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41.
PCR analysis of oilseed rape cultivars (Brassica napus L. ssp. oleifera) using 5′ -anchored simple sequence repeat (SSR) primers 总被引:8,自引:0,他引:8
Y. M. Charters A. Robertson M. J. Wilkinson G. Ramsay 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1996,92(3-4):442-447
Primers complementary to simple sequence repeats (SSRs) and with variable three-base anchors at their 5 end, were used in PCR analyses to compare pooled DNA samples from various Brassica napus and B. rapa cultivars. Amplification products were resolved on polyacrylamide gels and detected by silver-nitrate staining. The resulting banding patterns were highly repeatable between replicate PCRs. Two of the primers produced polymorphisms at 33 and 23 band positions, respectively, and could each discriminate 16 of the 20 cultivars studied. Combined use of both primers allowed all 20 cultivars to be distinguished. The UPGMA dendrogram, based on the cultivar banding profiles, demonstrated clustering on the basis of winter/spring growth habit, high/low glucosinolate content, and cultivar origin (i.e. the breeder involved). Intracultivar polymorphism was investigated using a minimum of ten individuals for each cultivar and was found to vary considerably between cultivars. It is concluded that anchored SSR-PCR analysis is a highly informative and reproducible method for fingerprinting oilseed rape populations, but that intra-cultivar variation should be investigated before using banding profiles from pooled samples for the identification of individuals. 相似文献
42.
Identification of molecular markers in soybean comparing RFLP,RAPD and AFLP DNA mapping techniques 总被引:16,自引:0,他引:16
Jhy-Jhu Lin Jonathan Kuo Jin Ma James A. Saunders Hunter S. Beard Margaret H. MacDonald William Kenworthy George N. Ude Benjamin F. Matthews 《Plant Molecular Biology Reporter》1996,14(2):156-169
Three different DNA mapping techniques—RFLP, RAPD and AFLP—were used on identical soybean germplasm to compare their ability
to identify markers in the development of a genetic linkage map. Polymorphisms present in fourteen different soybean cultivars
were demonstrated using all three techniques. AFLP, a novel PCR-based technique, was able to identify multiple polymorphic
bands in a denaturing gel using 60 of 64 primer pairs tested. AFLP relies on primers designed in part on sequences for endonuclease
restriction sites and on three selective nucleotides. The 60 diagnostic primer pairs tested for AFLP analysis each distinguished
on average six polymorphic bands. Using specific primers designed for soybean fromEco RI andMse I restriction site sequences and three selective nucleotides, as many as 12 polymorphic bands per primer could be obtained
with AFLP techniques. Only 35% of the RAPD reactions identified a polymorphic band using the same soybean cultivars, and in
those positive reactions, typically only one or two polymorphic bands per gel were found. Identification of polymorphic bands
using RFLP techniques was the most cumbersome, because Southern blotting and probe hybridization were required. Over 50% of
the soybean RFLP probes examined failed to distinguish even a single polymorphic band, and the RFLP probes that did distinguish
polymorphic bands seldom identified more than one polymorphic band. We conclude that, among the three techniques tested, AFLP
is the most useful. 相似文献
43.
Joan E. Vickers Glenn C. Graham Robert J. Henry 《Plant Molecular Biology Reporter》1996,14(4):363-368
Amplification of thebar gene usingTaq DNA polymerase in PCR is often not successful, possibly due tobar's high GC content. We describe a PCR protocol in which reliable amplification at a sensitivity of one gene copy per genome
(in this study, barley) present in the reaction was achieved using a novel pair of primers and Expandtm High Fidelity DNA polymerase mix (Boehringer Mannheim). This method should allow for rapid screening of plants putatively
transformed withbar. 相似文献
44.
Gavin R. Sills William Bridges Salah M. Al-Janabi Bruno W. S. Sobral 《Molecular breeding : new strategies in plant improvement》1995,1(4):355-363
Saccharum robustum Brandes & Jesw. ex Grassl has been suggested as the immediate progenitor species of cultivated sugarcane (S. officinarum L.) [4]. Chromosome pairing and assortment in these two species were previously studied by genetic analysis of single-dose DNA markers in parents in and 44 F1 progeny of a cross between euploid, meiotically regular 2n=80S. officinarum LA Purple andS. robustum Mol 5829 [2]. This same population was subsequently clonally propagated and evaluated in replicated trials for quantitative traits important to sugarcane breeders. Numbers of stalks, tasseled stalks, and stalks with smut, and the average diameter of two stalks were determined one day prior to harvest. At harvest, plant material from each plot was weighed and evaluated for pol (sucrose content) and fiber percentages. Clones were significantly different (P<0.01) for all traits analyzed. Associations of 83 single-dose arbitrarily primed PCR genetic markers with quantitative trait loci (QTL) of recorded traits was determined by single-factor ANOVA, and multiple regression. QTL analysis revealed markers significantly (P<0.05) associated with the expression of each trait analyzed. Markers associated with QTL after multiple regression were tested for digenic linear × linear epistatic interactions. The various multilocus models explained between 23% and 58% of the total phenotypic variation and 32% and 76% of the genotypic variation for the various traits. Digenic interactions were uncommon. Implications for marker-assisted selection in sugarcane and sugarcane domestication are discussed. 相似文献
45.
