Restriction Fragment Length Polymorphism Fragments Research Articles

CAPS Marker Linked to Tomato Hypocotyl Pigmentation

Tomato hypocotyl can generally be one of two colors, purple or green. Genetically, this trait is controlled by a single dominant gene. Hypocotyl tissue specific color expression is one of many visible genetic marker sources used to select tomato progeny. However, the visible marker does not show a clear distinction between homozygous genotype and heterozygous genotype from the breeding lines. Therefore, to identify a hypocotyl pigmentation related marker, we screened DNA polymorphisms in thirteen tomato lines showing purple or green hypocotyls. The markers used for screening consisted of primer set information obtained from anthocyanin related genes, conserved ortholog set II (COS II) marker sets localized near anthocyanin related genes, and restriction fragment length polymorphism (RFLP) markers localized near COS II markers, which produce polymorphisms between purple and green tomatoes. One primer from a RFLP fragment resulted in a polymorphism on agarose gel electrophoresis. From the RFLP fragment, a cleaved amplified polymorphic sequence (CAPS) marker was developed to distinguish between purple and green hypocotyls. The genotypes of 135 <TEX>$F_2$</TEX> individuals were analyzed using the CAPS marker, and among them, 132 individuals corresponded to the phenotypes of hypocotyl pigmentation.

Intra-specific rDNA-ITS restriction site variation and an improved protocol to distinguish species and hybrids in the Daphnia longispina complex

A collaborative research effort was undertaken to evaluate the robustness of a recently developed genetic tool for species identification of members in the morphologically variable Daphnia longispina species complex. This genetic method, based on restriction fragment length polymorphism (RFLP) of the internal transcribed spacer region (ITS) of nuclear ribosomal DNA (rDNA) with restriction enzymes Mwo I and Sau96 I [Billiones et al., 2004. Hydrobiologia 526: 43–53], was applied to many different European populations. Results were compared with two or more independently obtained characters (morphology, allozymes, mitochondrial DNA (mtDNA), or cloned rDNA-ITS sequences). Individuals of most taxa were readily identified, but unexpected ITS-RFLP patterns were found in many individuals indicated by other markers to be D. galeata or one of its hybrids. Among 43 investigated D. galeata populations (902 specimen analysed by ITS-RFLP), deviant RFLP fragment patterns occurred in 26 (i.e., more than half) of the populations. The deviant patterns could be attributed to the loss of one single restriction site in the ITS2 region. This loss made the distinction of D. galeata from other species unreliable, and F1 hybrids could not be identified. Future users should be aware of this shortcoming of the Billions et al. [2004. Hydrobiologia 526: 43–53] protocol. As a solution to this problem, we present an improved genetic identification protocol based on a simple double digestion of the rDNA-ITS region with the restriction enzymes BsrB I and EagI. Sequence analyses of rDNA-ITS clones and preliminary testing indicate that the new protocol is unaffected by the rDNA variation which troubled the Mwo I/Sau96 I protocol. Further, the new protocol identifies all European species of the D. longispina complex, as well as their F1 hybrids. However, a wider screening is required to verify its general utility for all species, since yet unknown variation may occur.

MHC class I variation associates with parasite resistance and longevity in tropical pythons

Using restriction fragment length polymorphism (RFLP) we identified 26 unique major histocompatibility complex (MHC) genotypes in 104 water pythons. We observed a significant independent association between reduced blood parasite load (Hepatozoon sp.) and python body length/age, presence of a specific RFLP fragment (C-fragment) and the overall number of fragments. The parasite has a negative impact on several python life-history traits such as growth, nutritional status and longevity. Thus, the C-fragment could be considered a 'good gene' (a fitness-enhancing genetic element). However, while the number of fragments affected parasite load, the association between level of parasitaemia and fragment number was not linear, and, hence, minimum parasite infection level was achieved at an intermediate number of fragments. Intermediate MHC fragment numbers were also observed among the largest/oldest pythons, suggesting that both a specific fragment and intermediate levels of MHC polymorphism enhanced python longevity. Thus, our results suggest python MHC is subject to both frequency-dependent and balancing selection.

