DNA Molecular Markers Research Articles

Biotechnology of mulberry (Morus L.) - A review

Mulberry (Morus sp.) is one of the economically important trees grown in Asian countries. It is cultivated to provide leaves for feeding the caterpillars of the silk producing insect (Bombyx mori L.). In addition, it adds value through production of edible fruits, timber and several pharmaceutically important chemicals. Improvement of mulberry through conventional breeding is limited due to high heterozygosity and long generation period. Attempts have recently been made to complement conventional breeding with modern biotechnological tools such as plant tissue culture, recombinant DNA technology and molecular markers to facilitate mulberry genetic improvement. The techniques of tissue culture have grown considerably in mulberry and encompassed areas including micropropagation, plant regeneration from leaf discs, and screening for stress tolerance. Recently, genetic engineering was adopted to enhance drought and salt tolerance in mulberry using HVA1 and Osmotin genes. Molecular markers such as Random amplified polymorphic DNA (RAPD), inter simple sequence repeats (ISSR) and simple sequence repeats (SSR) have been used for molecular characterization of mulberry germplasm, biodiversity analysis, genetic mapping and identification of molecular markers for growth and yield . However, still a number of issues such as resistance to fungal and bacterial diseases, combating infestation of pests and insects, and tolerance to drought and salinity are to be sorted out to achieve sustainable mulberry cultivation to meet the increasing demand of the silk industry. This review describes the developments of tissue culture, transgenesis and molecular markers in mulberry and highlights the current constraints and future prospects.

Isolation and characterization of polymorphic microsatellites in the genome of Yak (Bos grunniens)

The researches on yak genetics and breeding were extremely restricted due to lacking of reliable DNA molecular markers. The microsatellites with repeat motif (AC)n/(GT)n in yak genome were enriched by Dynal magnetic beads and the gene libraries containing (AC)n/(GT)n were constructed. Among the 92 identified and sequenced positive clones, 40 contained perfect repeats (43.48%), 41 contained imperfect repeats (44.57%) and 11 contained compound repeats (11.96%). As compared with the percentage of perfect repeats, no significant increases of imperfect repeats were observed in yak genome, which indicated that the level of adaptive evolution of the ability to repair damaged genomic DNA for yaks were high enough to endure the natural pressure of nucleotide substitution resulted from ultraviolet irradiation in high-altitude areas. Totally 19 polymorphic microsatellite loci were screened and genotyped on the basis of electropherograms on an ABI 310 Genetic Analyzer. All the loci exhibited moderate to high-level polymorphisms in a test population of Bos grunniens and the polymorphic information content ranged from 0.299 to 0.861 (mean 0.678). The newly isolated (AC)n/(GT)n repeats from yak genome will display their potential values in examining intra-population genetic structure and inter-population relationships, and also in investigating molecular markers for production and adaptive traits of individual/population.

Changes in allelic frequency over time in European bread wheat (Triticum aestivum L.) varieties revealed using DArT and SSR markers

A collection of 189 bread wheat landraces and cultivars, primarily of European origin, released between 1886 and 2009, was analyzed using two DNA marker systems. A set of 76 SSR markers and ~7,000 DArT markers distributed across the wheat genome were employed in these analyses. All of the SSR markers were found to be polymorphic, whereas only 2,532 of the ~7,000 DArT markers were polymorphic. A Mantel test between the genetic distances calculated based on the SSR and DArT data showed a strong positive correlation between the two marker types, with a Pearson’s value (r) of 0.66. We assessed the genetic diversity and allelic frequencies among the accessions based on spring- versus winter-wheat type as well as between landraces and cultivars. We also analyzed the changes in genetic diversity and allelic frequencies in these samples over time. We observed separation based on both vernalization type and release date. Interestingly, we detected a decrease in genetic diversity in wheat accessions released over the period from 1960 to 1980. However, our results also showed that modern plant breeding have succeeded in maintaining genetic diversity in modern wheat cultivars. Studying allelic frequencies using SSR and DArT markers over time revealed changes in allelic frequencies for a number of markers that are known to be linked to important traits, which should be useful for genomic screening efforts. Monitoring changes in the frequency of molecular DNA markers over time in wheat cultivars may yield insight into alleles linked to important traits that have been the subject of positive or negative selection in the past and that may be useful for marker-assisted breeding programs in the future.