Alec Breen Alan F. Rope Denise Taylor John C. Loper P. R. Sferra 《Journal of industrial microbiology & biotechnology》1995,14(1):10-16
Summary The use of DNA amplification fingerprinting (DAF) as a tool for monitoring mixed microbial populations in bioreactors was evaluated. Short (8-mer or 10-mer) oligonucleotides were used to prime DNA extracts from various biological reactors during polymerase chain reaction (PCR) amplification. The reactors examined in this study included two sets of anaerobic stirred tank continuous flow bioreactors. One set of anaerobic reactors was operated under methanogenic conditions and one set was operated under sulfate-reducing conditions. The anaerobic reactor communities in the methanol-fed reactors showed extensive DAF homology. DAF was also applied to a fixed-film azo dye degrading reactor to examine the degree of uniformity of colonization of the substratum in representative regions of the reactor. This method is a quick and relatively inexpensive means of monitoring microbial community structure during biological processes. Since no cultivation of the sample is involved, the genetic profile of the community is not biased by outgrowth conditions. DAF profiles may be useful for comparisons of population changes over time or of bench-scale vs pilot-scale reactors but not adequate for assessing community diversity. 相似文献
46.
47.
48.
Tatsuo Kawarasaki Tetsuya Kohsaka Masaru Sone Mitsutoshi Yoshida Kimio Bamba 《Molecular reproduction and development》1995,40(4):455-459
This study was carried out to determine whether Y-bearing porcine spermatozoa could be detected by in situ hybridization using a digoxigenin (Dig)-labelled DNA probe specific to the Y chromosome produced by polymerase chain reaction (PCR). A conventional PCR (with Dig-dUTP) was performed using a set of oligonucleotide primers (5′-AAGTGGTCAGCGTGTCCATA-3′ and 5′-TTTCTCCTGTATCCTCCTGC-3′) for 236 bp fragment of porcine male-specific DNA sequence and 1.25 × 104 template white blood cells obtained from a boar. When fluorescence in situ hybridization with the Dig-labelled DNA probe was applied to the metaphase chromosome spreads prepared from both boar and gilts, the fluorescein signal was only detected on the long arm of the Y chromosome. In addition, immunocytochemical detection with the Dig-labelled DNA probe and alkaline phosphatase-labeled anti-Dig was applied to both sperm nuclei pretreated with dithiothreitol and white blood cells; 51% of sperm nuclei and 96% of white blood cells obtained from boar were labelled, whereas none of white blood cells obtained from gilts were labelled with the Dig-labelled DNA probe. The results indicated that in situ hybridization with porcine male-specific DNA probe produced by PCR made possible the direct visualization of Y-bearing porcine spermatozoa by in situ hybridization. © 1995 Wiley-Liss, Inc. 相似文献
49.
Sex-related growth rate differences in preimplantation mouse embryos were investigated. In experiment I, Day 3 embryos were recovered from reproductive tracts, classified according to developmental stage, and cultured for 24 hr in CZB medium containing glucose. Each embryo was then reclassified and stained for measurement of number of nuclei and finally sexed using the polymerase chain reaction. In experiment II, Day 4 embryos were recovered, classified, stained, and sexed as in experiment I immediately after recovery. Morphologically, there were no differences between the sexes in either of the experiments on Day 4. However, based on number of nuclei, the data showed that in vitro conditions support the development of male embryos to the blastocyst stage compared to female embryos. Furthermore, growth rate differences were observed in vivo on Day 3, as females compacted earlier than males. These results suggest that the increased cell proliferation in cultured male embryos is an artifact caused by the in vitro environment. The variation may be due to sex differences in embryonal energy metabolism during the preimplantation stage. The growth difference implies different in vitro requirements of male and female embryos. © 1995 Wiley-Liss, Inc. 相似文献
50.
Evaluation of mass spectrometric techniques for charaterization of engineered proteins 总被引:7,自引:0,他引:7
Roepstorff Peter Schram Karl H. Andersen Jens S. Rafn Kate Baldursson Trausti Krøll Jenny Poulsen Kjeld Knudsen Jens Kristiansen Karsten 《Molecular biotechnology》1995,3(1):1-7
A simple and versatile method of in vitro site-specific mutagenesis based on polymerase chain reaction (PCR) is described.
The complete method required the use of three oligonucleotide primers and two PCRs. The product of the first PCR was used
as one of the primers (megaprimer) in the second PCR. Essentially 100% of the final product incorporated the desired mutation.
The various aspects of the procedure and its application is described in detail. 相似文献