Specific DNA Markers Research Articles

Isolation of a Variant Strain of Pleurotus eryngii and the Development of Specific DNA Markers to Identify the Variant Strain

A degenerated strain of Pleurotus eryngii KNR2312 was isolated from a commercial farm. Random amplified polymorphic DNA analysis performed on the genomic DNA of the normal and degenerated strains of this species revealed differences in the DNA banding pattern. A unique DNA fragment (1.7 kbp), which appeared only in the degenerated strain, was isolated and sequenced. Comparing this sequence with the KNR2312 genomic sequence showed that the sequence of the degenerated strain comprised three DNA regions that originated from nine distinct scaffolds of the genomic sequence, suggesting that chromosome-level changes had occurred in the degenerated strain. Using the unique sequence, three sets of PCR primers were designed that targeted the full length, the 5' half, and the 3' half of the DNA. The primer sets P2-1 and P2-2 yielded 1.76 and 0.97 kbp PCR products, respectively, only in the case of the degenerated strain, whereas P2-3 generated a 0.8 kbp product in both the normal and the degenerated strains because its target region was intact in the normal strain as well. In the case of the P2-1 and P2-2 sets, the priming regions of the forward and reverse primers were located at distinct genomic scaffolds in the normal strain. These two primer sets specifically detected the degenerate strain of KNR2312 isolated from various mushrooms including 10 different strains of P. eryngii, four strains of P. ostreatus, and 11 other wild mushrooms.

Free‐living nematodes as prey for higher trophic levels of forest soil food webs

Nematodes are the most abundant invertebrates in soils and are key prey in soil food webs. Uncovering their contribution to predator nutrition is essential for understanding the structure of soil food webs and the way energy channels through soil systems. Molecular gut content analysis of consumers of nematodes, such as soil microarthropods, using specific DNA markers is a novel approach for studying predator–prey interactions in soil. We designed new specific primer pairs (partial 18S rDNA) for individual soil‐living bacterial‐feeding nematode taxa (Acrobeloides buetschlii, Panagrellus redivivus, Plectus velox and Plectus minimus). Primer specificity was tested against more than 100 non‐target soil organisms. Further, we determined how long nematode DNA can be traced in the gut of predators. Potential predators were identified in laboratory experiments including nine soil mite (Oribatida, Gamasina and Uropodina) and ten springtail species (Collembola). Finally, the approach was tested under field conditions by analyzing five mite and three collembola species for feeding on the three target nematode species. The results proved the three primer sets to specifically amplify DNA of the respective nematode taxa. Detection time of nematode DNA in predators varied with time of prey exposure. Further, consumption of nematodes in the laboratory varied with microarthropod species. Our field study is the first definitive proof that free‐living nematodes are important prey for a wide range of soil microarthropods including those commonly regarded as detritivores. Overall, the results highlight the eminent role of nematodes as prey in soil food webs and for channelling bacterial carbon to higher trophic levels.

Reconstructing explicit mating schemes in poplar hybrids – a case study in the Populus nigra L. – Populus × canadensis Moench complex

AbstractIn the plant kingdom, a large percentage of taxa are known to interbreed. If these hybrids are fertile, introgressive gene flow may foster the development of hybrid swarms or even promote gene swamping. Our study focuses on the Eurasian black poplar (Populus nigra L.) which may be threatened by hybridization with the cultivated fertile Euramerican hybrid. Using a combination of taxa specific DNA markers from the chloroplast and the nuclear genome we set up a straightforward and cost efficient method for identification of all possible mating scenarios in the hybrid complex of P. nigra and its cultivar Populus × canadensis Moench. Within a mixed population, we analyzed seed collections from individual trees of both taxa as well as juveniles from natural regeneration for proportions of second-generation hybrids (F2 hybrids) and first generation backcrosses. While F2 hybrids were detected in the seeds only, first generation backcrosses occurred in seeds as well as in juveniles. Due to the meiotic segregation of alleles, a certain amount of such progeny may remain undetected. Based on Mendelian rules, we developed a scheme to adjust the observed proportion of hybrid progeny for these undetected cases. Moreover, the scheme can be used to iteratively add loci necessary to detect poplar hybrids beyond the second hybrid and first generation backcrosses. We questioned whether there is a risk of hybrid swarm formation or swamping of the P. nigra gene pool. We discuss the likelihood of such a scenario and draw conclusions for conservation issues while poplar plantations are increasingly appreciated as renewable resources.

