Repeat Primers Research Articles

Rep-PCR Analyses Reveal Genetic Variation of Ralstonia solanacearum Causing Wilt of Solanaceaous Vegetables in Bangladesh.

Ralstonia solanacearum, a soil-borne and seed-borne plant pathogenic bacterium, causes bacterial wilt to several important crop plants causing substantial economic losses. To provide population information on this pathogen for developing effective control strategies, Rep-PCR was used to analyze the genetic variation of 18 representative isolates of R. solanacearum collected in Bangladesh. Phenotypic analyses revealed that all eighteen isolates belong to biotype 3 with wide diversity in aggressiveness on eggplant, tomato, and chili. Rep-PCR studies utilizing the REP, ERIC, and BOXIR primers showed a wide variation at the genetic level among the R. solanacearum isolates used in this study. Dendrogram constructed using REP, ERIC, and BOXIR primers based on banding patterns implied that R. solanacearum isolates were genetically diversified and distributed in four clusters at 83%, 80%, and 63% similarity index, respectively. The genetic relationship assayed by rep-PCR highlighted a wide range of genetic variation but no relation among geographical origin, aggressiveness, and phylogenetic groups of R. solanacearum isolates. These results conceded that other molecular markers related to virulence gene(s) might reveal the complex relationship among geographical origin, aggressiveness, and phylogenetic groups.

Molecular Assessment of Genetic Diversity and Genetic Structure of Rhanterium epapposum Oliv. in Scarce Populations in Some Regions of Western Saudi Arabia.

Rhanterium epapposum Oliv. is a perennial medicinal shrub growing mainly in desert habitats in the Arabian Peninsula. In western Saudi Arabia, the remaining few populations of this species are exposed to many threats, including overcutting, overgrazing, and recently, increasing human activities. These threats are predicted to be exacerbated by the advancement of aridification caused by climate change. The conservation and recovering of the diminished populations of R. epapposum necessitate measurement of their genetic diversity and genetic differentiation. To accomplish this objective, we tested 150 simple sequence repeat (SSR) primer pairs, with which 40 polymorphic loci were identified. These polymorphic loci were used to determine the population genetics of 540 plant accessions sampled from a total of 45 populations of R. epapposum located in 8 sites in western Saudi Arabia: Wadi Khurieba, Wadi Al Khamas, Gebel Al Twaal, Al Asaafer, Wadi ALHamda, Wadi Al Nassayeif, Wadi Qaraba, Wadi Kuliayah, and Wadi Dahban. Low levels of genetic diversity were found in all populations (the values of the PPL ranged between 52.5 and 15) along with a declined value of HT (0.123) and a considerable inbreeding value (F = 0.942), which confirmed a noticeable shortage of heterozygotes. High genetic differentiation among the populations and a low value of gene flow are indicative of high isolation among the R. epapposum populations, which has caused a severe deficiency in gene migration. The data obtained herein inspire several recommendations for conservation and retrieval of the existing populations, including seed banks, restoration of diminished populations, and monitoring and prevention of cutting and grazing activities at threatened sites. All of these measures are urgently required to avoid imminent extinction.

Genetic characterization of cultivated apple (Malus x domestica Borkh.) in Morocco using microsatellite (SSR) markers