Efficiency of RAPD, ISSR, AFLP and ISTR markers for the detection of polymorphisms and genetic relationships in camote de cerro (Dioscorea spp.)

At present, species known as camote de cerro (Dioscorea spp.) are found only in the wilderness in Mexico, but their populations are extremely depleted because they are indiscriminately collected, it is urgent to evaluate the conservation status of these plants in order to design conservation genetics programs. In this study, genetic diversity parameters along with cluster analysis based on Jaccard's coefficient were estimated with the objective to assess the efficiency of Random Amplified Polymorphic DNA (RAPD), Inter Simple Sequence Repeat (ISSR), Amplified Fragment Length Polymorphism (AFLP) and Inverse Sequence Tagged Repeat (ISTR) molecular DNA markers in the Dioscorea genus. The polymorphic information contents were quite similar for all markers (≈ 0.48). Genetic variation of Dioscorea spp., in terms of average heterozygosity was lower with ISTR (0.36), and higher when other markers were used (RAPD = 0.43; ISSR = 0.45 and AFLP = 0.47). This indicates an important level of genetic differences despite the fact that the plant is asexually propagated. Based on the diversity statistics, any marker tested in present work can be recommended for use in large-scale genetic studies of populations. However, the low correlations among different molecular marker systems show the importance of the complementarity of the information that is generated by different markers for genetic studies involving estimation of polymorphism and relationships.

Molecular characterization of aflatoxigenic and non-aflatoxigenic Aspergillus flavus isolates collected from corn grains.

Twelve species from six fungal genera were found to be associated with corn (Zea mays L.) grain samples collected from three main regions of Saudi Arabia. The average frequencies of the most common genera were Aspergillus (11.4%), Fusarium (9.5%), Penicillium (5.1%), and Alternaria (5.8%). Fifteen isolates of Aspergillus flavus were screened by HPLC for their ability to produce aflatoxins (AF). The percentage of aflatoxigenic A. flavus isolates was 53%. Eight isolates produced AF, at concentrations ranging 0.7-2.9 ppb. Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) molecular markers were used to genetically characterize isolates of A. flavus and to discriminate between the aflatoxigenic and non-aflatoxigenic isolates. RAPD and ISSR analysis revealed a high level of genetic diversity in the A. flavus population, which was useful for genetic characterization. The clustering in the RAPD and ISSR dendrograms obtained was unrelated to geographic origin. The RAPD and ISSR markers could not discriminate between aflatoxigenic and non-aflatoxigenic isolates, but the ISSR primers were somewhat better.

Trichophyton tonsurans strains from Brazil: phenotypic heterogeneity, genetic homology, and detection of virulence genes

The objective of this study was to establish the phenotypical and molecular patterns of clinical isolates of Trichophyton tonsurans circulating in the state of Ceará, northeastern Brazil. For this purpose, 25 T. tonsurans strains isolated from independent cases of tinea capitis in children were phenotypically evaluated regarding their macro- and micro-morphological characteristics, vitamin requirements, urease production, and antifungal susceptibility. The molecular characterization was carried out with random amplified polymorphic DNA molecular markers and M13 fingerprinting. The presence of the genes CarbM14, Sub2, CER, URE, ASP, PBL, and LAC, which encode enzymes related to fungal virulence, was also evaluated. Finally, melanin production was assessed through specific staining. The data obtained demonstrated that these T. tonsurans strains have considerable phenotypical variation, although they showed a low degree of genetic polymorphism according to the markers used. The genes CarbM14, Sub2, CER, and URE were detected in all the analyzed strains. The gene LAC was also identified in all the strains, and melanin synthesis was phenotypically confirmed. The strains were susceptible to antifungals, especially itraconazole (GM = 0.06 μg/mL) and ketoconazole (GM = 0.24 μg/mL). Therefore, T. tonsurans strains can present great phenotypical heterogeneity, even in genetically similar isolates. Moreover, the presence of the LAC gene indicates the possible participation of melanin in the pathogenesis of these dermatophytes.