Development of DNA markers for identifying chrysanthemum cultivars generated by ion-beam irradiation

Four chrysanthemum cultivars were generated through (carbon) ion-beam irradiation of the original ‘Jimba’ (Chrysanthemum morifolium Ramat.). The new cultivars had acquired a number of superior cultivation traits, while remaining identical to the commercially available ‘Jimba’ in appearance. In this study, polymerase chain reaction (PCR) assays were used to detect the mutated region of each strain, thereby allowing clear identification at the molecular level. PCR assays were performed with 446 primer sets, including random amplified polymorphic DNA (RAPD) primer sets (10-mer RAPD), arbitrarily primed (AP)-PCR primers based on retrotransposon-like sequences and modified RAPD primers (15-mer RAPD). 15-mer RAPD primers generated a 1.49-fold increased band number at high annealing temperatures compared with the original 10-mer RAPD primers and could thus be effective for detection of polymorphic patterns. Our results provide information on the mutated regions of these ion-beam-irradiated chrysanthemum cultivars. Thus, specific DNA markers could be used to improve identification of new cultivars of chrysanthemum as well as other clonal cultivars of horticultural and agricultural crops.

Marker-assisted selection of high molecular weight glutenin alleles related to bread-making quality in Iranian common wheat (Triticum aestivum L.)

Bread-making quality in hexaploid wheats is a complex trait. It has been shown that the amount and composition of protein can influence dough rheological properties. The high-molecular-weight (HMW) glutenins are encoded by a complex locus, Glu-1, on the long arm of group-1 homoeologus chromosome of the A, B and D genomes. In this work we used PCR-based DNA markers as a substitution tool to distinguish wheat bread-making quality. We detected PCR-based DNA markers for coding sequence of Glu-A1x, Glu-B1x and Glu-D1x to be 2300 bp, 2400 bp and 2500 bp respectively. DNA markers related to coding sequence of Glu-A1y, Glu-B1y and Glu-D1y were; 1800 bp, 2100 bp and 1950 bp, however, the repetitive region of their coding sequence were shown to be about 1300 bp, 1500 bp and 1600 bp. The results demonstrate that the size variation was due to different lengths of the central repetitive domain. Good or poor bread-making quality in wheat is associated with two allelic pairs of Glu-D1, designated 1Dx5-1Dy10 and 1Dx2-1Dy12. The 1Bx7 allele has moderate-to-good quality score. The specific DNA markers, of 450 bp, 576 bp, 612 bp and 2400 bp respectively were characterized for 1Dx5, 1Dy10, 1Dy12 and 1Bx7 alleles. These markers are very important in screening of wheat for bread-making quality.

Development and characterisation of structural changes in chromosome 3Hch from Hordeum chilense in common wheat and their use in physical mapping

Structural changes in Hordeum chilense chromosome 3Hch were obtained in common wheat background by crossing tritordeum (the fertile amphiploid H. chilense–T. turgidum, 2n = 6x = 42, AABBHchHch) with the disomic addition line of chromosome 2Cc from Aegilops cylindrica in common wheat cv. ‘Chinese Spring’ (CS). Hybrid plants (2n = 43, AABBDHch + 2Cc) monosomic for the gametocidal chromosome were crossed with common wheat both CS and Anza. Rearranged 3Hch chromosomes were identified and cytologically characterized by FISH using both pAs1 repetitive sequence and genomic H. chilense DNA as probes. Two deletions on the short and long arm respectively, (del3HchS and del3HchL) and two reciprocal centromeric translocations involving chromosome 3Hch and 3D were identified. The breakpoints (FL) in the deleted chromosomes were determined as 56 % in del3HchS and 42 % in del3HchL, respectively. In order to obtain homozygous plants for rearranged 3Hch chromosomes the derived progenies were self-pollinated for four generations and the following lines were established: (1) disomic substitution DS-del3HchS(3D) (2n = 42) in CS; (2) disomic addition DA-del3HchL (2n = 44) in Anza; (3) homozygous translocation T3HchS·3DL (2n = 42) in CS, and (4) homozygous translocation T3DS·3HchL (2n = 42) in CS. These four lines were morphologically and agronomically characterized under field conditions. All lines were fertile, stable and vigorous. The four lines obtained were used to develop specific DNA markers for chromosome 3Hch in wheat. We studied the applicability of 162 barley chromosome 3H primers (55 genomic-SSRs, 17 genomic-STSs, 66 EST-SSRs and 24 EST-SNPs) to amplify markers showing polymorphism between H. chilense and common wheat. A total of 53 (32.7 %) markers gave reliable amplifications in H. chilense and among them 31 (19.1 %) were polymorphic between H. chilense and CS and Anza. Eighteen of the 31 polymorphic loci were mapped onto different chromosome sub-arm regions of chromosome 3Hch. Arm locations of these markers on barley chromosome 3H were determined using wheat–barley ditelosomic lines for 3HS and 3HL arms. The comparative positions of these markers in H. chilense and H. vulgare are discussed.