Genetic diversity of 27 apple genotypes from four geographical regions of Morocco was analyzed using 26 simple sequence repeat (SSR) primers. In total, 195 polymorphic SSR alleles were detected, with sizes ranging from 78 to 209 bp. The results obtained for the average number of alleles per locus (7.5), effective number of alleles (4.62), Shannon information index (1.66), expected heterozygosity (0.76), observed heterozygosity (0.74) and polymorphism information content (0.76) showed a moderate level of polymorphism, which suggests that Moroccan apple cultivars exhibit significant genetic diversity. Over the 26 SSR loci used, the probability of identity P (ID) was 4.62 10 −29 indicating that most markers were highly discriminating and powerful for cultivars characterization. The principal coordinate analysis and hierarchical classification suggested the presence of four well defined groups, which was confirmed later by model based population structure. Furthermore, strong genetic differentiation between these four groups was also detected (FST = 0.28) . Most of the individuals segregated into the four groups showing a membership coefficient greater than 0.80, which indicates their better genetic integrity. A parentage analysis, however, shows that cultivars from the four regions are minimally hybridized. After comparing SSR profiles and genotypes names, we conclude that the problem of homonyms and/or labeling errors appear in the studied cultivars.Thus, based on genetic profiles, two individuals (Beldi 1/Beldi 2 and Laakri 5/Laakri 6) found to be duplicated. Additionally, some cultivars with the same name are grouped in different clusters suggesting the existence of possible homonymy. Lastly, the present work confirms the usefulness of SSR markers for the elimination of duplications and characterization of diversity and hybrid characters of apple genotypes in Morocco.

Genetic diversity in populations of Girardinia diversifolia from Nepal Himalaya using ISSR markers

Girardinia diversifolia (Link) Friis from Urticaceae family is a perennial herb, commonly known as the Himalayan giant nettle. It has cultural, medicinal, and economic values among the indigenous peoples and local communities living in the mountains of Nepal and India. Prior knowledge of genetic diversity in plant species may contribute to conservation and sustainable utilization of important genotype. The aim of this study is to assess the genetic diversity within and among different populations of the 45 accessions of G. diversifolia collected from Far-western, Central, and Eastern regions of Nepal. The amplification of genomic deoxyribonucleic acid (DNA) with ten inter-simple sequence repeat (ISSR) primers yielded 131 clear DNA bands, of which 98.09% were found to be polymorphic. The mean effective number of alleles (ne), Nei's gene diversity (h) and Shannon's information index (I) were 1.598, 0.349, and 0.523, respectively. The total genetic diversity Ht (0.350 ± 0.04), low intrapopulation genetic diversity Hs (0.141 ± 0.03) and low estimated gene flow Nm (0.355 ± 0.11) reflected high genetic differentiation among population Gst (0.594 ± 0.08). The analysis of molecular variance among and within five populations of G. diversifolia showed that the value of genetic variation among the population was 60% whereas the value of genetic variation within the population was 40%. The results show that the ISSR markers are informative for the study of genetic diversity, which may help in the conservation and sustainable utilization of G. diversifolia and allied taxa.

Analysis of genetic diversity among Onobrychis accessions with high agronomic performance by simple sequence repeat (SSR) markers.

Onobrychis viciifolia Scop. is a short-lived perennial cool-season legume used for forage production. It is a common native species in Asia Minor, especially in Turkey, the districts of the Caucasus, and the Caspian fringes. It can grow well in a broad range of climatic and soil types found in Asia, Europe, and North America. It is a non-bloating crop, making it suitable for use in both hay and pasture. The aim was to assess the diversity of the 83 sainfoin genotypes selected based on their high agronomic performance from a germplasm collection evaluated in the experimental field of Tekirdag Namik Kemal University, Turkey. Ten nuclear simple sequence repeat (nSSR) primers (OVK036, OVK046, OVK094, OVK101, OVK125, OVK161, OVK174, OVM033, OVM061, and OVM125) were used in the study. All nSSR loci were found to be polymorphic and totally 92 alleles were detected. The mean observed number of alleles per locus was calculated as 9.2. Among the genetic diversity parameters, Shannon Index (I = 0.375), unbiased genetic diversity value (uh = 0.243), and mean polymorphic information content (PIC = 0.240) were calculated. The genetic distance value varied between 0.43 and 0.95. Based on the dendrogram built by the UPGMA clustering method using genetic distance values, it was observed that the studied sainfoin genotypes were divided into two main clusters, whereas the STRUCTURE analysis results had high support for three clusters. The results obtained from this study provide important information on the genetic structures of the studied sainfoin genotypes and their genetic relationship. Therefore acquired genetic data will be useful in designing more efficient polycross nurseries, allowing open pollination of best performing and genetically diverse genotypes in the isolated conditions, which will increase genetic gain in sainfoin breeding programs.

