Nuclear and chloroplast DNA variability and phylogeny of Iranian apples (Malus domestica)

This study was conducted to reveal genetic diversity among 23 local apple genotypes using nuclear (RAPD) and chloroplast DNA (PCR-cpRFLP) markers. Eleven RAPD primers and four cpDNA primer combinations were used in this study. RAPD primers produced a total of 77 polymorphic fragments with an average of seven bands per primer. The percentage of polymorphic bands (68.14 %) showed the efficiency of used RAPD primers in distinguishing all the genotypes considered. Genetic similarity between studied genotypes varied from 0.38 to 0.72 and cluster analysis showed the abundant diversity, indicating high intraspecific genetic variation between Iranian apple genotypes. From the four universal chloroplast primer pairs, three primer pairs amplified the fragments and their combinations showed polymorphic patterns and revealed intraspecific chloroplast variation. The information will facilitate germplasm identification, conservation and new cultivar development.     

RAPD and essential oil characterization of Turkish basil (Ocimum basilicum L.)

Sweet basil (Ocimum basilicum L., Lamiaceae), an important medicinal plant and culinary herb due to its delicate aroma and fragrance, shows great variation in both morphology and essential oil components. Genetic variation among basil accessions in Turkey has not been extensively examined with molecular markers. Genetic diversity was determined using random amplified polymorphic DNA (RAPD) markers of 14 genotypes of basil. A total of 375 bands were obtained from the RAPD analysis, and 273 of them (70.3 %) were polymorphic. The RAPD analysis allowed the grouping of samples into two main clusters. Genetic similarity values among the basil genotypes ranged between 0.46 and 0.87. Considerable genetic diversity was determined among basil genotypes. Essential oils were obtained by hydro-distillation and were characterized by gas chromatography. A total of 17 chemical components were identified. The evaluated genotypes of O. basilicum can be classified into seven chemotypes: (1) Linalool (7, 12, 16, 22, 25A and 33), (2) Methyl chavicol (6, 10A), (3) Citral/methyl chavicol (10L, 17), (4) Methyl eugenol (11), (5) Methyl cinnamate/linalool (23), (6) Linalool/methyl eugenol (25K), and (7) Methyl chavicol/linalool (Let). The chemical variability obtained from the essential oil composition of the genotypes in the study was remarkable. The chemical characterization of genotypes 10L and 17 was rich in citral (42.17 and 44.80 %) and methyl chavicol (30.56 and 32.03 %). Citral/methyl chavicol can be assessed as a new chemotype of basil cultivated in Turkey. The basil genotypes were grouped into two major clusters for both the RAPD analysis and chemical characterization with very few exceptions (genotype n. 6). A correlation analysis of the genetic distance matrix and the Euclidian distance matrix showed relatively low values (r = ?0.40). The results demonstrated a certain degree of correspondence between chemical and molecular data.     

Miscanthus: Genetic Diversity and Genotype Identification Using ISSR and RAPD Markers

Due to the limited number of molecular studies focused on European gene pool investigation, it is necessary to perform plant material recognition. Eighteen accessions of three Miscanthus species, namely, M. × giganteus, M. sinensis, M. sacchariflorus were evaluated with the use of molecular marker systems such as: inter simple sequence repeats (ISSRs), random amplified polymorphic DNA (RAPD), and by estimation of ploidy level based on flow cytometry. As a result, only one ISSR primer (ISSR1) and three RAPD primers (RAPD1, RAPD2, RAPD4) were required to identify all genotypes. Moreover, the use of the above mentioned molecular markers enable the proper species recognition of the interspecific hybrid M. × giganteus “Floridulus,” which has been previously mislabeled as M. floridulus. The highest genetic similarity coefficient (0.94) was observed between M. × giganteus clones, which indicates that the genetic diversity within this species was very low. Whereas M. sinensis genotypes represented a relatively wide diversity with similarity coefficient of 0.58. Cluster analysis using UPGMA grouped the 18 accessions in three clusters according to species affiliation including relabeled M. × giganteus “Floridulus,” which proved to be closely related to M.  × giganteus. Similar groupings were evident in the PCoA analysis.     