Community structure of the microbiota associated with nodal roots of rice plants along with the growth stages: estimation by PCR-RFLP analysis

Community structure of the microbiota in rice roots that developed from different nodes and at different growth stages were compared by using PCR (polymerase chain reaction)-RFLP (restriction fragment length polymorphism) analysis of 16S rDNA. This was the first study to have applied a molecular microbial technique for elucidating the rhizospheric microbial succession of rice roots. Rice plants were grown in submerged soil pots, and nodal roots were collected 5 times during the growing period of rice plants. The RFLP fragments digested by four restriction enzymes (Hinf I, Hae III, Sau3A I, and EcoR I) tended to increase along with the growth stage. A marked increase in the RFLP fragments coincided with the development of reduction in the rhizosphere soil. RFLP fragments that were associated with every nodal root irrespective to the sampling date and those specific to the early and late growth stages were identified. Systematic changes in RFLP patterns from higher (younger) nodal roots to lower (older) nodal roots were also observed at each sampling date, which indicated the succession of the microbial community from higher to lower nodal roots. Cluster analysis divided the RFLP patterns of the microbial community associated with nodal roots into four clusters depending on the growth stages of rice plants. The cluster of the early growth stage was further divided into two subclusters of higher and lower nodal roots. The specific RFLP fragments that contributed to the seasonal variation of the microbial community associated with nodal roots as well as those that characterized the aging of nodal roots were clarified by principal component analysis.

Quantitative assessment of apolipoprotein E genotypes by image analysis of PCR–RFLP fragments

Apolipoprotein E (APOE) genotyping usually involves polymerase chain reaction (PCR) and assessment of restriction fragment length polymorphism (RFLP) by gel electrophoresis. We made determination of HhaI restriction endonuclease digestive patterns more objective and improved diagnostic accuracy with a quantitative approach using sensitive DNA stain (SYBR Green) and image analysis of gel patterns. For distinguishing true and partially-digested restriction fragments, band ratios were calculated for the staining intensity of gel patterns from 116 sample runs of 63 human blood specimens. Each of these specimens was independently genotyped for APOE by at least two (and most of them by three) different PCR–RFLP methods. Based on the distribution of band ratios, decision levels were established and used for developing a program for computer-aided interpretation of APOE genotypes (Microsoft Excel software). Appropriateness of the decision levels for band ratios was validated by APOE genotyping of additional 61 specimens. The approach described here is applicable to a variety of other molecular diagnostic techniques that are based on PCR–RFLP or sequence-specific signal amplifications.

Polymorphism and transcription of Mhc class I genes in a passerine bird, the great reed warbler.

The class I genes of the major histocompatibility complex (Mhc) are here investigated for the first time in a passerine bird. The great reed warbler is a rare species in Sweden with a few semi-isolated populations. Yet, we found extensive Mhc class I variation in the study population. The variable exon 3, corresponding to the alpha2 domain, was amplified from genomic DNA with degenerated primers. Seven different genomic class I sequences were detected in a single individual. One of the sequences had a deletion leading to a shift in the reading frame, indicating that it was not a functional gene. A randomly selected clone was used as a probe for restriction fragment length polymorphism (RFLP) studies in combination with the restriction enzyme Pvu II. The RFLP pattern was complex with 21-25 RFLP fragments per individual and extensive variation. Forty-nine RFLP genotypes were detected in 55 tested individuals. To study the number of transcribed genes, we isolated 14 Mhc class I clones from a cDNA library from a single individual. We found eight different sequences of four different lengths (1.3-2.2 kilobases), suggesting there are at least four transcribed loci. The number of nonsynonymous substitutions (dN) in the peptide binding region of exon 3 were higher than the number of synonymous substitutions (dS), indicating balancing selection in this region. The number of transcribed genes and the numerous RFLP fragments found so far suggest that the great reed warbler does not have a "minimal essential Mhc" as has been suggested for the chicken.

