietal identification among 34 soybean genotypes. A total of 59 alleles with an average of 2.85 alleles per locus were detected. The polymorphic information content (PIC) values ranged from 0.208 on BE806308 to 0.741 on Satt411, with an average of 0.5870. The expected heterozygosity varied from 0.208 on BE806308 to 0.725 on Satt411, with an average of 0.548 per marker. The dendrogram constructed based on Jaccard’s genetic similarities among 34 soybean genotypes identified three major clusters, with six of the released varieties belonging to cluster I. The majority of elite genotypes including three recently released cultivars; Maksoy 4N, Maksoy 5N and Maksoy 6N were grouped in cluster II and III. The results showed moderate genetic variation among the soybean genotypes, which could accelerate genetic vulnerability. Therefore, there is need to widen the genetic base of the working germplasm through the use of techniques such as pre-breeding and novel biotechnology techniques such as mutation breeding and CRISPR to create genetic variation necessary to cope with the dynamics of biotic and abiotic stresses that affect soybean production in Uganda.   Key words: Glycine max, genetic relationship, microsatellite or simple sequence repeat (SSR), molecular markers, genetic vulnerability.

SSRMMD: A Rapid and Accurate Algorithm for Mining SSR Feature Loci and Candidate Polymorphic SSRs Based on Assembled Sequences.

Microsatellites or simple sequence repeats (SSRs) are short tandem repeats of DNA widespread in genomes and transcriptomes of diverse organisms and are used in various genetic studies. Few software programs that mine SSRs can be further used to mine polymorphic SSRs, and these programs have poor portability, have slow computational speed, are highly dependent on other programs, and have low marker development rates. In this study, we develop an algorithm named Simple Sequence Repeat Molecular Marker Developer (SSRMMD), which uses improved regular expressions to rapidly and exhaustively mine perfect SSR loci from any size of assembled sequence. To mine polymorphic SSRs, SSRMMD uses a novel three-stage method to assess the conservativeness of SSR flanking sequences and then uses the sliding window method to fragment each assembled sequence to assess its uniqueness. Furthermore, molecular biology assays support the polymorphic SSRs identified by SSRMMD. SSRMMD is implemented using the Perl programming language and can be downloaded from https://github.com/GouXiangJian/SSRMMD.

In Vitro Assessment of Kurdish Rice Genotypes in Response to PEG-Induced Drought Stress

Rice (Oryza sativa L.) is productively affected by different environmental factors, including biotic and abiotic stress. The objectives of this research were to evaluate the genetic distinction among Kurdish rice genotypes using the simple sequence repeats (SSRs) molecular markers and to perform in vitro tests to characterize the drought tolerance of six local rice genotypes. The polymorphic information content (PIC) varied from 0.38 to 0.84 with an average of 0.56. The genetic distance ranged from 0.33 to 0.88. Drought stress had a significant impact (p ≤ 0.05) on callus growth parameters. Enzymatic antioxidant systems were predicted and exhibited a significant variation. The findings revealed that proline levels increase in proportion to polyethylene glycol (PEG) concentrations. Kalar and Gwll Swr genotypes showed the worst performances in phenotypic and biochemical traits, while Choman and Shawre exhibited the best phenotypic and biochemical performances. A positive and substantial relationship between callus fresh weight (CFW) and callus dry weight (CDW) was found under stressful and optimized conditions. Callus induction (CI) was positively and significantly associated with the catalase activity (CAT) in all stressed treatments. Based on the results for callus growth and the biochemical parameters under stress conditions, a remarkable genotype distinction, based on the tolerance reaction, was noted as follows: PEG resistant > susceptible, Choman > Shawre > White Bazyan > Red Bazyan > Gwll Swr > Kalar. The CI and CAT characteristics were considered as reliable predictors of drought tolerance in rice genotypes.

Genome survey sequencing and identification of genomic SSR markers for Rhododendron micranthum.