Nuclear DNA assay of the wild endangered medicinal and aromatic Indian Himalayan Valeriana jatamansi germplasm with multiple DNA markers: implications for genetic enhancement,domestication and ex situ conservation

Population genetic analysis in the important endangered medicinal and aromatic plant species, Valeriana jatamansi, provided, first time, insights into the identification of novel sources of genetic variation as an aid for improvement and domestication, and for optimizing conservation strategies. The 75 genotypes of V. jatamansi were collected from 36 locations across northeast to northwest Indian Himalayas of ~1,000 km, harbouring variable climatic and ecological conditions and rugged rocky terrain. The known protocols for DNA extraction failed to yield quality DNA in good quantity. A new protocol was standardized for this purpose. All the three (RAPD, ISSR, AFLP) DNA markers were successful in detecting polymorphism in V. jatamansi genotypes, and the ISSR marker, vis-à-vis RAPD and AFLP markers, generated the highest level of polymorphism. The RAPD, ISSR and AFLP fingerprints with 23 and 15 primers and 8 primer combinations, respectively, revealed 85.8, 89.0 and 67.7 % polymorphism among 141, 91 and 37 genetic loci amplified from the 75 genotypes, respectively. The AMOVA analysis of AFLP (55.0, 8.3, 36.7 %), RAPD (57.4, 11.9, 30.6 %) and ISSR (76.0, 4.8, 19.1 %) data indicated that more variation existed in differences in genotypes within populations than between populations within a region and between regions, respectively. The present comprehensive input will assist in effective management and (or) devising conservation strategies of this important medicinal plant species. This study reports the start of a molecular biology programme targeting nuclear genome of V. jatamansi, the genetics of which is very little known.     

Genetic structure and inter-generic relationship of closed colony of laboratory rodents based on RAPD markers

Molecular genetic analysis was performed using random amplified polymorphic DNA (RAPD) on three commonly used laboratory bred rodent genera viz. mouse (Mus musculus), rat (Rattus norvegicus) and guinea pig (Cavia porcellus) as sampled from the breeding colony maintained at the Animal Facility, CSIR-Indian Institute of Toxicology Research, Lucknow. In this study, 60 samples, 20 from each genus, were analyzed for evaluation of genetic structure of rodent stocks based on polymorphic bands using RAPD markers. Thirty five random primers were assessed for RAPD analysis. Out of 35, only 20 primers generated a total of 56.88 % polymorphic bands among mice, rats and guinea pigs. The results revealed significantly variant and distinct fingerprint patterns specific to each of the genus. Within-genera analysis, the highest (89.0 %) amount of genetic homogeneity was observed in mice samples and the least (79.3 %) were observed in guinea pig samples. The amount of genetic homogeneity was observed very high within all genera. The average genetic diversity index observed was low (0.045) for mice and high (0.094) for guinea pigs. The inter-generic distances were maximum (0.8775) between mice and guinea pigs; and the minimum (0.5143) between rats and mice. The study proved that the RAPD markers are useful as genetic markers for assessment of genetic structure as well as inter-generic variability assessments.     

Is the insular endemic Psidium socorrense (Myrtaceae) at risk of extinction through hybridization?