Desaturase multigene families of Brassica napus arose through genome duplication

This paper reports the estimated gene copy number and restriction fragment length polymorphism (RFLP) map locations of five different desaturase cDNA clones from Brassica napus (oilseed rape). The desaturase enzymes encoded by four of these genes catalyze successive reactions that insert double bonds into lipid-linked fatty acid residues. Delta-12 (e2) and delta-15 (e3) desaturases are active in the endoplasmic reticulum, while omega-6 (p2) and omega-3 (p3) desaturases catalyze analogous desaturation reactions via a parallel pathway located in plastids. The fifth cDNA clone (b5) contains a desaturase-like domain bound to a cytochrome b5 segment. Estimates of gene copy number based on Southern blot analysis of 16 oilseed rape varieties and three different resynthesized Brassica napus lines indicated that e2 had 4–6 gene copies and e3, p2, p3 and b5 each had 6–8 gene copies per haploid genome. Estimates of the gene copy number for the two progenitor species, Brassica oleracea and Brassica rapa, supported the premise that all these genes were at least duplicated or triplicated in the two progenitor species before they combined to form B. napus. RFLP mapping results showed that the e2 probe detected 4 distinct loci, the e3 probe 6 loci and p2, p3 and b5 each detected 8 loci, with pairs of loci often mapping to homoeologous regions on 2 different linkage groups. The 28 mappable loci were distributed across 12 linkage groups of the B. napus map (Parkin et al. 1995) and were usually represented by single RFLP fragments. A collinear segment containing the e2 and p3 loci was positioned on B. napus linkage groups N1, N11, N3, N13, N5 and N15. This segment was collinear with a 30-cM region of Arabidopsis thaliana chromosome 3 that contains the homologous fad2 (e2) and fad7(p3) genes. This suggests that the desaturase multigene families arose as the result of duplication of large chromosome segments rather than duplication of individual genes.

A comparative analysis of the genetic relationships between rye cultivars using RFLP and RAPD markers

The genetic similarities of eight closely related rye cultivars were estimated using two molecular marking techniques: restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD). Cultivars were evaluated for variation by 11 random cDNA and genomic clones used in combination with four restriction enzymes and 40 decamer primers. A total of 53 polymorphic RFLP fragments and 94 polymorphic RAPD fragments were observed. Based on the presence/absence of fragments, two genetic similarity matrices were calculated which were then used in cluster analysis. Differences between pair of cultivars were observed in RFLP and RAPD dendrograms. RFLP analysis produced estimates of genetic relationships more in accordance with the partially known pedigree of the cultivars than did RAPD analysis. The use of bulk samples of DNA in these analyses affected the sensitivity of RAPD assays more strongly. Dendrograms which took into account all fragments produced, either by RFLP or RAPD, reflected better the relationships between cultivars than did dendrograms based on only one type of marker. This reflects the importance of the number of markers used in determining the genetic relationships between genotypes.

The Genetic Relationship Between Mycoplasma‐like Organisms Causing Diseases in the Sudan and Different Continents Revealed by Polymerase Chain Reaction (PCR) Amplification of the 16S rRNA Gene Followed by Restriction Fragment Length Polymorphism Analysis

AbstractThe genetic relationship between faba bean (Vicia faba L.) phyllody and other mycoplasma‐like organism (MLO) diseases has been studied by amplification of the conserved region of the 16S rRNA gene followed by restriction fragment length polymorphism (RFLP) analysis using Alu I restriction endonuclease. The restriction patterns produced by faba bean phyllody MLO were smilar to that of Crotalaria saltiana phyllody MLO which persists throughout the year in the Sudan. These, and serological results clearly confirmed that C. saltiana is a reservoir of faba bean phyllody MLO in the Sudan. Moreover, restriction patterns have also shown that MLOs of other diseases have the same RFLP fragment pattern as faba bean phyllody MLO, including C. juncea witches'broom (Thailand) and tomato big‐bud (Australia), which differs from the other selected MLO diseases (Gladiolus aster yellow, clover phyllody and yellow decline of lavender, aqll from France).Fragment patterns also revealed the existence of genetically diverse MLO strains in the Sudan. Faba bean phyllody may be placed in group III including WX, apricot chlorotic leaf roll, golden flaveswcence dorée of grapevine, plum leptonecrosis of Prunus salciana, peachy yellow leaf roll, sunnhemp phyllody from Thailand, and blueberry witches' broom.

REMI-RFLP mapping in the Dictyostelium genome.