Using DArT Markers to Monitor Genetic Diversity throughout Selection: A Case Study in Nebraska's Winter Wheat Breeding Nurseries

ABSTRACTAssessing the genetic relationship among wheat (Triticum aestivum L.) lines in the intermediate generations of a breeding program has important consequences on conserving genetic variability for selection in later generations. The objectives of this study were to (i) determine the breadth of the genetic base of two F3:6 nurseries and (ii) to monitor the effect of selection on the genetic diversity. Two independent F3:6 nurseries (2010 and 2011 seasons) were used; the first nursery contained 276 lines and two local check cultivars genotyped using 1925 polymorphic diversity array technology (DArT) markers while the second nursery contained 278 lines plus the same local check cultivars genotyped using 2236 polymorphic DArT markers. The F3:6 nurseries, 2010 and 2011, were each grouped into three main clusters. Overall, the results suggested that, in both years, the difference among clusters was significant, and the genetic diversity in the F3:6 nurseries exceeded that between the two check cultivars. The results for the two nurseries showed that each cluster in the F3:6 nurseries was represented in the F3:7 and F3:8 with at least one line, except for one cluster in the 2011 nursery which was not represented in F3:8. We concluded that much of the genetic diversity was maintained while advancing lines from F3:6 to F3:7; however, as the selection intensity increases, for example from F3:7 to F3:8, the effect of selection and the importance of monitoring genetic diversity using DNA molecular markers increases.

Heredity of Cognitive Functions and Personality in Twins

The inheritance of cognitive functions and personality is still a problem under investigation. A classical method, investigation of twins, is often used to find relative contributions from genetics and the environment to quantitative traits. The aim of this study was to investigate a possible influence of heredity on cognitive functions (by applying the Raven test) and personality traits (according to the Eysenck's theory) in twins. In total, 139 pairs of same-sex twins were investigated. The zygosity of the twin pairs was confirmed through genotyping with 15 molecular DNA markers. Constructive thinking was assessed using the Raven test. Personality assessment was conducted using the Eysenck Personality Questionnaire (EPQ) in Lithuanian. The difference in the total number of incorrect solutions between monozygotic (MZ) and dizygotic (DZ) twin pairs younger than 15 years was not significant; however, in the group older than 15 years, this difference was found to be significant. Based on the total number of incorrect solutions, the concordance in the MZ twins was greater than in the DZ twins. The same tendency was found with cumulative EPQ scores on the extraversion scale. The results imply that the quality of cognitive functions and personality traits (extraversion) can be influenced by heredity. This confirms previous findings on the heritability of cognitive factors and personality traits.

Effect of X-ray irradiation on olive shoot culture evaluated by morphological measurements, nuclear DNA content and SSR and AFLP markers

This article presents the first investigation of X-ray irradiation treatments of olive shoot culture followed by detailed genetic analysis of induced mutation events. This was achieved by comparing morphological measurements, studies of nuclear DNA content and molecular marker (SSR and AFLP) analysis. Traditional olive cultivars often need minor phenotypic corrections, which can be assessed using mutation breeding. We investigated the effects of X-ray irradiation at 0, 10, 30 and 60 Gy delivered to in vitro grown shoots of the olive variety ‘Canino’. Morphological measurements showed that growth parameters did not decrease at a 10-Gy dose, that rooting ability was diminished at a 30-Gy dose and that shoot growth was inhibited at a 60-Gy dose. Measurements of acclimatized plants showed that nuclear DNA content decreased with irradiation dose from 2.972 to 2.963 and 2.935 pg/2C nucleus of control, for 10 and 30 Gy treated plants, respectively. SSR profiling using 6 microsatellite loci revealed no polymorphism. AFLP analysis generated in total 630 scorable fragments within 13 primer combinations, of which 179 (28.4 %) were polymorphic. Although no major morphological changes were observed at a 10-Gy dose, a high number of absent/novel fragments were detected by AFLP analysis, indicating that mutation events can also be detected for doses at which growth parameters are not affected.