Rhododendron micranthum is an evergreen shrub species widely distributed in China that has high ornamental and medicinal value. However, there is a lack of molecular and genomic data for this plant, which severely restricts the development of its relevant research. The objective of the present study was to conduct a first genomic survey of R. micranthum and determine its whole-genome sequencing scheme. Next-generation sequencing (Illumina Hi-Seq Xten) was used to measure the genome size of R. micranthum, K-mer analysis were employed to investigate its genomic profile. Finally, we conducted bioinformatics methods to performed SSR (simple sequence repeat) prediction based on the genomic data. The genome size of R. micranthum was estimated to be 554.22 Mb. The heterozygosity ratio was 0.93%, and the sequence repeat ratio was calculated to be 49.17%. The clean reads of R. micranthum were assembled into 2281551 scaffolds with a N50 value of 916 bp. A total of 479724 SSR molecular markers were identified in the R. micranthum genome, and 871656 pairs of primers designed for application. Among of them, 100 primer pairs were validated, and 71 primer pairs were successfully amplified. In summary, the R. micranthum genome is complex with high heterozygosity and low repeated sequences. In future whole-genome research in R. micranthum, higher-depth ‘2+3’ (Illumina+PacBio) sequencing may yield better assembly results.

Efficient plant regeneration from in vitro leaves and petioles via shoot organogenesis in Sapium sebiferum Roxb.

Sapium sebiferum Roxb. is a widespread and economically important multipurpose tree due to its high value in ornamental, and biodiesel production as well as medicine. A highly efficient in vitro plant regeneration system through direct shoot organogenesis was established for the first time from leaves and petioles of S. sebiferum. The results showed that plant growth regulators (PGRs), mechanical damage, explant orientation, explant source, and developmental stage had a strong influence on the in vitro morphogenesis of S. sebiferum. For shoot organogenesis from leaves, the highest adventitious shoot induction rate (96.67%) with 25.67 shoots per explant was obtained when mechanically damaged leaves (the first three leaf explants at the top, leaf #1–3) were cultured with the abaxial surface placed down on Murashige and Skoog (MS) medium containing 0.5 mg L−1 thidiazuron (TDZ). For in vitro morphogenesis of petioles, the combination of 1-naphthylacetic acid (NAA) and 6-benzylainopurine (6-BA) played a key role in cell fate determination. All of the in vitro petioles produced adventitious shoots on MS medium containing 1.0 mg L−1 6-BA and 0.1 mg L−1 NAA, while they produced green calli on medium fortified with 0.5 mg L−1 6-BA and 1.0 mg L−1 NAA. The shoots were subcultured in medium fortified with 0.5 mg L−1 6-BA and 0.1 mg L−1 NAA for multiplication and elongation. The elongated shoots successfully rooted on half-strength MS (1/2 MS) medium fortified with 0.5 mg L−1 indole-butyric acid (IBA) and 0.25 mg L−1 indole-3-acetic acid (IAA), and the regenerated plantlets successfully acclimatized with a survival rate of 92.56% in the greenhouse. The genetic fidelity of in vitro regenerated plants was evaluated using inter simple sequence repeat molecular markers. The in vitro regenerated plants were found to be the true to their mother plant. This study will be beneficial for the large-scale propagation as well as the genetic improvement of S. sebiferum.

Genetic diversity and population structure of Colletia paradoxa (Rhamnaceae) a medicinal species threatened with extinction in Brazil

In order to obtain a better knowledge about the endangered species Colletia paradoxa (Rhamnaceae), which appears in fragmented populations, a study was carried out with six populations from different physiographic regions of Rio Grande do Sul and Santa Catarina states, Brazil. The objective of this study is to investigate the real conditions of genetic diversity in populations, through the use of inter simple sequence repeat (ISSR) molecular marker, and to evaluate the distribution of genetic diversity among and within populations, in order to plan conservation and management strategies. Five selected primers produced 112 DNA fragments in 46 samples from individuals that were evaluated. The result of the analyses showed 100% polymorphism. Nei’s genetic diversity index (H) was 0.3083. Shannon’s information index (I) was 0.4880. Bayesian inference of clustering performed by Structure revealed the most likely K = 4 to C. paradoxa. The analysis of molecular variance among all populations showed that the greatest genetic diversity occurs within the populations (82%), while among them there is less differentiation (18%), although in some populations good levels of genetic migration occur (Nm = 1.0465) indicating that genetic exchanges among populations of C. paradoxa are still occurring.

High efficiency plant regeneration and genetic fidelity of regenerants by SCoT and ISSR markers in chickpea (Cicer arietinum L.)