Natural hybridization between an insular endemic species and a widely distributed congener may endanger the endemic through genetic assimilation or outbreeding depression. Furthermore, hybrids can exhibit complex morphological variation, causing taxonomic problems in the identification of the involved taxa. In this work, we used a combination of leaf morphological and molecular markers (RAPD) to establish the differentiation between Psidium sp. aff. sartorianum and the insular endemic P. socorrense. It was also determined if hybridization between these taxa occurs in the southern slope of Isla Socorro, Mexico. Plant collection was carried along an altitudinal gradient (100–800 m). We collected eight populations separated 100 m a.s.l. apart from each other; 25 individuals were collected per population. Psidium socorrense and P. sp. aff. sartorianum differed significantly in all but two morphological characters measured. Also, a high number of diagnostic RAPD markers were found for each taxon. These results suggest that two Psidium species occur at Isla Socorro. Furthermore, both morphological and RAPD markers revealed a hybrid zone located in the southern slope of Isla Socorro (400–700 m a.s.l.) with an asymmetrical pattern of gene flow towards P. socorrense. We suggest that the disturbance caused by the sheep population in the mixed stand favors the establishment of hybrids. We further discuss whether hybridization represents a threat to the insular endemic P. socorrense.     

Variation in Saffron (Crocus sativus L.) accessions and Crocus wild species by RAPD analysis

Crocus, a genus of Iridaceae is mostly grown in areas with Mediterranean environment as the best region. Saffron (C. sativus) is a perennial plant and is cultivated as an industrial crop in several regions of Iran. In this research, five accessions of cultivated Saffron from five areas in Khorasan, and Esfahan including Gonabad, Ferdows, Torbat-e-Heyidariye, Estahbanat, Gopayegan were used. Other nine species of saffron were grown naturally in Iran; so we collected two wild species (C. caspius and C. speciosus) from north of Iran (Gilan Province). RAPD markers were used to classify these species and to find their relationships. In the results of this study, the cluster analysis showed two distinct groups. Also, the maximum similarity was seen between C. caspius and C. speciosus (0.82) and the minimum was between Estahbanat, Ferdows accessions and C. speciosus (0.33). Finally, this method as a convenience procedure could be used to separate different accessions and species of Crocus as well.     

Differentiation of tortoises of the genera Testudo and Agrionemys (Testudinidae) based on the polymorphism of nuclear and mitochondrial markers

Based on polymorphism of the 12S rRNA gene and RAPD markers, differentiation of 122 tortoise individuals belonging to the three species of genus Testudo (T. kleinmanni, T. marginata, and T. graeca), six subspecies of T. graeca (T. g. nikolskii, T. g. pallasi, T. g. armeniaca, T. g. zarudnyi, T. g. terrestris, T. g. ibera), and two subspecies of the Central Asian tortoise Agrionenemys horsfieldii (A. h. horsfieldii, A. h. kazakhstanica) was performed. For comparison, 32 known sequences of 12S rRNA gene (392 bp) from tortoises of the two genera inhabiting the territories of Europe, Asia, and Africa were used. In the populations of A. horsfieldii, a total of six haplotypes, including three newly described variants, were identified. In the examined tortoises of the genus Testudo, eleven 12S rRNA haplotypes were identified. One new haplotype was detected in T. kleinmanni. Among the eight subspecies of T. graeca, eight haplotypes were identified, with four newly described ones. The reported RAPD markers generally supported the reconstructions obtained with the use of the mitochondrial marker. Similarly to the 12S rRNA-based reconstructions, two independent clusters included representatives of the two genera, Agrionemys and Testudio. Among the latter, representatives of T. marginata and T. kleinmanni, as well as T. graeca, with high statistical support values, formed two reciprocally monophyletic groups. Compared to the mitochondrial markers, RAPDs more statistically significantly discriminated the sample of T. g. terrestris and the four subspecies, T. g. ibera, T. g. armeniaca, T. g. pallasi, and T. g. nikolskii. In almost all cases except the representatives of T. g. ibera, the representatives of each of four subspecies formed individual subclusters. The geographical haplotype distribution patterns and possible evolutionary scenario of the origin and dispersal of tortoises of the two genera are discussed.     