Evaluation of a DNA microarray for the rapid detection of extended-spectrum -lactamases (TEM, SHV and CTX-M), plasmid-mediated cephalosporinases (CMY-2-like, DHA, FOX, ACC-1, ACT/MIR and CMY-1-like/MOX) and carbapenemases (KPC, OXA-48, VIM, IMP and NDM)

Carbapenem-resistant Gram-negative bacilli are reported increasingly and represent an emerging public health concern. Laboratory detection of extended-spectrum β-lactamase (ESBL), plasmid-mediated cephalosporinase (pAmpC) and carbapenemase producers remains a challenge for microbiology laboratories and is important to avoid clinical failure due to inappropriate antimicrobial therapy and to prevent nosocomial outbreaks. We evaluated a novel microarray, the 'Check-MDR CT103 array' test (Check-Points, Wageningen, The Netherlands), that employs highly specific DNA markers to identify the β-lactamase genes of ESBLs (TEM, SHV and CTX-M, and discriminates between ESBL and non-ESBL TEM and SHV variants), of pAmpC (CMY-2-like, DHA, FOX, ACC-1, ACT/MIR and CMY-1-like/MOX) and of carbapenemases (KPC, OXA-48, VIM, IMP and NDM). One-hundred-and-eighty-seven well-characterized Gram-negative bacilli isolates possessing different bla genes were tested. Total DNAs were extracted using a Qiagen DNA mini kit. The 'Check-MDR CT103 array' was used as recommended by the manufacturer. The system correctly identified representatives of the three ESBL gene families tested, including differentiation between non-ESBL and ESBL TEM and SHV variants. All bla(CTX-M) genes were classified into the appropriate family group (i.e. CTX-M-1 group, CTX-M-2 group, CTX-M-9 group and CTX-M-8/25/26 group). In addition, the clinically relevant plasmid-encoded cephalosporinase and carbapenemase genes were also reliably detected. Specificities and sensitivities of 100% were recorded for most bla genes. The 'Check-MDR CT103 array' is a powerful high-throughput tool for rapid identification of ESBL, pAmpC and carbapenemase producers in culture. Because of its rapid performance, this platform is a valuable tool for epidemiological or infection control studies.

Methylated RASSF1a Is the First Specific DNA Marker for Minimal Residual Disease Testing in Neuroblastoma

PCR-based detection of minimal residual disease (MRD) in neuroblastoma (NB) is presently based on NB-specific transcripts. However, the expression of these targets varies between patients and upon treatment, and only PHOX2B is truly specific. RASSF1a is methylated (RASSF1a(M)) in NB, and we investigated whether it can serve as a specific and stable DNA MRD marker. The RASSF1a(M)-specific quantitative real-time PCR was tested on control bone marrow (BM; n = 50), on 71 NB tumors, and on 159 clinical BM samples at diagnosis and at follow-up of 77 patients. Results were compared with a panel of RNA markers and correlated with prognosis. RASSF1a(M) was present in all stage 4 and 4s tumors (n = 50) and in 86% stages 1 to 3 tumors (n = 21). The level of methylation in stage 4 NB was correlated with overall survival (P = 0.02). RASSF1a(M)-PCR was highly specific (only 1 amplification in 50 control samples tested in triplicate) and had a similar sensitivity as the RNA-based PCRs, as shown on clinical samples. Moreover, RASSF1a(M) enabled accurate quantification without need for the original tumor. RASSF1a(M) is a novel, highly specific DNA marker for MRD detection in NB, equal to PHOX2B in specificity and sensitivity, and better suitable for MRD quantification. We propose to include RASSF1a(M) in further prospective MRD studies in NB alongside RNA MRD markers. In addition, this assay might also be applicable for detection of circulating tumor cells in patients with other cancers withRASSF1a(M) such as breast or lung cancer.