Characterization and Genetic Diversity of Pseudomonas syringae pv. syringae Isolates Associated with Rice Bacterial Leaf Spot in Heilongjiang, China.

Simple SummaryIn Northeast China, rice bacterial brown leaf spot caused by Pseudomonas syringae pv. syringae is among the most damaging rice diseases. This disease results in significant yield losses. This study focuses on a comprehensive analysis of the pathogen, population structure, and genetic diversity within the species based on biochemical tests and genetic characterizations. Our results indicate high genetic heterogeneity in Pseudomonas syringae pv. syringae isolates, and clustering of testing isolates and reference strains are related with the genomospecies 1. This work contributes to the physiological classification of the P. s. pv. syringae isolated from Heilongjiang Province, China and the results present new data concerning the phylogeny and genetic diversity. Such studies have not been reported about P. s. pv. syringae from this region yet.In China, rice is one of the most important cereal crops. Rice bacterial brown leaf spot caused by P. s. pv. syringae is among the most damaging rice diseases in the Heilongjiang Province of China and results in substantial yield losses. In this study, a comprehensive analysis of the pathogen, population structure, and genetic diversity within the species was performed. For this purpose, 176 bacterial isolates of P. s. pv. syringae collected from 15 locations were characterized by using biochemical tests such as the LOPAT test, and genetic characterizations such as multilocus sequence analysis (MLSA) and repetitive PCR, using BOX, REP and ERIC primers. Biochemical testing and detection of syrB genes confirm the presence of P. s. pv. syringae, genetic characterization by MLSA and genetic fingerprinting by repetitive PCR confirmed that high genetic heterogeneity exists in the P. s. pv. syringae isolates, and clustering of the tested isolates and reference strains are related with the same genomospecies 1. This work contributes to the physiological classification of the P. s. pv. syringae isolated from Heilongjiang Province, China, and the results present new data concerning the phylogeny and genetic diversity. This type of study about P. s. pv. syringae has been not reported from this region until now.

Susceptibility assessment and genetic population structure associated with Rhizoctonia solani AG3-PT - Potato stem canker disease

Rhizoctonia solani Kühn AG3-PT causes stem canker (SCD), and significantly impacts potato, Solanum tuberosum L. production globally. However, up to date, there are few resistant or tolerant potato cultivars to R. solani. Given the importance of the subject, we screened ninety-two potato genotypes to SCD, R. solani in the field and greenhouse conditions. Of which, fifty genotypes were subjected to genetic diversity analyzing using 20 Inter Simple Sequence Repeats (ISSR) primers. SCD severity on genotypes ranged from 5 to 73% in the field, and out of 20 used ISSR primer pairs, 14 primers generated 485 loci, of which 216 (45%) showed polymorphic bands. The highest percentage (67%) of polymorphisms was in uBC834 primer. Polymorphism information content value varied from 0.262 to 0.459 with an average of 0.338. Nei's measure of average population genetic diversity per locus ranged from 0.06 to 0.5. Effective number of alleles, Nei's genetic diversity and Shannon's Information Index were 1.530, 0.266, and 0.392, respectively. uBC834 and uBC811 primers identified the most polymorphic loci. According to simple-matching coefficient, the highest similarity (99%) was among “KSG64”, “KSG82”, ‏“KSG81”, ‏“KSG21”, “KSG31”, and “KSG109”, and lowest similarity (54%) in “8708/177” and “KSG300”. Thus, resistant sources can play a significant role in disease management in an integrated pest management planning program. Moreover, diverse potato resistant/susceptible genotypes reported in this study will be useful to generate mapping populations for potato and work in breeding programs for developing and releasing new resistant cultivars for SCD, R. solani infection.

