Assessment of genetic diversity among and within Carthamus species using sequence-related amplified polymorphism (SRAP) markers

Due to precise evaluation of genetic diversity of Carthamus species, sixty-two genotypes consisting fifty-two from five wild (C. oxyacanthus M. Bieb, C. lanatus L., C. dentatus Vahl, C. boissieri Halácsy, C. glaucus M.B.) and ten from cultivated species (C. tinctorius L.) were selected for evaluation of the genetic diversity in Carthamus species. A total of 238 (81.2 %) polymorphic bands were detected by 12 SRAP primer combinations with an average of 22 bands per combination. Me4-Em1 and Me5-Em2 primer combinations were known as the most informative SRAP markers based on the PIC values (0.34) where they distinguished all studied Carthamus species. Cluster analysis classified all accessions into five main groups among which clusters containing cultivated individuals were distinctly separated from those containing wilds. The most and the least genetic variation based on analysis of molecular variance, were detected within (76.90 %) and among (22.84 %) groups, respectively. The obtained results suggested that C. dentatus, C. glaucus and C. boissieri species may be classified in one section including C. dentatus in one and C. glaucus and C. boissieri in another subsection. The results also revealed high genetic similarity between C. oxyacanthus and C. tinctorius despite their different morphological characteristics.     

Genetic characterization of Iranian native Bombyx mori strains using amplified fragment length polymorphism markers

Genetic relationships within and among seven Iranian native silkworm strains was determined by DNA fingerprinting by using amplified fragment length polymorphism (AFLP) markers. In total, 189 informative AFLP markers were generated and analyzed. Estimates of Nei's gene diversity for all loci in individual strains showed a higher degree of genetic similarity within each studied strain. The highest and the least degrees of gene diversity were related to Khorasan Pink (h = 0.1804) and Baghdadi (h = 0.1412) strains, respectively. The unweighted pair-group method with arithmetic average dendrogram revealed seven strains of silkworm, Bombyx mori (L.), resolving into two major clusters. The highest degree of genetic similarity was related to Baghdadi and Harati White, and the least degree was related to Guilan Orange and Harati Yellow. The genetic similarity estimated within and among silkworms could be explained by the pedigrees, historical and geographical distribution of the strains, effective population size, inbreeding rate, selection intensity, and gene flow. This study revealed that the variability of DNA fingerprints within and among silkworm strains could provide an essential basis for breeders in planning crossbreeding strategies to produce potentially hetrotic hybrids in addition to contributing in conservation programs.     

Genetic characterization of <Emphasis Type=Italic>Meloidogyne incognita</Emphasis> isolates from Turkey using sequence-related amplified polymorphism (SRAP)

Southern root knot nematode Meloidogyne incognita is the most widespread-species, causing serious yield losses in protected vegetables fields in the West Mediterranean region of Turkey. The knowledge of genetic variation within M. incognita is required for disease management and improvement of resistant varieties by breeding programs. In the present study, the isolates were classified into different groups based on sequence-related amplified polymorphism (SRAP) fingerprints. To our knowledge, this is the first study carried out on the characterization of M. incognita isolates using SRAP. The schematic diagram by tested primers to differentiate of M. incognita isolates was formed in discrimination of nematodes as an effective molecular tool since it is cost effective and easiness. Data presents a genetic variation on root-knot nematode species. These selected SRAP markers can be used to follow genetic structure and differentiation on M. incognita isolates in a certain region.     

Assessment of genetic diversity of cultivated and wild Iranian grape germplasm using retrotransposon-microsatellite amplified polymorphism (REMAP) markers and pomological traits

Molecular Biology Reports - Understanding the genetic diversity and relationships between genotypes is an effective step in designing effective breeding programs. Insertional polymorphisms of...     