GENE SEQUENCE ANALYSIS FOR THE MOLECULAR DETECTION OF PSEUDOMONAS SYRINGAE pv. ACTINIDIAE: DEVELOPING DIAGNOSTIC PROTOCOLS

SUMMARY Bacterial canker of kiwifruit caused by Pseudomonas syringae pv. actinidiae (Psa) is seriously damaging Actinidia deliciosa and A. chinensis in central Italy. Since this severe outbreak of the disease may reflect on the trade of kiwifruit pollen and fruits, standardized protocols are needed for the extraction of the bacterium from different matrices and for its detection and identification. Current PCR detection of Psa is aspecific, as the amplified product has the same size as that of P. syringae pv. theae. To improve the specificity of molecular detection of Psa, a gene-sequence analysis was done to identify new specific DNA markers. This enabled us to develop a duplex-PCR that distinguishes Psa from P. syringae pv. theae and from other genetically related P. syringae pathovars. This method was also successfully applied to detect Psa directly in infected kiwifruit matrices such as leaves, wood, flowers and in experimentally contaminated pollen and fruits. We propose two protocols for Psa extraction and detection from pollen and fruits. These protocols can be used for epidemiological studies, to establish whether symptomless fruits or pollen can harbour Psa, and can help diagnostic laboratories in the analysis of these type of materials.

Co-infection with Opisthorchis viverrini and Haplorchis taichui detected by human fecal examination in Chomtong district, Chiang Mai Province, Thailand

Diseases caused by the liver fluke, Opisthorchis viverrini and the minute intestinal fluke, Haplorchis taichui, are clinically important, especially in the Northeast and North regions of Thailand. It is often difficult to distinguish between these trematode species using morphological methods due to the similarity of their eggs and larval stages both in mixed and co-infections. A sensitive, accurate, and specific detection method of these flukes is required for an effective epidemiological control program. This study aimed to determine the prevalence of O. viverrini and H. taichui infections in human feces by using formalin–ether sedimentation and high annealing temperature random amplified polymorphic DNA (HAT-RAPD) PCR methods. Fecal specimens of people living along the Mae Ping River, Chomtong district were examined seasonally for trematode eggs using a compound microscope. Positive cases were analyzed in HAT-RAPD, DNA profiles were compared with adult stages to determine the actual species infected, and specific DNA markers of each fluke were also screened. Our results showed that out of 316 specimens, 62 were positive for fluke eggs which were pre-identified as O. viverrini and H. taichui. In addition, co-infection among these two fluke species was observed from only two specimens. The prevalence of H. taichui infections peaked in the hot-dry (19.62%), gradually decreased in the rainy (18.18%), and cool-dry seasons (14.54%), respectively. O. viverrini was found only in the hot-dry season (6.54%). For molecular studies, 5 arbitrary primers (Operon Technologies, USA) were individually performed in HAT-RAPD-PCR for the generation of polymorphic DNA profiles. The DNA profiles in all 62 positives cases were the same as those of the adult stage which confirmed our identifications. This study demonstrates the mixed infection of O. viverrini and H. taichui and confirms the extended distribution of O. viverrini in Northern Thailand.

English

In this study, we identified novel family- and genus-specific DNA markers in Mugilidae fish. Genomic DNA was isolated from the blood of fish of 15 families and eighty (80) random primers were used for random amplified polymorphic DNA (RAPD) fingerprinting. When the primer OPAV04 was employed, a novel specific PCR product was observed in the Mugilidae family. In addition, another novel specific PCR product was also observed in the Liza genus while using primer OPAV10. Sequencing analysis revealed that the novel family- and genus-specific DNA fragments were 857 and 419 bp, respectively, and no similar sequences were found in GenBank. Two primers sets were designed based on the family- and genus-specific sequences to confirm the RAPD results and the 571 and 187 bp predicted bands were successfully amplified by PCR. Intriguingly, these two novel specific DNA markers were also effectively used for terrestrial and aquatic animal discrimination. Therefore, the novel family- and genus-specific DNA markers identified in this study can be used as an effective tool for rapid and accurate determination of the Mugilidae family and Liza genus, and even for cross-species identification.   Key words: Mugilidae, family- and genus-specific sequences, DNA markers.