Enzyme activity and population genetic structure analysis in wheat associated with resistance to Bipolaris sorokiniana-common root rot diseases

Common root rot disease (CRR) caused by Bipolaris sorokiniana (Sacc.) (Pleosporaceae), is an important fungal disease of wheat, Triticum aestivum (Poaceae), causing considerable yield losses globally. Incorporating genetic resistance in cultivated crops is considered the most efficient and sustainable solution to counter root rot diseases. Moreover, resistance to CCR is quantitative in nature, and thus the mechanism is poorly understood. To this aim, we analyzed the activities of defense-related enzymes; peroxidase (POX), superoxide dismutase (SOD), polyphenol oxidase (PPO), catalase (CAT), phenylalanine ammonia-lyase (PAL), β-1,3-glucanase (GLU) and chitinase (CHI), as well as total phenol content (TPC) to CRR on the three known resistant wheat ‘Alvand’ and ‘Bam’, ‘Mehregan’ at different time points (wpi) following CRR pathogen, B. sorokiniana inoculation. Of which, were selected out of 33 wheat cultivars previously screened for resistance to CRR. We also analyzed the genetic variability of the entire germplasm, 33 wheat cultivars using seven simple sequence repeat (SSR) primer pairs. The activity of antioxidant enzymes was increased in the related resistant genotypes. Of which, ‘Bam’ had the highest increase in PPO, and GLU activities, followed by ‘Alvand’ in SOD, PAL, and CHI significantly. Whereas, ‘Mehregan’ showed the highest level of TPC, POX, and CAT activities. In addition, five out of seven used SSR primers produced a total of 20 polymorphic bands, of which the number of alleles in each gene locus varied within 3–7 bands. The polymorphism information content (PIC) value also ranged from 0.44 to 0.81, with the mean of 0.65, Shannon Information Index (I) between 0.29 and 0.63 with an average of 0.47 per locus, and Nei's gene diversity (h) value varied from 0.16 to 0.44 with an average of 0.32. The average number of effective alleles was 1.52, ranging between 1.21 and 1.8. The gene locus Xgwm 140 showed the highest diversity in the population genetic structure, which explains the ability of the primers to resolve the assayed germplasm. Thus, resistance to CRR in wheat was mainly related to the enhancement of antioxidant enzymes, although the specific metabolic pathways require further study. This study presents new insights for understanding resistance mechanisms of the selected wheat cultivars to CRR, thus improving wheat yield in the future.

Morphological and Molecular Characterization of Cocoyam (Colocasia esculentum and Xanthosoma sagittifolium) (L) Schott germplasm from Nigeria using Simple Sequence Repeat-SSR marker

Colocasia esculentum (L) Schott and Xanthosoma sagittifolium (L) Schott, commonly called cocoyam is one of the orphan crops with great potentials, but neglected by researchers and growers despite their high demands by consumers in recent times. Consequently, some of the local varieties have become almost extinct; this calls for the need to revive interest in these crops. This study was undertaken to characterize six cocoyam accessions collected from Cross River and Akwa Ibom States, Nigeria, using 13 qualitative and 6 quantitative Agro-morphological descriptors as well as simple sequence repeat (SSR) primers. The crops were laid out in a field using the Randomized Complete Block Design (RCBD) in 4 replications. Analysis of variance (ANOVA) test showed that the 6 accessions were not significantly different (P>0.05) in crop span, crop height, number of leaves, leaf width and leaf length. This indicates that these features cannot be used to distinguish between Colocasia and Xanthosoma spp. The qualitative traits were more useful in distinguishing between the two cocoyam species. Three of the primers used for molecular studies successfully amplified a few of the cocoyam accessions, indicating their suitability for genetic diversity studies in Colocasia esculenta and Xanthosoma spp.