Long-term exposure to a pulsed magnetic field (1.5 mT, 25 Hz) increases genomic DNA spontaneous degradation

The aim of this work is to investigate whether long-term pulsed magnetic field (MF) has genotoxic activity by induction of DNA damage on DNA molecules in vitro, in the absence of repair mechanisms. Yeast genomic DNA prepared by phenol extraction from S. cerevisiae cultures and the commercial DNA molecular weight marker Hyperladder I (HL-I) were exposed to 1.5 mT peak, pulsed 25 Hz MF, 8 h/day, 16 days. The total content of DNA (undamaged and damaged DNA) decreased during the exposure of genomic DNA to MF. On day 16 of exposure the DNA content was 41 ± 8.1%. In addition, the undamaged DNA decreases until 6.2 ± 3.1% for unexposed control samples and until 0.3 ± 0.1% for pulsed MF-treated samples at day 16 of exposure. Therefore, the pulsed MF induced at day 16 an increase of 20.7-fold more degradation of DNA molecules >10 000 bp (undamaged DNA) than that observed for unexposed control samples. However, no effect was observed for HL-I DNA marker exposures. We conclude that long-term exposure to a pulsed MF (1.5 mT peak, 25 Hz, 8 h/day, 16 days) induces an increment in the DNA spontaneous degradation of yeast genomic DNA.

Genetic structure of Mexican Mestizos with type 2 diabetes mellitus based on three STR loci

BackgroundThe aims of this population genetics study were: 1) to ascertain whether Mexicans with type 2 diabetes mellitus (DM) were genetically homogeneous and 2) to compare the genetic structure of this selected population with the previously reported data of four random populations (Nuevo León, Hispanics, Chihuahua, and Central Region of Mexico). MethodsA sample of 103 unrelated individuals with DM and whose 4 grandparents were born in five zones of Mexico was interviewed in 32 Medical Units in the Mexican Institute of Social Security (IMSS). The non-coding STRs D16S539, D7S820, and D13S317 were analyzed. ResultsGenotype distribution was in agreement with Hardy–Weinberg expectations for all three markers. Allele frequencies were found to be similar between the selected population and the four random populations. Gene diversity analysis suggested that more than 99.57% of the total gene diversity could be attributed to variation between individuals within the population and 0.43% between the populations. ConclusionsAccording to the present and previous studies using molecular and non-molecular nuclear DNA markers not associated with any disease, the Mexican Mestizo population is found to be genetically homogeneous and therefore the genetic causes of DM are less heterogeneous, thereby simplifying genetic epidemiological studies as has been found in a previous study with the same design in Mexican women with breast cancer.

Molecular variants in populations of Bryconamericus aff. iheringii (Characiformes, Characidae) in the upper Paraná river basin

There are evidences that Bryconamericus aff. iheringii represents a species complex. DNA molecular markers have been effective in studies on phylogeny, taxon...

Assessment of introduced Kappaphycus (Solieriaceae, Rhodophyta) species relationships in China with molecular markers

Due to morphological plasticity and paucity of diagnostic morphological characters, the taxonomy of Kappaphycus gets more and more confused with the expanding of commercial cultivation. In this study, the phylogenetic relationship of 13 strains of introduced Kappaphycus species in China was defined using DNA molecular markers, such as 18S rDNA, rbcL and cox2-cox3 spacer region. The resolutions obtained by three different molecular markers were compared: both cox2-cox3 spacer region and rbcL sequences are eligible in inter- species identification of Kappaphycus, whereas cox2-cox3 spacer region is more variable than rbcL sequence. There is several basepairs' discrepancy among 18S rDNA sequences, while it is 100% identical among both cox2-cox3 spacer region and rbcL sequences of the ten strains of K. alvarezii. We suppose that 18S rDNA sequence can provide more information in biogeography study of Kappaphycus than other two DNA sequences.

Advances and perspectives on the gene mapping of root rot resistance in common beans

Common bean was originated in Mexico, and it is the most important protein source for millions of Mexicans, mainly those with low economic incomes. Grain yield of this crop is mainly affected by several fungal, bacterial and viral diseases. Among them, there are root rot diseases caused by the genera Macrophomina and Fusarium sp. Currently, some molecular biology tools such as DNA molecular markers are being used successfully to develop genetic maps where QTLs and major molecular markers are located, which are linked to genes that confer resistance to these fungi. The impor - tance of the development of genetic maps is based on the fact that they are the previous step for Marker-Assisted Selection (MAS). In this paper we review the current overview about the studies of root rot diseases mapping of resistance genes in common beans caused by the fungi M. phaseolina and Fusarium sp. We also report on those successful cases where results are translated to future applications in MAS in order to increase the efficiency of common bean breeding to both fungi based on costs, working and effectiveness. Finally, we analyze the perspectives of biotechnological tools on common bean breeding with special emphasis in Mexico.