Another CAPS DNA marker for sex identification in jojoba seedlings

Jojoba [Simmondsia chinensis (Link) Schneider] plant has immense industrial value. Jojoba is grown for its seed oil, which is a unique liquid wax consisting of esters formed from acids and alcohols with chain lengths of 20 or 22 carbon atoms. Higher seed yield is obtained when well balanced male and female plants are established in jojoba orchard. However, the sex of jojoba plants cannot be determined with morphological characters until the plants reach reproductive maturity. DNA markers are commonly used for plant genetic studies [1,2,3,4], Up-to-date several male and female specific DNA markers have been developed for sex identification in jojoba seedlings [5,6]. This study reports another cleavage-amplified polymorphic sequence (CAPS) assay, which easily identify male individuals. Genomic DNAs of 16 male and 16 female jojoba plants growing in an orchard were extracted using a DNA extraction protocol [4]. A touch-down PCR approach was used to amplify genomic DNAs of jojoba using J818F 5'-AGGGGATAAATGAGCCGAAT-3' and J818R 5'-GACCCAGAGGATGAGGAATG-3' primer pair [6]. Amplified products were digested using Hind III restriction enzyme. Electrophoresis separation of the digested products as shown in Figure 1 indicated that male and female jojoba plant can be easily identified. CAPS marker reported in this study could be used in breeding studies and in the sex allocation of seedlings in seed orchard establishment.

Cytomorphological and molecular characterization of interspecific F1 hybrid of Momordica dioica Roxb. Momordica subangulata subsp. renigera (G. Don) de Wilde

Interspecific hybrid (F1) between spine gourd (Momordica dioica) and teasle gourd (Momordicasubangulata Blume subsp. Renigera (G. Don) WJJ de Wilde) produced through bagging and hand pollination showed vigorous growth habit with less fertility. Morphological parameters along with meiotic, mitotic chromosome behaviors and Randomly Amplified Polymorphic DNA (RAPD) analysis, confirmed interchanges of genetic characters in the new hybrids. The interspecific hybrid showed a vigorous foliage with intermediate flower characters than that of parental species. Self pollination and backcrossing of F1plants to either parent confirmed presence of both male and female sterility, that might be due to improper meiotic chromosome pairing. The characters like petal spot and time of anthesis (early morning) were similar to that of pollen parent while the diameter and size of leaves and flowers, pedicel length and plant growth were intermediate as compared to parents. Somatic chromosome analysis revealed 2n = 28 in M.dioica – the female parent, 2n = 56 in M. subangulata ssp. renigera - the male parent and 2n = 42 in F1hybrid of M. dioica × M. subangulata ssp. renigera. RAPD analysis of both the parents and F1 hybrid confirmed more genetic affinity (60.95%) with female parent than the male parent (32.35%). Primer specific DNA markers confirmed the introgression of male genetic elements into the newly developed amphidiploids. Incorporation of male specific DNA markers into the F1 hybrid is discussed. Preliminary evaluation indicates that the F1 plants sprouted early, which is lacking in the mother parent and this character could be exploited by restoring of fertility by chromosome doubling. Key words: Momordica, cytomorphology, F1 hybrid, karyotype, RAPD markers.