SELECTION OF ISSR MOLECULAR PRIMERS FOR STUDIES OF GENETIC DIVERSITY IN Handroanthus impetiginosus (MART. EX DC.) MATTOS

ABSTRACT Handroanthus impetiginosus is a tree species with ecological and economic potential. Despite that, in the Brazilian market, its wood is heavily exploited in the illegal trade. Therefore, studies on genetic diversity are necessary in order to propose strategies for conservation of this species. Thus, the aim of this study was to select Inter Simple Sequence Repeat (ISSR) primers for genetic diversity studies applied to the forest population of H. impetiginosus. For this, 30 ISSR molecular primers were tested in 30 individuals, evaluating the total number of loci, polymorphism rate and polymorphic information content, as well as marker index and resolving power. Eight primers were selected for having a better amplification pattern, which provided 62 loci. The polymorphic information content of the primers ranged from 0.34 to 0.49, while the marker index (MI) averaged 3.20, with resolving power (RP) of 2.40, Nei’s diversity (He) of 0.35 and Shannon index (I) of 0.52. The results show that the primers UBC 807, 809, 818, 824, 857, 860, 873 and 881 are efficient for quantifying the genetic diversity of H. impetiginosus. These results can contribute to supporting strategies aimed at the conservation of this species and selection of parent trees.

Identification of QTL Underlying Plant Height and First Branch Height of Cassava

This study sought to develop a genetic map of cassava based on 91 progenies derived from a cross between “Rayong 60” (R60) and “Piroon 1” (PR1) cultivars. A total of 4,515 simple sequence repeat (SSR) primer pairs were screened against the parental lines and six progenies resulting in 656 informative polymorphic markers, which were then used to genotype the F1 population. A total of 314 linked SSR loci were identified that were distributed across 40 linkage groups (LGs) at a logarithm of the odds (LOD) score of 4.0. The total distance of the linkage map was 2,026.68 cM with an average of 13.85 cM between markers and 7.85 loci per LG. The SSR loci on each LG were categorized into the 18 chromosomes of the cassava genome. Two major quantitative trait loci (QTL) and one minor QTL controlling first branch height (FBH) were detected with phenotypic variability explained (PVE) ranging from 10.2–18.9% and 8.2%, respectively. In addition, two major QTL for plant height (PH) with PVE ranging from 16.5–24.3% were detected in the F1 population. The QTL controlling PH and FBH detected in this study will be useful for future identification of QTL and genes underlying economically important traits to accelerate molecular breeding of cassava variety improvement via marker-assisted selection.

Morphological and molecular characterization of garlic (Allium sativum L.) genotypes sampled from Turkey.

Garlic is a vegetable widely used both in food and as a pharmaceutical raw material in the world due to its contents. Although morphological differences are observed in garlic, which is obligatory apomictically propagated, clonal propagation causes narrowing variation, a genetic bottleneck. This situation complicates breeding programs aiming improvements in preferred agronomic characteristics. For this reason, determining the morphological and molecular differences between garlic genotypes originating from Turkey is important for breeding studies. In this study, morphological and molecular characteristics of 39 garlic genotypes, which are widely cultivated in Turkey, were determined. Kahramanmaraş4 genotype was different from other genotypes in terms of some morphological features (fresh weight, dry weight, and bulb diameter). In the molecular characterization study, 10 Inter-Simple Sequence Repeats (ISSR) primers were used, and it was determined that the genotype TekDiş31 of Tunceli region was different from other garlic genotypes. Genetic similarity coefficient was found to be high (0.85–1.0) in genotypes except for TekDiş31 garlic genotype. In general, some garlic clones (Maraş3 and Kayseri30, Urfa33 and Topaklı35, Kastamonu22 and Kastamonu28, Urfa10 and Kastamonu14, Kastamonu29 and Bademci23) were completely similar to each other, while few differences were found among others. In conclusion, this study revealed that the garlic plant, despite its clonal propagation, consisted of some level of morphological and partially molecular variation. Due to its mode of reproduction (vegetative), this variation may largely be due to point or chromosomal mutation. Furthermore, the 10 identified ISSR primers can generate valuable information for genetic diversity for use by garlic breeders.