The level of genetic variability of cells in prolonged suspension culture of Arabidopsis thaliana

The level of heterogeneity and genetic variability of cells in a suspension of Arabidopsis thaliana cultured in vitro for more than seven years was studied. The considerable heterogeneity of the suspension in cell size was shown. As revealed by nuclear DNA cytophotometry, the suspension culture was mixoploid and the amount of DNA in the cells varied from 4 to 16 C. However, PCR with 6 RAPD- and 4 ISSR-primers and their intragroup combinations showed the lowest degree of variability of DNA markers. The genetic distances of clones obtained from a suspension culture of the parent plant were only 1.5%. Differences between the clones were identified with only one pair of 31 primer combinations tested, indicating low level of genetic heterogeneity of the suspension. The results showed that variations in the amount of DNA in the suspension culture cells are not accompanied by significant changes in the DNA sequence.     

Selecting with markers linked to the PPVres major QTL is not sufficient to predict resistance to Plum Pox Virus (PPV) in apricot

Sharka is one of the most serious viral diseases affecting stone fruit species and, in apricot, resistance to its viral agent, the Plum Pox Virus (PPV), is conferred by one major quantitative trait locus (QTL), named PPVres for PPV resistance. Previous studies indicated that PPV-resistant cultivars and breeding progenies can be selected by using a set of SSR markers (named PGS) targeting the PPVres locus. However, before these markers can be employed for marker-assisted selection, they were validated in a wide range of genetic backgrounds and environments. We used a total of 11 mapping populations issued from three distinct environments to confirm that this marker set located within the QTL adequately predicted PPV resistance. In this study, we show that selection of PPV-resistant material based only on markers co-localizing with the PPVres major locus is not fully reliable. Indeed, genotype-phenotype discrepancies were observed depending on the progeny and the PPV-resistant/susceptible parents. While most of the PPV-resistant individuals displayed the resistant alleles, a significant number of PPV-susceptible individuals showed the same resistant haplotype. An effect of the PPV strain used for phenotyping was also demonstrated. We thus hypothesize that the presence of other factors or genes involved in the mechanism of resistance to sharka in apricot could explain these unexpected results. Our work indicates that the current PGS marker set is not broadly applicable for MAS and that marker-assisted breeding based on the sole PPVres locus is not sufficient to unambiguously select PPV-resistant apricot cultivars.     

Exploring genetic variability within lentil (Lens culinaris Medik.) and across related legumes using a newly developed set of microsatellite markers

Lentil (Lens culinaris Medik.) is an economically important grain legume, yet the genetic and genomic resources remain largely uncharacterized and unexploited in this crop. Microsatellites have become markers of choice for crop improvement applications. Hence, simple sequence repeat (SSR) markers were developed for lentil through the construction of genomic library enriched for GA/CT motifs. As a result 122 functional SSR primer pairs were developed from 151 microsatellite loci and validated in L. culinaris cv. Precoz. Thirty three SSR markers were utilized for the analysis of genetic relationships between cultivated and wild species of Lens and related legumes. A total of 123 alleles were amplified at 33 loci ranging from 2–5 alleles with an average of 3.73 alleles per locus. Polymorphic information content (PIC) for all the loci ranged from 0.13 to 0.99 with an average of 0.66 per locus. Varied levels of cross genera transferability were obtained ranging from 69.70 % across Pisum sativum to 12.12 % across Vigna radiata. The UPGMA based dendrogram was able to establish the uniqueness of each genotype and grouped them into two major clusters clearly resolving the genetic relationships within lentil and related species. The new set of SSR markers reported here were efficient and highly polymorphic and would add to the existing repertoire of lentil SSR markers to be utilized in molecular breeding. Moreover, the improved knowledge about intra- and inter-specific genetic relationships would facilitate germplasm utilization for lentil improvement.     

Mutagenic effects of heavy-ion beam irradiation on in vitro nodal segments of Artemisia annua L.