Abstract LB-312: Methylated RASSF1a is the first specific DNA marker for minimal residual disease testing in neuroblastoma

Abstract Purpose PCR-based detection of minimal residual disease (MRD) in neuroblastoma is presently based on neuroblastoma-specific transcripts. However, the expression of these targets varies between patients and upon treatment, and only PHOX2B is truly specific. RASSF1a is methylated (RASSF1aM) in neuroblastoma and we investigated whether it can serve as a specific and stable DNA MRD marker. Patients and methods The RASSF1aM specific real-time quantitative (RQ)-PCR was tested on control bone marrow (BM) (n=50), on 71 neuroblastoma tumors and on 159 clinical BM samples at diagnosis and at follow up of 77 patients. Results were compared to a panel of RNA markers and correlated with prognosis. Results RASSF1aM was present in all stage 4 and 4s tumors (n=50) and in 86% stage 1–3 tumors (n=21). The level of methylation in stage 4 neuroblastoma was correlated with overall survival (p=0.02). RASSF1aM-PCR was highly specific (only one amplification in 50 control samples tested in triplicate) and had a similar sensitivity as the RNA-based PCRs as shown on clinical samples. Moreover, RASSF1aM enabled accurate quantification without need for the original tumor. Conclusion RASSF1aM is a novel, highly specific DNA marker for MRD detection in neuroblastoma, equal to PHOX2B in specificity and sensitivity, and better suitable for MRD quantification. We propose to include RASSF1aM in further prospective MRD studies in neuroblastoma alongside RNA MRD markers. In addition, this assay can be used for sensitive detection and quantification of circulating cells in all cancers with hypermethylated RASSF1a. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-312. doi:10.1158/1538-7445.AM2011-LB-312

Genetic variability among male populations of a minor mangrove Excoecaria agallocha L. as evident by chromosome morphology and DNA markers

Genetic diversity analysis was done in five male populations of Excoecaria agallocha L. (Family Euphorbiaceae) obtained from east coast of India. Karyotype analysis could not differentiate five male populations studied here which reports high numbers of small sized somatic chromosomes (2n = 130). A total of 303 amplicons were generated from RAPD and ISSR primers of which 43.6% were polymorphic. Cluster analysis obtained from RAPD and ISSR profile exhibited prominent patterns of inter-population relationships. A high degree of genetic divergence was evident among the populations II, IV and V; however, a low genetic variation was detected between populations I and III. A prominent RAPD marker of ∼1,000 bp of population III using OPN-04 primer and two such population specific DNA markers ∼1,100 and ∼2,000 bp with OPD-12 primer could differentiate populations I and III. ISSR 2 primer (CT)8A produced unique bands of ∼650, ∼900 and ∼1,250 bp in populations III, V and I, respectively. ISSR 5 primer (GGAGA)5 separate population III with the help of marker DNA bands of ∼600 and ∼1,200 bp. Population I from Gopalpur and population III from Chilka showed lower genetic divergence as compared to rest of the examined populations (P < 0.05). AMOVA test revealed 23.6% and 53.5% genetic variability among populations and within populations, respectively. Genetic differentiation among the populations from Chandipur (population II), Paradeep (population IV) and Bhitarkanika (population V) were significant. This study suggests that ocean currents, land barriers and limited dispersal ability of seeds play important role in the occurrence of genetic diversity in sexually reproduced and dioeciously represented species E. agallocha.

Genetic dissimilarities between wild olives by random amplified polymorphic DNA (RAPD) assay

Olive, being a fruit of subtropical climate and recognized to have significant importance in terms of human health, is being investigated with different aspects. Wild olives are valuable in the sense that they contain the sources of stability and they can be separated by using random amplified polymorphic DNA (RAPD) technique. In this study, a total of 59 primers were used for the purpose of determining the DNA fingerprints of 12 wild olives obtained from different provinces of Aegean region, and 92 polymorphic bands were yielded. Mean number of polymorphic bands per individual was calculated as 7.67 and number of polymorphic bands per scorable primer was calculated as 4.6. Specific DNA markers are important for determining the genetic relation among wild subspecies in olives. The purpose of this study is to identify the wild olives by using RAPD analysis. In conclusion, the genetic relation between wild olives were determined through Jaccard, Sorensen Dice, Simple Matching coefficients, (unweighted pair group method with arithmetic averages) UPGMA and neighbor joining method by using the data obtained with RAPD.

Development of amplified fragment length polymorphism-derived functional strain-specific markers to assess the persistence of 10 bacterial strains in soil microcosms.