Molecular characterization of Aspergillus flavus and aflatoxin contamination of wheat grains from Saudi Arabia

Twelve species belonging to six fungal genera were found to be associated with wheat (Triticum aestivum L.) grain samples collected from three main regions in Saudi Arabia. The most common genera (average frequency) were Aspergillus (14.3%), Fusarium (29.1%), Penicillium (9.3%), and Alternaria (8.2%). Nineteen isolates of Aspergillus flavus were screened for their ability to produce aflatoxins using HPLC. Thirteen isolates produced aflatoxins ranging from 0.5 to 2.6 µg/kg. Inter-simple sequence repeats (ISSR), and random amplified polymorphic DNA (RAPD) molecular markers were used, with the aim of genetically characterizing strains of A. flavus to discriminate between aflatoxigenic and non-aflatoxigenic isolates. RAPD and ISSR analysis revealed a high level of genetic diversity in the A. flavus population, useful for genetic characterization. Clustering based on RAPD and ISSR dendograms was unrelated to geographic origin. RAPD and ISSR markers were not suitable to discriminate aflatoxigenic and non-aflatoxigenic isolates, but ISSR primers were better compared to RAPD.

Genetic Diversity and Population Genetics of Mosquitoes (Diptera: Culicidae:Culexspp.) from the Sonoran Desert of North America

The population genetics and phylogenetic relationships of Culex mosquitoes inhabiting the Sonoran Desert region of North America were studied using mitochondrial DNA and microsatellite molecular markers. Phylogenetic analyses of mitochondrial cytochrome c oxidase subunit I (COI) from mosquitoes collected over a wide geographic area, including the Baja California peninsula, and mainland localities in southern Arizona, USA and Sonora, Mexico, showed several well-supported partitions corresponding to Cx. quinquefasciatus, Cx. tarsalis, and two unidentified species, Culex sp. 1 and sp. 2. Culex quinquefasciatus was found at all localities and was the most abundant species collected. Culex tarsalis was collected only at Tucson, Arizona and Guaymas, Sonora. The two unidentified species of Culex were most abundant at Navojoa in southern Sonora. Haplotype and nucleotide diversities in the COI gene segment were substantially lower in Cx. quinquefasciatus compared with the other three species. Analysis of molecular variance revealed little structure among seven populations of Cx. quinquefasciatus, whereas significant structure was found between the two populations of Cx. tarsalis. Evidence for an historical population expansion beginning in the Pleistocene was found for Cx. tarsalis. Possible explanations for the large differences in genetic diversity between Cx. quinquefasciatus and the other species of Culex are presented.

Genetic diversity of spineless Cereus jamacaru accessions using morphological and molecular markers

This is the first study to examine the genetic diversity of mandacaru cactus (Cereus jamacaru P. DC.). Plants of spineless mandacaru are commonly found in gardens and parks of urban areas in northeastern Brazil. In addition to exploring their ornamental potential, morphological, and genetic characterization may contribute to the development of plant materials that can be used as a source of macromolecules of potential economic interest. The goal of this study was to estimate the genetic variability of spineless mandacaru accessions using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) molecular markers, and to characterize their morphology. Ten samples of newly emitted shoots with differentiated areolas and ribs were collected from each accession from the Cactaceous Germplasm Collection of Embrapa Agroindústria Tropical, in Fortaleza, CE. Shoot shape and aspects of spine primordia (presence, location, grouping, and size of spines) were evaluated. The morphological analysis showed that the spineless mandacaru presented spine primordia. Twenty-six RAPD and 15 ISSR primers were polymorphic. A total of 262 markers were obtained, 129 of which were polymorphic. The average polymorphism of ISSR markers was higher than that of RAPD markers. The dendrograms for both analyses showed differentiation between accessions. Nevertheless, the molecular markers detected higher levels of diversity and a different pattern of diversity than those found using morphological markers. The molecular results revealed significant genetic variability both within and between groups.