Intraspecific Identification of Lingzhi Medicinal Mushroom, Ganoderma lingzhi (Agaricomycetes), Using ISSR Markers.

This study aimed to obtain a set of specific inter simple sequence repeat (ISSR) primers and establish a stable and accurate intraspecific identification method for Ganoderma lingzhi. A total of 117 G. lingzhi strains were identified using internal transcribed spacer sequences from 147 strains determined as G. lingzhi via simple morphological identification. Based on the sequences obtained, specific ISSR primers for G. lingzhi were screened and validated, and 15 specific ISSR primers showed polymorphic banding pattern with clear band resolution. Subsequently, ISSR PCRs of the 15 specific primers were performed for the 117 G. lingzhi strains. As expected, DNA analysis of the ISSR markers could distinguish G. lingzhi strains, with similarity coefficients ranging from 0.11 to 0.89. Thus, the 15 specific ISSR primers can be used for intraspecific identification and polymorphism analysis of G. lingzhi.

Genetic characterization of remaining populations of paratecoma peroba, an endangered and endemic species of the atlantic forest

Background: The objective of this study was the genetic characterization of remaining populations of the species Paratecoma peroba occurring in fragments of the Atlantic Forest, by estimating parameters of diversity and genetic structure. The study was carried out in two forest fragments, the Atlantic Forest Environmental Education Pole (area 1) and the Pacotuba National Forest (area 2), where 93 adult trees were identified. Results: Ten Inter Simple Sequence Repeats (ISSR) primers were used in genotyping, obtaining 112 amplified bands with 87.5% polymorphism. The genetic diversity estimated for the populations from the Nei (H*) and Shannon (I*) indices was higher for area 1. For the joint data, moderate genetic diversity was observed, referring to the average values of 0.26 and 0.40, obtained for the H* and I* indices, respectively. Molecular variance analysis resulted in moderate differentiation between populations (Φ ST = 0.143), while gene flow analysis (N m = 6.69) revealed the occurrence of similar alleles between them. However, the predominance of a single genetic group in area 2, revealed from Bayesian approach, indicates that its genetic structure, possibly generated by the current scenario of fragmentation of the Atlantic Forest and the distancing of populations, affecting the contemporary gene flow. Conclusion: Despite the moderate genetic diversity of the species for the area 2 population, actions toward inclusion of seedlings obtained from seeds from neighboring fragments, including area 1, and the increase in the connectivity of forest fragments through ecological corridors, could assist in augmenting its genetic variation.

Population genetics of Hyphaene thebaica Mart., in Rabigh province, Saudi Arabia: Implications for conservation

Hyphaene thebaica is a perennial plant distributed in desert and subtropical regions of the world. In Rabigh Province, western Saudi Arabia, the few persisting populations of this species are exposed to many threats, including overcutting and, recently, human habitation. These threats are predicted to be exacerbated with the advancement of aridification caused by climate change. The conservation and revival of the diminished populations of H. thebaica requires an assessment of their genetic diversity and genetic differentiation. To accomplish this objective, we applied 10 simple sequence repeat (SSR) primer pairs, with which all are polymorphic loci. These polymorphic loci were used to determine the population genetics of 60 plant accessions sampled from 5 populations of H. thebaica located in five sites in Rabigh Province: Wadi EL Khaneg, Wadi Al Johfa, Wadi Al Hakak and Wadi Khurieba and Wadi Kuliayah . Low to moderate levels of genetic diversity were found in all populations (the values of the PPL% ranged between 52.5% and 22.5%) along with a decreased value of HT (0.388) and a considerable inbreeding value (F= 0.4552), which verified an obvious shortage of heterozygotes. High genetic differentiation among the populations and a low level of gene flow suggest isolation among the H. thebaica populations, which caused a severe deficiency in gene migration. The data obtained herein will inspire several recommendations for conservation the existing populations, including seed preservation, and management of human activities. All of these actions are urgently needed to prevent imminent extinction.