Molecular characterization and genetic mapping of random amplified microsatellite polymorphism in barley

 This study has analyzed the molecular basis and genetic behaviour of the polymorphism generated by the amplification of barley genomic DNA with primers complementary to microsatellites. Primers anchored at the 5′ end, used alone or in combination with arbitrary sequence primers, generated random amplified microsatellite polymorphisms (RAMPs). Unanchored primers were also used as single primers in a microsatellite primed-PCR (MP-PCR). Twenty six randomly selected RAMP DNA fragments which showed polymorphism between the cultivars Steptoe and Morex were cloned and sequenced. All sequences showed the expected repeated motif at the end of the insert, with the number of repeats ranging from five to ten. Genomic sequences containing low numbers of microsatellite motifs were preferentially amplified; therefore, only a fraction of the polymorphism could be attributed to variation in the number of microsatellite motifs at the priming site. Some sequences contained either cryptic simple sequences or members of families of repeated DNA. Polymorphism at the internal cryptic simple sequences was detected by RAMP bands inherited as co-dominant markers. Four MP-PCR bands were cloned and sequenced. A number of repeats identical to the primer itself were found at each end of the insert. Two allelic bands were polymorphic for an internal microsatellite. The potential use of cloned bands as fingerprinting tools was investigated by employing them as hybridization probes in Southern blots containing digested barley DNA from a sample of cultivars. RAMP probes produced complex hybridization band patterns. MP-PCR probes produced either a highly variable single locus or low-copy number loci. Segregations for 31 RAMPs and three MP-PCR bands were studied in a population of 70 doubled-haploids from the Steptoe/Morex cross. One third of all markers were co-dominantly inherited. Markers were positioned on an RFLP map and found to be distributed in all barley chromosomes. The new markers enlarged the overall length of the map to 1408 cM. Received: 6 May 1998 / Accepted: 20 July 1998     

Molecular characterization of Fusarium head blight resistance in Wangshuibai with simple sequence repeat and amplified fragment length polymorphism markers.

Molecular mapping of Fusarium head blight (FHB) resistance quantitative trait loci (QTL) and marker-assisted selection of these QTL will aid in the development of resistant cultivars. Most reported FHB resistance QTL are from 'Sumai 3' and its derivatives. 'Wangshuibai' is a FHB-resistant landrace that originated from China and is not known to be related to 'Sumai 3'. A mapping population of 139 F(5:6) recombinant inbred lines was developed from a cross of 'Wangshuibai' and 'Wheaton'. This population was developed to map the FHB-resistant QTL in 'Wangshuibai' and was evaluated twice for Type II FHB resistance. A total of 1196 simple sequence repeat and amplified fragment length polymorphism markers were screened on this population, and four FHB resistance QTL were detected. A major QTL near the end of 3BS explained 37.3% of the phenotypic variation. Another QTL on 3BS, located close to the centromere, explained 7.4% of the phenotypic variation. Two additional QTL on 7AL and 1BL explained 9.8% and 11.9% of the phenotypic variation, respectively. The simple sequence repeat and amplified fragment length polymorphism markers closely linked to these QTL may be useful for stacking QTL from 'Wangshuibai' and other sources to develop cultivars with transgressive FHB resistance.     

Molecular characterization of sawtooth oak (Quercus acutissima) germplasm based on randomly amplified polymorphic DNA

Sawtooth oak (Quercus acutissima) is a predominant tree species in the deciduous broad-leaved forest in China. It distributes in a large landscape area and can disperse in various ecology types. Molecular study on sawtooth oak can provide valuable information about the genetic diversity level and genetic relatedness on this important tree species. Insight into the genetic structure also provides resources of a species with its current feature and future evolutionary potential. The genetic structure of sawtooth oak was investigated by randomly amplified polymorphic DNA (RAPD). Twelve RAPD markers were used to assess genetic diversity of 408 individuals from 17 provenances enveloping most of the current distribution area of sawtooth oak. A total of 66 amplification products were detected, of which 49 bands (74.24 %) were polymorphic. Nei’s gene diversity, 0.2409, indicated a relatively high level of genetic variation in sawtooth oak germplasm. Analysis of molecular variance showed that most of the genetic diversity (87 %) was allocated within provenances. A combination of UPGMA dendrogram and STRUCTURE analysis was employed to estimate the genetic relationships of sawtooth oak germplasm; interestingly, the two methods presented similar grouping pattern with few discrepancies. Results revealed that 16 out of 17 provenances were clustered into one group, while the other 1 (LQ provenance) constituted a separate cluster. The data presented in this study suggested that the RAPD method was a valuable tool for estimation of genetic diversity and genetic relatedness of sawtooth oak germplasm. The present study also gave useful implications for germplasm conservation and new cultivar development for this promising energy tree species.     