A set of 147 Dictyostelium discoideum strains was constructed by random integration of a vector containing rare restriction sites. The strains were generated by transformation using restriction enzyme-mediated integration (REMI) which results in the integration of linear DNA fragments into randomly distributed genomic restriction sites. Restriction fragment length polymorphism (RFLP) was generated in a single genomic site in each strain. These REMI-RFLP strains were used to confirm gene linkages previously supported by two other physical mapping techniques: yeast artificial chromosome (YAC) contig construction, and megabase-scale restriction mapping. New linkages were uncovered when two or more hybridization probes identified the same RFLP fragments. Probes for 100 genes have marked 53% of the RFLPs, representing greater than 22 Mb of the 40 Mb Dictyostelium genome. Alignment of these and other large fragments along each chromosome should lead to a complete physical map of the Dictyostelium genome.

Validation Studies on the Forensic Analysis of Restriction Fragment Length Polymorphism (RFLP) on LE Agarose Gels Without Ethidium Bromide: Effects of Contaminants, Sunlight, and the Electrophoresis of Varying Quantities of Deoxyribonucleic Acid (DNA)

This study was designed to analyze the effects of sunlight, various contaminants (those found typically in forensic samples) and the electrophoresis of varying quantities of DNA on the restriction fragment length polymorphism (RFLP) patterns produced from DNA isolated from blood and semen stains. The DNA RFLP patterns were obtained following Hae III restriction enzyme digestion, low electroendosmotic (LE) agarose gel electrophoresis (in the absence of ethidium bromide), Southern transfer, hybridization with DNA probes detecting highly polymorphic variable number of tandem repeats (VNTRs) and autoradiography. Computer assisted image analyses were used to detect variations in RFLP band sizes in relation to control samples. Comparisons between the samples were made for the presence of high molecular weight DNA, the ability to achieve a complete restriction digestion, and the RFLP fragment sizes obtained. The results demonstrate that high molecular weight DNA can be obtained when blood and semen stains are subjected to environmental and contaminating factors. The RFLP allele sizes were not significantly affected by environmental conditions, contamination factors or by loading varying amounts of DNA. This study serves to further document the reliability and validity of DNA typing for forensic applications.

HCMV strain comparison by restriction analysis of genomic virus DNA and of a PCR-amplified DNA fragment

background: Human cytomegalovirus (HCMV) is ubiquitous and a major cause of disease in the immunocompromised which include the unborn fetus. HCMV strains are closely related and may be difficult to differentiate. Objectives: To analyze new HCMV isolates and highly passaged strains by conventional restriction fragment length polymorphism (RFLP) analysis of HCMV DNA and restriction analysis (RA) of a PCR-amplified a-sequence fragment to identify epidemiological relatedness. Study design: DNA from 14 low-passaged clinical isolates and the high-passaged strains S348 and AD169 were analyzed by RFLP analysis of BamHI, PstI or HpaII digestion as well as by PCR a-sequence amplification. The laboratory results were compared according to epidemiological findings. Results: Of the 14 clinical isolates 13 could be distinguished from each other by RFLP analysis. Isolates from an infant and its mother had identical restriction fragment patterns. Eight of 13 clinical isolates were differentiated from each other by comparing a-sequence PCR fragments according to length and the presence of cleavage sites for 6 different restriction enzymes. The mother-infant isolates were identical by the a-sequence PCR criteria. All 14 low-passaged isolates differed from the high-passaged S384 and AD169 strains by both distinguishing approaches. Conclusions: Both RFLP and PCR-amplified a-sequence fragment analysis clearly differentiated between low- and high-passaged HCMV isolates and are useful epidemiological tools for determining viral strain relatedness.

RFLP mapping of the ym4 virus resistance gene in barley

RFLP (restriction fragment length polymorphism) mapping of a recessive gene (ym4) conferring resistance to barley yellow mosaic and barley mild mosaic virus was performed using progeny of 86 F1 anther-derived doubled haploid lines. Two closely linked RFLP markers that flank the gene at a distance of 1.2 centiMorgans were identified. Using one of these markers (MWG10) we obtained a clear differentiation between resistant and susceptible German cultivars. An analysis of a series of unrelated barley lines with probe MWG10 did not reveal additional RFLP fragments. The use of this probe for both marker-assisted selection and the generation of a high-density map around the resistance locus is discussed.