A simple and cost-effective method for DNA extraction suitable for PCR in “sucupira branca”

“Sucupira branca” is a plant found in the Brazilian Cerrado and is adapted to low fertility soils, and its fruit extract has anti-inflammatory, healing, antiulcerogenic, antimicrobial, cercaricidal, leishmanicidal and antioxidant activities. Furthermore, it provides protection against oxidative stress, is a natural biocontrol agent of Aedes aegypti, has very resistant wood, is a melliferous plant and has been used in reforestation programs. The development of conservation strategies is important for maintaining diversity in natural populations of “sucupira branca” since these populations are in the process of genetic erosion. Molecular biology techniques, which are important for characterizing the genetic diversity of plants to develop conservation strategies, require sufficient high-quality genomic deoxyribonucleic acid (DNA). This study aimed to compare five methods to extract DNA from “sucupira branca”. The quality and concentration of DNA were revealed by agarose gel electrophoresis, and only the protocols of Dellaporta, Wood and Hicks et al. (1983) and Khanuja et al. (1999) did not result in satisfactory quantities of DNA. When PCR (Polymerase Chain Reaction) was performed with three inter-simple sequence repeat (ISSR) primers, DNA was successfully amplified from extractions performed with the protocols proposed by Doyle and Doyle (1987), Romano and Brasileiro (1998) and Ferreira and Grattapaglia (1995), which are less expensive than commercial purification kits. These protocols resulted in DNA of sufficient quality and quantity after the amplification reactions were performed.

Picloram-induced enhanced callus-mediated regeneration, acclimatization, and genetic clonality assessment of gerbera

BackgroundGerbera jamesonii Bolus ex Hooker f. (African daisy) is listed among the top five most important ornamental plants in the global floricultural industry. To satisfy its demand, the floriculture industry relies on reproducible and effective propagation protocol while retaining the genetic uniformity of G. jamesonii. The present study, for the first time, reports the potential of picloram for enhanced induction of organogenic calli from leaves of G. jamesonii and its high-frequency indirect regeneration. ResultsThe fastest induction of calli with maximum fresh and dry weight was recorded in the Murashige and Skoog (MS) semisolid medium supplemented with 1 mg/l picloram. In addition, callus induction was observed in 2,4-dichlorophenoxy acetic acid- and α-napthaleneaceticacid-supplemented media but with delayed response and reduced fresh and dry weight. The proliferated calli were transferred to shoot induction media containing MS salt and 0.5–1 mg/l N6-benzylaminopurine, kinetin, or thidiazuron. A mean number of ~6 shoots per callus were developed after 5 days of culture in the MS medium supplemented with 1 mg/l kinetin, with a mean length of 5.2 cm. Successful rooting of shoots was achieved in the MS medium fortified with 1.5 mg/l indole-3-acetic acid, wherein the earliest root initiation (~5 days), as well as the maximum number (~9) and length (~4.8 cm) of roots, were recorded. Complete plantlets were primarily acclimatized in sand before being transferred to a mixed substrate (of soil, sand, tea leaf waste, and cow urine) that secured >90% survival and further growth of the plantlets. Eventually, clonal fidelity of the in vitro regenerants assessed via inter-simple sequence repeats (ISSR) primers exhibited a monomorphic banding patterns that suggested genetic integrity within the plantlets as well as with their mother plant. ConclusionsThe results of the present study should be of interest for commercial propagation and mutagenesis- as well as genetic transformation-related research.

Identification and Fine Mapping of a Locus Related to Leaf Up-Curling Trait (Bnuc3) in Brassica napus.