Molecular characterization of Lebanese olive germplasm

Lebanon, an East Mediterranean country, does not have a comprehensive reference national olive collection. A report published 30 years ago indicated the presence of four Lebanese varieties, but the confusion regarding these varieties was discussed then and is still prevalent today hindering prospects for conserving and exploring the national germplasm. This study assessed the existing in situ diversity of old Lebanese olive groves using simple sequence repeat (SSR) and amplified fragment length polymorphisms (AFLP). Trees (140) were collected from 14 groves located in the four traditional olive areas. Analysis was based on 22 polymorphic alleles produced from six SSR primers, and on 135 polymorphic AFLP fragments. SSR and AFLP did not yield consistent results in terms of level of polymorphism, with SSR detecting higher variation than AFLP. On the other hand, both clustered trees based on geographic provenance. AFLP coefficient of similarity between trees ranged between 0.70 and 0.99 indicating the possible presence of varieties since some values fall within reported intervarietal ranges of 0.65 to 0.84. SSR unveiled the presence of nine different genotypes: four corresponding each to a provenance and five consisting of single trees characterized by the presence of less frequent alleles with an average of 0.33. Results also revealed a high incidence of clones greater than 90% in three of the four provenances. The findings of this study point for the need to plan for a sampling strategy that takes into consideration geographic provenances.     

Multi-locus DNA fingerprinting and genetic diversity in jute (Corchorus spp.) based on sequence-related amplified polymorphism

Sequence-Related Amplified Polymorphism (SRAP) markers were used for genetic diversity assessment and cultivar identification among 31 cultivars of jute belonging to two cultivated species Corchours olitorius L. and C. capsularis L. Forty-three primer-pairs produced a total of 394 bands with an average of 9 bands per primer pair and 89% bands were polymorphic across the genotypes of two species. Average genetic diversity in the cultivars of C. olitorius and C. capsularis was 7.2% (range 2.8–12.3%) and 7.6% (range 2.2–13.1%), respectively. Jute cultivars JRC 698, JRC 7447, TJ 40, S19 and JRO 3690 were more diverse compared to rest of the cultivars. UPGMA cluster analysis grouped all cultivars into two clusters which were representative of C. olitorius and C. capsularis species. All the cultivars could be unequivocally differentiated from one another based on the pooled profile of 43 primer-pairs, however, 24 of 31 cultivars could be identified uniquely. The probability of chance identity of any two cultivars based on SRAP markers was very low and was 6.95?×?10^?07 and 2.23?×?10^?07 for cultivars of C. capsularis and C. olitorius, respectively. Primer-pairs EM1-ME5, EM4-ME1, EM8-ME1 and EM10-ME1 were found to be useful for genetic diversity and cultivar identification. Our results show that SRAP markers could be effectively used for genetic diversity analyses in jute. For poor genetic diversity and resulting narrow genetic base, these markers will prove to be highly useful for identifying elite germplasm in a jute breeding program.     

Molecular evolution and diversity in Bacillus anthracis as detected by amplified fragment length polymorphism markers.