Artemisia annua L. is a commercial source of artemisinin. Nevertheless, artemisinin content within the plant is relatively low and varies depending on genotype and environment. To broaden the genetic variability, the mutation effect of ¹²C-ion beam irradiation on A. annua was examined. Irradiation at 2.5 Gy had a slight lethal effect to nodal segments while a noticeable lethal effect was observed at 5 and 10 Gy. Furthermore, at higher doses (20 and 50 Gy), a severe lethal effect was observed. Mutations at the DNA level of axillary bud-derived shoots were performed by RAPD. The mutation frequency at 10 Gy was about 1.7 and 2.1 times higher than that at 2.5 and 5 Gy, respectively. After growth and artemisinin production observation of 72 irradiated mutants, around 14 and 7 % of them showed higher artemisinin content and artemisinin yield compared to the controls, respectively. The highest artemisinin content in a mutant was 1.43 % DW, which was 3.2-fold higher than the original wild type. Additionally, the highest artemisinin yield in mutants was 3.68 mg/plant, which was around 1.4-fold higher than in the wild type. Moreover, irradiated mutants exhibited antibacterial activity against S. aureus, but the wild types did not. This study presents an effective application of heavy ion beam irradiation to create variations and improve artemisinin production in A. annua.     

Identification of a RAPD Marker Linked to Fusarium Wilt Resistant Gene in Wild Watermelon Germplasm (Citrullus lanatus var. citroides

Random amplified polymorphic DNA(RAPD) was employed to detect a molecular marker linked to Fusarium wilt resistant gene in the wild watermelon ( Citrullus lanatus (Thunb.) Mansfeld var. citroides ) germplasm P1296341. The resistance to race 1 Fusarium wilt of PI296341 was controlled by one dominant gene. A RAPD marker OPPO1/700 was proved to be linked to the resistant gene. The genetic distance is 3.0 cM (centimorgan). This work has provided a solid basis for molecular marker-assisted selection (MAS) for disease resistance, and made location and cloning of disease resistant genes possible.     

Genome-Wide Analysis of Simple Sequence Repeats in Marine Animals—a Comparative Approach

Tandem simple sequence repeats (SSRs) are one of the most popular molecular markers in genetic analysis owing to their ubiquitous occurrence,high reproducibility, multiallelic nature, and codominant mode. High mutability makes SSRs play a role in genome evolution and correspondingly show different patterns. Comparative analysis of genomic SSRs in different taxonomic groups usually focuses on land species, while marine animals have been neglected. This study examined the abundance of genomic SSRs with repeated unit lengths of 1–6 bp in 30 marine animals including nine taxonomic groups and further compared with the land species. More than thousands of SSRs were discovered in every organism which provided a huge resource for the development of molecular markers. Thirty marine animals showed profound differences in SSR characteristics, but some group-specific trends were also found. Both similarities and differences of repeat patterns were discovered between the land and marine species. Two taxon-specific SSR types were discovered: the pentanucleotides motif AGAGG in Euteleostei and the hexanucleotide repeats of ATGTAC in Porifera and Echinodermata. Gene ontology (GO) enrichment analysis of two representative species (Amphimedon queenslandica for Porifera and Strongylocentrotus purpuratus for Echinodermata) revealed functional preference of the ATGTAC motif associated genes, and this might hint at evolutionary significance.     

与西瓜野生种质抗枯萎病基因连锁的RAPD标记

运用ＲＡＰＤ技术,采用混合分组分析（ｂｕｌｋｅｄｓｅｇｒｅｇａｎｔａｎａｌｙｓｉｓ,ＢＳＡ）方法进行了西瓜（Ｃｉｔｒｕｌｌｕｓｌａｎａｔｕｓ（Ｔｈｕｎｂ．）Ｍａｎｓｆｅｌｄｖａｒ． ｃｉｔｒｏｉｄｅｓ） 野生种质ＰＩ２９６３４１ 抗枯萎病基因连锁的分子标记研究。研究结果表明：西瓜野生种质Ｐ１２９６３４１ 抗枯萎病生理小种１ 的抗性由单显性基因控制,ＲＡＰＤ标记ＯＰＰＯＬ／７００ 与其抗病基因连锁,其遗传距离为３０ ｃＭ（ｃｅｎｔｉｍｏｒｇａｎ）。这为进行抗病分子标记辅助选择,以及最终定位与克隆其抗病基因打下了良好基础。     