To augment the information on commercial microbial products, we investigated the persistence patterns of high-priority bacterial strains from the Canadian Domestic Substance List (DSL). Specific DNA markers for each of the 10 DSL bacterial strains were developed using the amplified fragment length polymorphism (AFLP) technique, and the fates of DSL strains introduced in soil were assessed by real-time quantitative PCR (qPCR). The results indicated that all DNA markers had high specificity at the functional strain level and that detection of the target microorganisms was sensitive at a detection limitation range from 1.3 × 10² to 3.25 × 10⁵ CFU/g of dry soil. The results indicated that all introduced strains showed a trend toward a declining persistence in soil and could be categorized into three pattern types. The first type was long-term persistence exemplified by Pseudomonas stutzeri (ATCC 17587) and Pseudomonas denitrificans (ATCC 13867) strains. In the second pattern, represented by Bacillus subtilis (ATCC 6051) and Escherichia hermannii (ATCC 700368), the inoculated strain populations dropped dramatically below the detection threshold after 10 to 21 days, while in the third pattern there was a gradual decrease, with the population falling below the detectable level within the 180-day incubation period. These patterns indicate a selection effect of a microbial community related to the ecological function of microbial strains introduced in soil. As a key finding, the DSL strains can be quantitatively tracked in soil with high sensitivity and specificity at the functional strain level. This provides the basic evidence for further risk assessment of the priority DSL strains.

Association between the A1 allele of the DRD2 gene and reduced verbal abilities in adolescence and early adulthood

Behavioral genetic research has consistently revealed that genetic factors explain at least one half of the variance in measures of cognitive skills. However, the specific DNA markers involved in the etiology of cognitive abilities have remained elusive. The current study examined the association between the TaqI polymorphism in the dopamine D2 receptor gene (DRD2) and verbal skills. Analysis of data drawn from the National Longitudinal Study of Adolescent Health (Add Health) revealed that the A1 allele was associated with group membership in high-, average-, and low-verbal skills groups for Caucasians. The results suggest that DRD2 may be related to domain specific cognitive abilities, such as verbal skills.

Sequence variation at the rice blast resistance gene Pi-km locus: Implications for the development of allele specific markers

The recently cloned blast resistance ( R) gene Pi-km protects rice crops against specific races of the fungal pathogen Magnaporthe oryzae in a gene-for-gene manner. The use of blast R genes remains the most cost-effective method for an integrated disease management strategy. To facilitate rice breeding we developed a Pi-km specific DNA marker. For this purpose, we initially explored the existing sequence diversity for alleles of the two genes responsible for the Pi-km specificity. The analysis of 15 rice cultivars revealed that the majority of nucleotide polymorphisms were associated with the Pi-km1 gene. Interestingly, the correspondent amino acid variation was localized within the predicted coiled-coil domain of the putative Pi-km1 protein. In contrast, the sequence of Pi-km2 alleles was highly conserved even within distantly related cultivars. Furthermore, disease reactions of the selected cultivars to five M. oryzae isolates, as well as their determined Pi-km1 allele, showed a good correlation with the known Pi-k genes (- k/- kh/- km/- ks/- kp) historically reported for these cultivars. Based on these findings, specific primer sets have been designed to discriminate among the various Pi-km alleles. The new markers should simplify the introgression of the valuable blast resistance associated with the complex Pi-k locus into rice cultivars.

A reliable gender diagnostic PCR assay for jojoba (Simmondsia chinensis (Link) Schneider)

Jojoba (Simmondsia chinensis (Link) Schneider) is an obligate cross-pollinated shrub native to the Sonora desert. The most valuable product of the jojoba seeds is the liquid wax or jojoba oil which is marketed extensively in the cosmetic industry. Differing from the most of the cultivated crop species, jojoba has slow-growing habit, difficulties in the determination of sex at the early seedling stages, a male-biased ratio and low seed yield. In jojoba, the development of molecular strategies for the identification of sexes at early stages has been a priority in plantations and breeding programs. Two previous studies reported two candidate male-specific jojoba DNA markers. However, present study indicated that these markers were not useful in jojoba sex identification. A reliable gender diagnostic marker for jojoba is, therefore, needed. In the present study a novel jojoba male-specific touch-down polymerase chain reaction based DNA marker (JMS900) was reported using a total of 120 individual jojoba plants bulked into 16 samples. This sex specific DNA marker may have considerable theoretical and practical applications in the establishment of jojoba plantation and breeding studies.