Bacillus anthracis causes anthrax and represents one of the most molecularly monomorphic bacteria known. We have used AFLP (amplified fragment length polymorphism) DNA markers to analyze 78 B. anthracis isolates and six related Bacillus species for molecular variation. AFLP markers are extremely sensitive to even small sequence variation, using PCR and high-resolution electrophoresis to examine restriction fragments. Using this approach, we examined ca. 6.3% of the Bacillus genome for length mutations and ca. 0.36% for point mutations. Extensive variation was observed among taxa, and both cladistic and phenetic analyses were used to construct a phylogeny of B. anthracis and its closest relatives. This genome-wide analysis of 357 AFLP characters (polymorphic fragments) indicates that B. cereus and B. thuringiensis are the closest taxa to B. anthracis, with B. mycoides slightly more distant. B. subtilis, B. polymyxa, and B. stearothermophilus shared few AFLP markers with B. anthracis and were used as outgroups to root the analysis. In contrast to the variation among taxa, only rare AFLP marker variation was observed within B. anthracis, which may be the most genetically uniform bacterial species known. However, AFLP markers did establish the presence or absence of the pXO1 and pXO2 plasmids and detected 31 polymorphic chromosomal regions among the 79 B. anthracis isolates. Cluster analysis identified two very distinct genetic lineages among the B. anthracis isolates. The level of variation and its geographic distribution are consistent with a historically recent African origin for this pathogenic organism. Based on AFLP marker similarity, the ongoing anthrax epidemic in Canada and the northern United States is due to a single strain introduction that has remained stable over at least 30 years and a 1,000-mile distribution.     

Molecular detection of cell line cross-contaminations using amplified fragment length polymorphism DNA fingerprinting technology

Summary We have tested amplified fragment length polymorphism (AFLP) technology, in comparison with isoenzyme analysis, for the simultaneous detection of inter-and intraspecific cell line cross-contaminations (CCCs) in the cell line collection held at the Istituto Zooprofilattico della Lombardia e dell’Emilia Romagna. Isoenzyme analysis identified four cases of interspecific CCCs. In a single expreiment, AFLP was able to identify the species of origin of all cell lines for which a reference genomic deoxyribonucleic acid was available and to detect five interspecific contaminations. Four CCCs confirmed data on isoenzymes, whereas the fifth CCC was detected in a species for which isoenzyme analysis was noninformative. In addition, AFLP was able to identify the putative source of the contaminations detected. The utility of the technology in the detection of intraspecific cell line contaminations, depends on the number of cell lines that have to be distinguished in a specific species and on the availability of highly informative fingerprinting systems. In mice, a single AFLP primer pair produced 16 polymorphisms and distinguished all the 15 strains of mouse cell lines analyzed. In humans, 18 AFLPs identified 83 different profiles in the 159 cell lines analyzed. Amplified fragment length polymorphism can conveniently be applied for cell line fingerprinting in species for which hypervariable markers are not available. In species for which a highly informative multiplex of microsatellite markers is available, AFLP can still provide a useful and cheap tool for simultaneously testing inter-and intraspecific contaminations.     

Molecular characterization of UV-treated sugar beet somaclones using RFLP markers

Sugar beet plants regenerated from UV-treated calluses were examined by restriction fragment length polymorphism (RFLP) analysis to determine the extent of somaclonal variation occurring at the DNA level. In total, 50 random sugar beet DNA sequences were used to screen 42 somaclones for genetic alterations. Three polymorphisms were detected among the 7 644 alleles analysed. From these data a mutation frequency of 0.03 ± 0.02% per allele was estimated. This frequency is in agreement with similar studies of somaclonal DNA variation using molecular markers and lies in the upper range of the spontaneous gene mutation frequencies found in plants. The two probegenotype combinations showing independent polymorphisms, were further analysed using the restriction enzymes Bam HI, Eco RI, Eco RV and Hind III. Both polymorphisms are likely to result from structural rearrangements rather than from point mutations. Differences in methylation among 10 of the investigated somaclones were tested for by comparing Hpa II and Msp I generated RFLP patterns. The somaclones showed extensive methylation, but no differences in their degree of methylation. Cytological analysis revealed 34 diploid, 8 tetraploid, but no aneuploid plants.     