High efficient and repeatable in vitro regeneration protocol was established from embryo axis, half-seed, axillary meristem, and cotyledonary node explants of chickpea. Various concentrations and combinations of various plant growth regulators (PGRs) were employed to induce multiple shoots, shoot elongation and rooting of shoots to obtain complete plantlets of chickpea. The pretreatment of seeds with 6-benzyl aminopurine (BAP) at 1.0 mg l−1 was found to significantly increase the multiple shoot regeneration from the all explants tested. Among three PGRs such as BAP, kinetin (KIN) and thidiazuron (TDZ) tested for multiple shoot induction; BAP at 2.0 mg l−1 produced the maximum number of shoots in all tested explants. The maximum number of shoots (48.80 shoots/explant) was attained from the embryo axis explant followed by half-seed (32.76 shoots/explant), axillary meristem (28.34 shoots/explant) and cotyledonary node explant (18.47 shoots/explant) on medium augmented with 2.0 mg l−1 BAP along with 0.05 mg l−1 Indole-3-butyric acid (IBA). The optimum percentage of shoot elongation response was recorded (96.68%) on medium fortified with IAA (0.05 mg l−1), GA3 (1.0 mg l−1) and BAP (1.0 mg l−1) with an average shoot length of 8.82 cm. The elongated shoots were successfully rooted in medium augmented with 2.0 mg l−1 IBA. The complete plants were acclimatized in the greenhouse with a survival rate of 72%. The plantlets regenerated from four explants appeared to be morphologically similar to mother plants. The genetic fidelity of in vitro regenerated plants was evaluated using Start Codon Targeted and Inter simple sequence repeats molecular markers. The in vitro regenerated plants from all four explants were found to be the true to type with their mother plant. The in vitro protocol presented in the study should offer as a feasible system for chickpea genetic transformation. An efficient and reproducible in vitro regeneration protocol was established for chickpea. Application of different concentrations and combinations of PGRs was found to enhance multiple shoot induction, shoot elongation, rooting and acclimatization of in vitro regenerated plants in field conditions, and further evaluated genetic fidelity using molecular markers.

Mapping Quantitative Trait Loci Associated With Resistance to Aflatoxin Accumulation in Maize Inbred Mp719.

Aflatoxins are carcinogenic and toxic compounds produced principally by fungal species Aspergillus flavus (Link: Fries) and A. parasiticus (Speare), which are common contaminants of food and feed. Aflatoxins can be found at dangerously high levels and can readily contaminate pre-harvest maize (Zea mays L.) grain. Sources of resistance to aflatoxin accumulation in maize have been identified, however, the highly quantitative nature and complex inheritance of this trait have limited the introgression of aflatoxin accumulation resistance into agronomically desirable lines. Mapping of quantitative trait loci (QTL) was performed on a bi-parental population comprised of 241 F2:3 families derived from the cross of inbred lines Mp705 (susceptible) × Mp719 (resistant). The mapping population was phenotyped in replicated field trials in three environments for resistance to aflatoxin accumulation under artificial inoculation with an A. flavus spore suspension. The genetic linkage map was constructed with 1,276 single nucleotide polymorphism (SNP) and simple sequence repeat (SSR) molecular markers covering a total genetic distance of 1,642 cM across all ten maize chromosomes. Multiple interval mapping revealed that majority of the aflatoxin-reducing alleles and the source for the larger effect QTL identified in this study were contributed from Mp719, the resistant parent. Two QTL identified on chromosome 1 (bin 1.06–1.07) and chromosome 3 (bin 3.09) were the most stable across different environments and when combined, explained 24.6% of the total phenotypic variance across all three environments. Results from the study showed that these chromosomal regions harbor important QTL for influencing aflatoxin accumulation, which is consistent with previous reports with other different mapping populations. These stable QTL were the most promising for controlling aflatoxin accumulation in maize grain. Identifying beneficial alleles derived from Mp719 and closely linked molecular markers through QTL analysis for implementation of MAS could accelerate breeding efforts to reduce aflatoxin accumulation in maize.

Development of SSR markers in Paeonia based on De Novo transcriptomic assemblies.