ASSESSMENT OF GENETIC DIVERSITY OF TOMATO (Lycopersicon esculentum L.) GERMPLASM USING MOLECULAR MARKERS (RAPD AND ISSR)

Two DNA molecular marker systems, RAPD and ISSR were used to assess genetic diversity among nine tomatos (Lycopersicon esculentum L.), one tomatille (Physalis philadelphica) and one cherry tomato (Solanum lycopersicum var. cerasiforme) accessions, collected from different regions in Egypt. Accurate and unambiguous identification of these accessions is essential for germplasm preservation and use. Genomic DNA from the 11 accessions was screened with 18 ISSR primers and nine RAPD primers. A total of 293 and 180 clear fragments were amplified by ISSR and RAPD, respectively. On the other hand, unique positive markers were detected for ‘Tomatillo’ and for ‘Super marmande’, by 4 and 3 RAPD primers, respectively. Moreover, 18 primers of ISSR produced unique positive markers for Tomatille and Castle Rock, respectively. The ISSR technology proved useful in describing genetic diversity among tomato accessions and studies the phylogenetic relationships between cultivars. Cluster analysis using the UPGMA method placed all tomato accessions and cultivars into a single group, while the Tomatille and cherry tomato accessions were placed in a second group.

Comparative analysis of genetic diversity ofDosidicus gigasfrom eastern Pacific Ocean based on molecular markers of mitochondrial DNA

The worldwide climate and marine environmental changes have significant impacts on the distributions and fishery resources abundance fluctuations of Dosidicus gigas in the East Pacific Ocean. It is important to investigate genetic variance and population genetic structure of D. gigas in order to exploit and manage this squid scientifically. In this study,the genetic diversity and the genetic differentiation betw een tw o geographical populations of D. gigas w ere analyzed based on tw o mitochondrial DNA molecular markers. The results show ed that the number of haplotype,haplotype diversities,nucleotide diversities and average nucleotide differences w ere 7,( 0. 397 ± 0. 079),( 0. 001 09 ± 0. 000 96) and 0. 600 respectively in all Cytb sequences of tw o geographical populations,and haplotype diversity in population off the coast of Peru w as only( 0. 282 ± 0. 101) w hich w as significantly low er than that of equatorial w aters. On the other hand,the number of haplotype,haplotype diversities,nucleotide diversities and average nucleotide differences in all COⅠ sequences w ere 17,( 0. 787 ± 0. 051),( 0. 002 90 ± 0. 001 38) and 1. 802 respectively. The pairw ise fixation index F st and median netw ork revealed lack of genetic structure betw een populations of equatorial w aters and off the coast of Peru( Cytb: F st= 0. 055 23,P 0. 05; CO Ⅰ: F st= 0. 005 91,P 0. 05),and this might be caused by equational oceanographic current. The minimum spanning netw ork tree,mismatch distribution analysis and neutrality tests suggested that D. gigas experienced a recent population expansion,possibly 138 900 to 167 900 years ago.

Inter-Simple Sequence Repeat (ISSR) Markers to Study Genetic Diversity Among Cotton Cultivars in Associated with Salt Tolerance

Developing salt-tolerant crops is very important as a significant proportion of cultivated land is salt-affected . Screening and selection of salt tolerant genotypes of cotton using DNA molecular markers not only introduce tolerant cultivars useful for hybridization and breeding programs but also detect DNA regions involved in mechanism of salinity tolerance. To study this, 28 cotton cultivars, including 8 Iranian cotton varieties were grown in pots under greenhouse condition and three salt treatments were imposed with salt solutions (0, 70 and 140 mM NaCl). Eight agronomic traits including root length, root fresh weight, root dry weight, chlorophyll and fluorescence index, K + and Na + contents in shoot (above ground biomass), and K + /Na + ratio were measured. Cluster analysis of cultivars based on measured agronomic traits, showed ‘Cindose’ and ‘Ciacra’ as the most tolerant cultivars, and ‘B-557’ and ‘43347’ as the most sensitive cultivars of salt damage. A total of 65 polymorphic DNA fragments were generated at 14 inter-simple sequence repeat (ISSR) loci. Plants of 28 cultivars of cotton grouped into three clusters based on ISSR markers. Regression analysis of markers in relation with traits data showed that 23, 33 and 30 markers associated with the measured traits in three salt treatments respectively. These markers might help breeders in any marker assisted selection program in order to improving cotton cultivars against salt stress.