Molecular characterization and genetic structure of Quercus acutissima germplasm in China using microsatellites

Quercus acutissima is native to eastern Asia. It has a wide distribution in China and China is an important component in understanding the ecology and genetic structure of this species. Q. acutissima attained high economic value for hardwood product and can be managed as an energy tree species. To investigate the genetic variation of Q. acutissima provenances, 12 microsatellite primer pairs were used to analyze 672 trees sampled from 28 provenances of Q. acutissima in China. All of the tested microsatellite loci proved to be effective for the studied Q. acutissima provenances. The results revealed that allele numbers varied from 5 to 13 per locus, with an average of 8 alleles per locus. The mean observed heterozygosity and expected heterozygosity were 0.4927 and 0.7023, respectively. The relatedness of the provenances was studied using the arithmetic mean algorithm based on Nei’s genetic distance and principal coordinates analysis. Interestingly, both approaches revealed two main groups: one consisted of the eastern Chinese provenances, and the other comprised of the western Chinese provenances. An analysis of molecular variance indicated that most genetic variation was contained within populations (84 %). The two microsatellite markers developed in this study may be employed for genetic characterization of other oak species. Considering the management or breeding programs of Q. acutissima provenances in China, we should treat each main group as a single gene resource.     

Construction of a rat genetic map by using randomly amplified microsatellite polymorphism (RAMP) markers

Many rat strains have been employed in the genetic study of quantitative traits such as blood pressure. In such genetic studies, it is essential to prepare rat genetic maps fine enough to identify the genes regulating quantitative traits. However, it is not an easy task to isolate a sufficient number of genetic markers polymorphic between a particular pair of rat strains. In this study, we applied the randomly amplified microsatellite polymorphism (RAMP) method, a simple method to identify co-dominant markers (Wu et al. Nucleic Acids Res 22, 3257, 1994), to isolate markers polymorphic between the stroke-prone spontaneously hypertensive rat and the Wistar-Kyoto rat, a genetically hypertensive strain and its normotensive control strain, which share a common genetic background. We successfully identified 111 RAMP markers distributed throughout the rat genome after screening 3046 sets of primers. We also showed that we could isolate ordinary simple-sequence-length-polymorphism markers by cloning RAMP markers. The RAMP method is a simple and efficient way to identify co-dominant genetic markers on mammalian genomes. Received: 10 October 1997 / Accepted: 16 March 1998     

Genetic diversity in European pigs utilizing amplified fragment length polymorphism markers

The use of DNA markers to evaluate genetic diversity is an important component of the management of animal genetic resources. The Food and Agriculture Organisation of the United Nations (FAO) has published a list of recommended microsatellite markers for such studies; however, other markers are potential alternatives. This paper describes results obtained with a set of amplified fragment length polymorphism (AFLP) markers as part of a genetic diversity study of European pig breeds that also utilized microsatellite markers. Data from 148 AFLP markers genotyped across samples from 58 European and one Chinese breed were analysed. The results were compared with previous analyses of data from 50 microsatellite markers genotyped on the same animals. The AFLP markers had an average within-breed heterozygosity of 0.124 but there was wide variation, with individual markers being monomorphic in 3-98% of the populations. The biallelic and dominant nature of AFLP markers creates a challenge for their use in genetic diversity studies as each individual marker contains limited information and AFLPs only provide indirect estimates of the allelic frequencies that are needed to estimate genetic distances. Nonetheless, AFLP marker-based characterization of genetic distances was consistent with expectations based on breed and regional distributions and produced a similar pattern to that obtained with microsatellites. Thus, data from AFLP markers can be combined with microsatellite data for measuring genetic diversity.     