Peony is a famous ornamental and medicinal plant in China, and peony hybrid breeding is an important means of germplasm innovation. However, research on the genome of this species is limited, thereby hindering the genetic and breeding research on peony. In the present study, simple sequence repeat (SSR) locus analysis was performed on expressed sequence tags obtained by the transcriptome sequencing of Paeonia using Microsatellite software. Primers with polymorphism were obtained via polymerase chain reaction amplification and electrophoresis. As a result, a total of 86,195 unigenes were obtained by assembling the transcriptome data of Paeonia. Functional annotations were obtained in seven functional databases including 49,172 (Non-Redundant Protein Sequence Database: 57.05%), 38,352 (Nucleotide Sequence Database: 44.49%), 36,477 (Swiss Prot: 42.32%), 38,905 (Clusters of Orthologous Groups for Eukaryotic Complete Genomes: 45.14%), 37,993 (Kyoto Encyclopedia of Genes and Genomes: 44.08%), 26,832 (Gene Ontology: 31.13%) and 37,758 (Pfam: 43.81%) unigenes. Meanwhile, 21,998 SSR loci were distributed in 17,567 unigenes containing SSR sequences, and the SSR distribution frequency was 25.52%, with an average of one SSR sequence per 4.66 kb. Mononucleotide, dinucleotide, and trinucleotide were the main repeat types, accounting for 55.74%, 25.58%, and 13.21% of the total repeat times, respectively. Forty-five pairs of the 100 pairs of primers selected randomly could amplify clear polymorphic bands. The polymorphic primers of these 45 pairs were used to cluster and analyze 16 species of peony. The new SSR molecular markers can be useful for the study of genetic diversity and marker-assisted breeding of peony.

Mapping Quantitative Trait Loci for Resistance to Fall Armyworm (Lepidoptera: Noctuidae) Leaf-Feeding Damage in Maize Inbred Mp705.

The fall armyworm, Spodoptera frugiperda (J. E. Smith), is an agronomically important pest that severely limits maize (Zea mays (Linnaeus) [Poales: Poaceae]) production. This migrant insect devastates maize plants in many countries threatening the livelihood of millions. Quantitative trait loci (QTL) were mapped to identify chromosomal regions that control resistance to fall armyworm leaf-feeding and to identify molecular markers linked to the target loci for use in marker-assisted selection (MAS). A bi-parental mapping population, comprising 243 F2:3 families from the cross Mp705 (resistant) × Mp719 (susceptible), was evaluated for fall armyworm leaf-feeding damage under artificial infestation over 3 yr. A linkage map comprised of 1,276 single-nucleotide polymorphism and simple sequence repeat molecular markers was constructed. Quantitative trait loci analyses identified two major QTL in bins 4.06 and 9.03 that when combined, explained 35.7% of the phenotypic variance over all environments. Mp705 was responsible for the leaf-feeding damage reducing alleles for both large effect QTL and most of the small effect QTL identified in this study. The QTL identified in bin 9.03 co-locates with a previously identified QTL that controls resistance to leaf-feeding damage in maize by fall armyworm and other lepidopteran insects. The QTL in bin 4.06 is a new source of resistance identified in this study. Beneficial alleles derived from Mp705 for the application of an integrated QTL-MAS approach could accelerate breeding efforts to minimize fall armyworm leaf-feeding in maize.

Diagnostic fingerprints ISSR/SSR for tropical leguminous species Stylosanthes capitata and Stylosanthes macrocephala

ABSTRACT: Stylosanthes capitata Vogel and Stylosanthes macrocephala M.B.Ferreira & Sousa Costa are two forage leguminous species of agronomic importance for animal husbandry in tropical environments. The physical mixture of both species (80 % S. capitata and 20 % S. macrocephala) comprises the commercial cultivar “Estilosantes Campo Grande”. However, proximity of fields for seed production may contaminate seed lots, compromising seeds quality. The combined use of dominant and co-dominant molecular markers is an appropriate strategy to certificate genetic purity and perform diversity studies of cultivars. In this research, a set of ISSR (Inter-Simple Sequence Repeat polymorphic DNA) and SSR (Simple Sequence Repeat polymorphic DNA) molecular markers were standardized to characterize S. capitata and S. macrocephala species and evaluate the genetic purity of commercial samples. Four ISSR markers (UBC 2, 864, 885, 886) and SSR marker SC18-01 G4B showed precise species-specific electrophoretic fingerprints for both species. Electrophoretic patterns of ISSR molecular markers should be displayed first to confirm the sample identification. The structure analysis showed that the less contaminated sample was S. capitata with 97 % of its genetic composition assigned to a single genetic cluster vs. 95 % for S. macrocephala. S. capitata has greater genetic diversity (ISSRHe:0.292; SSRHe:0.57) than S. macrocephala (ISSRHe:0.285; SSRHe:0.16); however, this difference was only significant with SSR molecular markers. As these genetic resources have considerable ecological, agronomic and economic importance, tools for accurate species identification and genetic studies are essential for further seed multiplication, as well as for improvement and conservation of cultivars.