Leaf trait is an important target trait in crop breeding programs. Moderate leaf curling may be a help for improving crop yield by minimizing the shadowing by leaves. Mining locus for leaf curling trait is of significance for plant genetics and breeding researches. The present study identified a novel rapeseed accession with up-curling leaf, analyzed the up-curling leaf trait inheritance, and fine mapped the locus for up-curling leaf property (Bnuc3) in Brassica napus. Genetic analysis revealed that the up-curling leaf trait is controlled by a single dominant locus, named BnUC3. We performed an association study of BnUC3 with single nucleotide polymorphism (SNP) markers using a backcross population derived from the homozygous up-curling leaf line NJAU-M1295 and the canola variety ‘zhongshuang11’ with typical flat leaves, and mapped the BnUC3 locus in a 1.92 Mb interval of chromosome A02 of B. napus. To further map BnUC3, 232 simple sequence repeat (SSR) primers and four pairs of Insertion/Deletion (InDel) primers were developed for the mapping interval. Among them, five SSR markers and two InDel markers were polymorphic. By these markers, the mapping interval was narrowed to 92.0 kb using another F2 population. This fine mapping interval has 11 annotated genes among which BnaA02T0157000ZS were inferred to be candidate casual genes for up-curling leaf based on the cloned sequence analysis, gene functionality, and gene expression analysis. The current study laid a foundational basis for further elucidating the mechanism of BnUC3 and breeding of variety with up-curling leaf.

Genetic Structure and Geographical Differentiation of Larix sibirica Ledeb. in the Urals

The Ural Mountains and the West Eurasian Taiga forests are one of the most important centers of genetic diversity for Larix sibirica Ledeb. Forest fragmentation negatively impacts forest ecosystems, especially due to the impact of their intensive use on the effects of climate change. For the preservation and rational use of forest genetic resources, it is necessary to carefully investigate the genetic diversity of the main forest-forming plant species. The Larix genus species are among the most widespread woody plants in the world. The Siberian larch (Larix sibirica, Pinaceae) is found in the forest, forest-tundra, tundra (Southern part), and forest-steppe zones of the North, Northeast, and partly East of the European part of Russia and in Western and Eastern Siberia; in the Urals, the Siberian larch is distributed fragmentarily. In this study, eight pairs of simple sequence repeat (SSR) primers were used to analyse the genetic diversity and population structure of 15 Siberian larch populations in the Urals. Natural populations in the Urals exhibit indicators of genetic diversity comparable to those of Siberia populations (expected heterozygosity, He = 0.623; expected number of alleles, Ne = 4017; observed heterozygosity, Ho = 0.461). Genetic structure analysis revealed that the examined populations are relatively highly differentiated (Fst = 0.089). Using various algorithms for determining the spatial genetic structure, the examined populations formed three groups according to geographical location. The data obtained are required for the development of species conservation and restoration programs, which are especially important in the Middle Urals, which is the region with strong forest fragmentation.

Morphological and genetic diversity of Senecio vulgaris L. (Asteraceae) in Iran

Senecio vulgaris L., an annual herb belonging to the Asteraceae, is widely distributed in different regions of the world. There is no information on the intraspecific variations of the morphological and molecular features of this species. In the present investigation, we studied the morphological and genetic diversity of 81 accessions of S. vulgaris collected from 10 geographical populations. Eleven inter simple sequence repeat (ISSR) primers were used for the examination of genetic variations among the populations. Analysis of molecular variance (AMOVA) and GST analyses revealed significant differences among the investigated populations. A significant correlation between genetic distance and geographical distance was revealed by the Mantel test. However, reticulation analysis indicated the occurrence of gene flow among most of the populations studied. Principal component analysis (PCA) plot showed that the number of capitula, length of the cauline leaf and plant height were the most variable morphological characters. Principal coordinates analysis (PCoA) plot revealed two groups of populations, according to molecular and morphological data. The results suggested the existence of possible intraspecific taxonomic ranks within this species.