Molecular characterization of Mauritanian date palm cultivars using plasmid-like DNAs markers

Mauritanian date palm cultivars and progenies of two controlled crosses were analyzed according to the identity of mitochondrial plasmid-like DNAs. Starting from total genomic DNA and appropriate primers, polymerase chain reaction was designed to amplify either a 373-bp or a 265-bp fragments corresponding to the S and the R-plasmid respectively. Data proved that 5 cultivars out of 10 studied have exhibited the R-plasmid suggesting their resistance to the fusariosis. The existence of intra-cultivar variability has also been revealed in the cv. Ahmar. In addition, analysis throughout progenies of two controlled crosses suggested the strict maternal transmission of the date palms’ mitochondrial genome.     

Molecular characterization of Syrian date palm cultivars using plasmid-like DNA markers

Date palm (Phoenix dactylifera L.) is one of the most important domesticated fruit trees in the Near East and North African countries. This tree has been, for several decades, in serious threat of being completely destroyed by the "Bayoud" disease caused by Fusarium oxysporum f. sp. albedinis. In this study, 18 Syrian date palm cultivars and four male trees were analyzed according to the identity of mitochondrial plasmid-like DNAs. A PCR strategy that employs plasmid-like DNAs-specific primer pair was used. These primers amplify a product of either 373-bp or 265-bp that corresponds to the S-(Bayoud-susceptible) or the R-plasmid (Bayoud-resistant), respectively. Generated data revealed that only six cultivars ('Medjool', 'Ashrasi', 'Gish Rabi', 'Khineze', and yellow- and red-'Kabkab') have the S-plasmid, suggesting their susceptibility to the fusariosis, while the remaining 12 cultivars and the four male trees contain the R-plasmid, suggesting their resistance to the fusariosis. The PCR process applied here has been proved efficient for the rapid screening for the presence of the S and R DNAs in Syrian date palm. PCR markers developed in this study could be useful for the screening of date palm lines growing in the field. The availability of such diagnostic tool for plasmid characterization in date palm would also be of great importance in establishing propagation and breeding programs of date palm in Syria.     

Molecular characterization of Syrian date palm cultivars using plasmid-like DNA markers

Date palm (Phoenix dactylifera L.) is one of the most important domesticated fruit trees in the Near East and North African countries. This tree has been, for several decades, in serious threat of being completely destroyed by the “Bayoud” disease caused by Fusarium oxysporum f. sp. albedinis. In this study, 18 Syrian date palm cultivars and four male trees were analyzed according to the identity of mitochondrial plasmid-like DNAs. A PCR strategy that employs plasmid-like DNAs-specific primer pair was used. These primers amplify a product of either 373-bp or 265-bp that corresponds to the S- (Bayoud-susceptible) or the R-plasmid (Bayoud-resistant), respectively. Generated data revealed that only six cultivars (“Medjool”, “Ashrasi”, “Gish Rabi”, “Khineze”, and yellow- and red-“Kabkab”) have the S-plasmid, suggesting their susceptibility to the fusariosis, while the remaining 12 cultivars and the four male trees contain the R-plasmid, suggesting their resistance to the fusariosis. The PCR process applied here has been proved efficient for the rapid screening for the presence of the S and R DNAs in Syrian date palm. PCR markers developed in this study could be useful for the screening of date palm lines growing in the field. The availability of such diagnostic tool for plasmid characterization in date palm would also be of great importance in establishing propagation and breeding programs of date palm in Syria.     

Amplification of reproducible allele markers for amplified fragment length polymorphism analysis.

A procedure for amplification by PCR of reproducible allele markers for amplified fragment length polymorphism (Amp-FLP) analysis is presented. We have prepared markers for the allelic products of the VNTR loci D1S80 (MCT118) and D17S30 (YNZ22) and for the hypervariable VNTR locus close to the 3' end of the apolipoprotein B gene (apoB) by re-amplifying a mixture of PCR products from individuals with known alleles. These allele markers allow precise and discrete determination of the VNTR alleles at these loci using the Amp-FLP technique that should prove suitable in forensic analyses, paternity testing and population studies.