Genetic diversity and variability among pigmented rice germplasm using molecular marker and morphological traits

Exploring the genetic aspects of germplasm using genotypic and phenotypic analysis is more reliable for the development of rice varieties that could lead to the attainment of food security. This is because genetic variations provide a space for recombinants, which is an important factor in new varietal development. This study was conducted to evaluate genetic variability and diversity among 32 coloured rice accessions using agro-morphological characteristics and simple sequence repeats (SSR) markers. Quantitative traits (morphological, grain quality and antioxidant properties) and 34 SSR molecular markers were used as tools for determining cultivar identities and genetic diversity. Most of the quantitative traits showed significant differences (p ≤ 0.01) among all rice accessions. Clustering analysis from quantitative traits categorised the accessions into four groups. Similarly, the 32 accessions were grouped into 4 cluster groups based on the analysis of 34 SSR markers. The accessions YTM15, Pulut Merah 3, Padi Randau, Ringan Bawang, DNJ128 and DV 107 can be potentially selected for development of new varieties for local cultivation. Finally, these accessions can be used as parents in further breeding programmes.

Genetic Similarity of Local Mandarin Accessions (Citrus reticulata) Resulted from Endosperm Culture According to ISSR and Microsatelite Markers

Citrus is the third most important fruit commodity in Indonesia, especially seedless citrus. One technique to get seedless citrus is by endosperm culture possessing triploidy sets of the chromosome. Plants that have triploidy sets of the chromosome will usually become sterile or seedless plants. This research aimed to 1) compare ratio of the length and width of the leaves between three types of local mandarin (Citrus reticulata) as mother plants and mandarin accessions grown from endosperm cultures, 2) identify mother plant and mandarin accessions from endosperm cultures with Inter Simple Sequence Repeats (ISSR) markers and 3) identify mother plant and mandarin accessions from endosperm cultures with Simple Sequence Repeats (SSR) markers. The research was conducted at Indonesian Citrus and Subtropical Fruits Research Institute (ICSFRI) Batu started from October 2018 until January 2019. The plants used in the study were Garut mandarin and seven accessions grown from endosperm cultures. Identification of plants with morphological markers has several disadvantages due to environmental factors. Identification with molecular markers is needed to complete the morphological information obtained. SSR markers are more suitable to be used in this study compared to ISSR markers because the results are in accordance with the objectives of this study, which is not expected to have high genetic variation. Identification with SSR molecular markers is more suitable because it showed low genetic diversity or no genetic diversity between mother plants and plants endosperm cultures when compared to ISSR markers. The desired change in the plants produced by endosperm cultures is only in ploidy properties.

Genetic variation of Eruca sativa L. genotypes revealed by agro-morphological traits and ISSR molecular markers

Taramira (Eruca sativa L.) is an oilseed crop with extensive industrial and pharmaceutical applications. The present study was carried out to identify genetic diversity in 60 different genotypes of E. sativa L. using 19 inter simple sequence repeats (ISSR) molecular markers and 12 agro-morphological traits, collected from different geographical regions of the world. The results of analysis of variance (ANOVA) indicated that there was a significant difference among genotypes in terms of all agronomic traits (except for capitula length and 1000-seed weight). In this study, genotype 161 (with Iranian origin) had the highest grain yield per plant (519.57 gm−2). The number of 19 ISSR primers generated a total of 286 polymorphic bands which polymorphism information content (PIC) value varied from 0.17 (in UBC827 primer) to 0.31 (in UBC825 primer) with average of 0.25. Genetic diversity statistics showed that, the UBC840 primer had the most significant role in differentiating genotypes. Taramira genotypes were divided into three subgroups in cluster analysis of molecular traits using unweighted pair group method with arithmetic mean (UPGMA) method and the STRUCTURE methods. There was a considerable similarity between the geographic origin of genotypes and molecular classification pattern. Therefore, the selection of genotypes with a high genetic distance in terms of the molecular marker, along with desirable agronomic traits, can be effective in future breeding programs of taramira in order to produce new superior hybrids.

Molecular characterization of a germplasm bank of Platonia insignis Mart.: a fruit tree

Platonia insignis Mart. is a fruit species native from Brazil, known as “bacurizeiro” and commonly used in food culture in the northern region of Brazil. The exploitation of the fruit is important for local commercialization and especially for family farmers that use it as a source of income. The objective of this work was to characterize the genetic diversity conserved in a germplasm bank based on inter simple sequence repeats (ISSR) molecular markers. We used 31 accessions collected in different sampling localities in the State of Para, Brazil and genotyped with eleven ISSR primers. The eleven primers amplified 85 loci, of which 67 loci were polymorphic. The dendrogram generated by the UPGMA method and based on the genetic similarities of Jaccard did not show formation of groups according to the sampling localities. Bayesian analysis identified the presence of two genetic groups (K = 2), and the distribution of the genetic groups between the accessions was also not related to the sampling locality. The analysis of the main coordinates (PCoA) showed that the first two components analyzed explained 30.14% of the total variation between the accessions, and accessions did not cluster according to sampling locality. AMOVA indicated a smaller portion of genetic variation between (2%) than within the sampling localities (98%), with ΦPT of 0.023 among sampling localities.