Genetic variability among populations of Fusicladium species from different host trees and geographic locations in the USA

Peach and almond scab caused by Venturia carpophila and pecan scab caused by Fusicladium effusum result in yield loss, downgrading of fruit, defoliation and subsequent decline of an orchard. To understand the levels of genetic diversity and divergence of pathogens from different hosts and locations 51 isolates were genotyped and analyzed using 10 RAPD and 5 UP-PCR markers, including 18 isolates of V. carpophila from peach trees in the southeastern United States, 12 isolates of V. carpophila from almond trees in California, and 21 isolates of F. effusum (a related species) from pecan trees in the southeastern United States. The combined marker results showed a low incidence of polymorphisms among the peach isolates (4.2 % of markers), but a higher incidence of polymorphisms among the almond isolates (42.0 %) and the pecan isolates (61.0 %). The Dice coefficient of similarity ranged from 0.932 to 1.000 for the peach V. carpophila isolates, 0.214 to 0.976 for the almond V. carpophila isolates, and 0.528 to 0.920 for the pecan F. effusum isolates. UPGMA bootstrap values indicated that UP-PCR data were slightly more robust and, based on the combined data, the UPGMA bootstrap analysis (1,000 runs) gave a high node value (100 %) differentiating all the isolates of V. carpophila from F. effusum and a moderate node value differentiating the peach and almond isolates of V. carpophila (68 %). The results suggest some divergence between the V. carpophila populations of almond trees in California and peach tree populations in the southeastern United States, and different levels of genetic diversity within the two populations.     

Identification of an STMS marker for the double-podding gene in chickpea

Chickpea, a self-pollinating diploid annual with 2n = 2x = 16 chromosomes, is an important food legume crop throughout the world and especially in developing countries. A gene that confers double-podding, with the symbol "s", is considered important for breeding higher yielding cultivars. Positive effects of "s" on yield stability were found by comparing single- and double-podded near-isogenic lines (NILs) derived from a cross of CA-2156 (single-podded) with JG-62 (double-podded). Considering the significant effects on seed yield, the NILs were used to identify molecular markers closely linked to "s". Sequence tagged microsatellite site (STMS) markers developed for chickpea, random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) markers were used to analyze the NILs. Out of 400 RAPD, 100 STMS and 100 ISSR markers, one STMS marker (TA-80) was polymorphic and was used to evaluate a recombinant inbred line population developed from the cross of Surutato-77 (single-podded) 2 JG-62 (double-podded) for co-segregation of the locus with "s". Our results indicated that the marker and "s" were 4.84 cM apart. This marker may be used by breeders for marker-assisted selection (MAS) to monitor the incorporation of the double-podding gene into improved germplasm.     

Comparative study of different molecular markers for classifying and establishing genetic relationships in Coffea canephora

The genetic variability characterization of the accessions of the germplasm collection, using molecular markers, is being applied as a complementary strategy to the traditional approaches to redefine the plant genetic resources. In this study, we compared the informativeness and efficiency of the molecular markers RAPD, AFLP and SSR in the analysis of 94 accessions of Coffea canephora germplasm held by the breeding program of the Brazilian Agricultural Research Corporation (Embrapa), Rondônia State, Brazil. For this, we considered the marker’s discriminatory power and level of polymorphism detected and also the genetic relationships and clustering (dendrogram) analysis. The RAPD marker yielded low-quality data and problems in the discrimination of some accessions, being less recommended for genetic studies of C. canephora. The SSRs had a higher level of information content and yielded high-quality data, while AFLP was the most efficient marker system because of the simultaneous detection of abundant polymorphism markers per few reactions. Our results indicate that AFLP and SSR, allies to the intrinsic characteristics of each technique, are the most suitable molecular markers for genetic studies of C. canephora. However, the choice of AFLP or SSR in the species characterization should be made in agreement with some characteristics that are discussed in this work.