Field Evaluation and Molecular Analysis of Three Early Flowering Canola Mutants under Natural Salinity-Stressed Environment

Mutagenesis is a good way to create new plant lines having desirable traits. Therefore, this study was conducted to evaluate three gamma ray-induced early flowering canola mutants, their parent (Serw4) and chick verity (Serw6). Some agronomic traits were evaluated at M6 and M7 generations, under natural saline conditions. The results revealed that all the mutants were earlier and had more seed yield production than their parent and the chick in both two generations and combined. Otherwise, the mean performance of the other studied traits is significantly decreased. Furthermore, the ability of the mutants for salt stress tolerance was evaluated in vitro by treating seedlings with different concentrations of NaCl. All the mutants were the most salt tolerant. On the other hand, genetic diversity and similarity relationships among all genotypes were measured by inter simple sequence repeat (ISSR) molecular marker. The percentage of polymorphism was 50%, while the highest similarity was 0.90 between Serw4 and Serw6, but the lowest similarity (0.79) was detected between Serw6 and M11-4-1 mutant. Moreover, the dendrogram classified all the genotypes into two main clusters, the mutant's parent and the chick variety were gathered in one cluster, while all the mutants were collected in the other one. These induced earlier flowering mutants having higher seed yield production and salinity tolerance, suggested that these mutants could be new great promising mutant lines.

Molecular investigation in few spices of Dacocephalum in Iran: Species relationship, reticulation and divergence time

It is believed that Lamiaceae or the Mint family has numerous paraphyletic genera and the relationship between the species has not been fully determined in several cases. The genus Dracocephalum is one of the genera, which has not been investigated thoroughly. In the family Lamiaceae, Dracocephalum is the second largest genus consisting of about 186 species. Eight Dracocephalum species were reported of Iran belonging to the Irano-Turanian phytogeographical region. The present study has attempted to investigate Dracocephalum in terms of the molecular phylogeny for the first time and gather data on their species relationship, divergence time, and show how they are distributed geographically. To report the results, Chloroplast DNA (ribosomal protein L16 (rpL16)) sequences and Inter Simple Sequence Repeat (ISSR) molecular markers have been used. According to the results, the genus Dracocephalum is paraphyletic and has close affinity with Lallemantia. The results of horizontal gene transfer (HGT) analysis confirmed the inter-specific hybridization in the genus. Chronogram obtained by Bayesian Evolutionary Analysis Sampling Trees (BEAST) date revealed Dracocephalum species divergence time back to 2–10 million years ago(MYA). These findings extend the knowledge on the evolution of Lamiaceae.

Using genome conservation between Lotus japonicus and agronomically important Lotus species for discovering drought tolerance QTLs

Lotus tenuis is a perennial forage legume species closely related to the model legume L. japonicus. Genetic relatedness between cultivated and model Lotus species has not been exploited so far. Furthermore, L. tenuis drought tolerance cultivars for marginal environments are in high demand. Marker-assisted selection is a modern breeding tool that allows to dissect genetically complex traits such as drought tolerance and to select for favorable alleles. However, L. tenuis is considered an orphan crop because no genome sequencing effort has been made. The objective of this work was to perform association mapping analyses on a L. tenuis population (LtAM) comprised of 100 genotypes for identifying genomic regions (QTLs) controlling drought tolerance. The LtAM population was established on a two-water regime (rainfed and irrigated) under a Mediterranean climate condition. Phenotyping was carried out during two growing seasons and included the measurement of important physiological and agronomical traits. Genetic characterization was performed with 88 transferable simple sequence repeats (SSR) molecular markers from L. japonicus genome. Association mapping analyses allowed to identify 40 marker-trait associations, almost 50% of them were found on chromosomes two and six of L. japonicus genome. For seven SSR-markers, 11 putative candidate genes were found. Based on annotations from the Medicago truncatula and Arabidopsis thaliana genomes, these genes could plausibly be candidates of causative polymorphism altering biochemical and physiological functions involved in the abiotic stress responses.