Restriction Fragment Length Polymorphism Clone Research Articles

Genetic Relatedness among Soybean Genotypes Using DNA Amplification Fingerprinting (DAF), RFLP, and Pedigree

Numerous methods now exist for plant breeders and others to estimate genetic relationships among different germplasm. Two of the most commonly used methods have been restriction fragment length polymorphism (RFLP) and pedigrees. The objective of this study was to compare a relatively new method, DNA amplification fingerprinting (DAF), which is a short, arbitrary‐primer, polymerase chain reaction (PCR)‐amplification method, to RFLP and pedigree methods for genetic distance estimation among soybean [Glycine max (L.) Merr.] genotypes. The genotypes consisted of agronomically important soybean lines Archer, BSR101, Forrest, Hartwig, 9171, 9341, 9392, 9593, S8330 and the plant introduction, PI437.654. The 10 genotypes were evaluated by means of 53 RFLP clones, a subset of 12 RFLP clones, seven DAF primers, and available pedigrees. The DAF data set and the subset of 12 RFLP clones each consisted of 18 markers. For each method, the data were used to calculate the 45 pairwise genetic‐distance estimates among the varieties using the average linkage algorithm in SAS. The genetic‐distance estimates were correlated from 0.63 to 0.89 among the four methods. Dendrograms based on each method were therefore considered similarly reliable. For varietal identification, the DAF and RFLP markers distinguished the varieties with similar efficiency. We conclude that both DAF and RFLP marker technologies and pedigrees are useful tools for estimating genetic distances in soybean.

Genetic diversity in Eastern U.S. soft winter wheat (Triticum aestivum L. em. Thell.) based on RFLPs and coefficients of parentage

Genetic diversity in a set of 11 red and 11 white wheat lines from the Eastern U.S. soft wheat germplasm pool was measured using restriction fragment length polymorphism (RFLP) assay and coefficients of parentage (COP) analysis. On average, 78% of all bands revealed by three enzymes with 48 RFLP clones were monomorphic. Average pairwise genetic similarity (GS) was 0.97 when data from all enzymes were pooled. Probe Polymorphic Information Content (PIC) indexes ranged from 0 to 0.73 with a mean of 0.2. Fewer than 55% of the probes revealed any polymorphism. The frequency of polymorphism in the Eastern U.S. soft white winter (SWW) wheat gene pool was much lower than that observed in the Eastern U.S. soft red winter (SRW) wheat gene pool. SWW lines formed a single group on a dendrogram based on cluster analysis of RFLP-derived GS estimates, while SRW lines did not form a single group. COP values for all pairs of the Eastern U.S. soft wheat lines ranged from 0.02 to 0.9 with a mean of 0.21. SWW wheat lines traced to 53 ancestral lines and had an average COP of 0.51. The SRW wheat gene pool had more complex parentages (mean COP=0.15 and a total of 65 ancestral lines). COPs were correlated with RFLP-based GS for all line pairs (r=0.73, P<0.01). However, correlations between the two similarity measures were substantially lower when the SRW and SWW wheat gene pools were considered individually (r values of 0.23 and 0.28, respectively). The actual GS among unrelated lines in the U.S. Eastern soft wheat gene pool appears to be higher than that observed for unrelated landraces from Southwest Asia (0.96 vs. 0.905), suggesting that the ancestral landrace parents of this gene pool were themselves drawn from a base population where inbreeding, i.e., F, was greater than zero.

Transfer of sequence tagged site PCR markers between wheat and barley

Transfer of mapping information between related species has facilitated the development of restriction fragment length polymorphism (RFLP) maps in the cereals. Sequence tagged site (STS) primer sets for use in the polymerase chain reaction may be developed from mapped RFLP clones. For this study, we mapped 97 STS primer sets to chromosomes in wheat and barley to determine the potential transferability of the primer sets and the degree of correspondence between RFLP and STS locations. STS products mapped to the same chromosome group in wheat and barley 75% of the time. RFLP location predicted STS location 69% of the time in wheat and 56% of the time in barley. Southern hybridizations showed that most primer sets amplified sequences homologous to the RFLP clone, although additional sequences were often amplified that did not hybridize to the RFLP clone. Nontarget sequences were often amplified when primer sets were transferred across species. In general, results suggest a good probability of success in transferring STSs between wheat and barley, and that RFLP location can be used to predict STS location. However, transferability of STSs cannot be assumed, suggesting a need for recombinational mapping of STS markers in each species as new primer sets are developed. Key words : sequence tagged sites, PCR, wheat, barley.

Estimating Nuclear DNA Content in Peach and Related Diploid Species Using Laser Flow Cytometry and DNA Hybridization

Using laser flow cytometry, nuclear DNA amounts were estimated for 12 Prunus species, representing three subgenera [Prunophora (Prunus), Amygdalus, and Cerasus (Lithocerasus)], two interspecific hybrids, four cultivars, and a synthetic polyploid series of peach consisting of haploids, diploids, triploids, and tetraploids (periclinal cytochimeras). Peach nuclear DNA content ranged from 0.30 pg for the haploid nuclei to 1.23 pg for the tetraploid nuclei. The diploid genome of peach is relatively small and was estimated to be 0.60±0.03 pg (or 5.8×108 nucleotide base pairs). The polyploid series represented the expected arithmetic progression, as genome size positively correlated with ploidy level (i.e., DNA content was proportional to chromosome number). The DNA content for the 12 diploid species and two interspecific diploid hybrids ranged from 0.57 to 0.79 pg. Genome size estimates were verified independently by Southern blot analysis, using restriction fragment length polymorphism clones as gene-copy equivalents. Thus, a relatively small and stable nuclear genome typifies the Prunus species investigated, consistent with their low, basic chromosome number (× = 8).

Polymerase chain reaction mediated localization of RFLP clones to microisolated translocation chromosomes of barley.

A new strategy has been devised and used for the physical localization of genetically mapped restriction fragment length polymorphism (RFLP) clones to barley chromosomes. Morphologically distinct translocation chromosomes from synchronized root-tip meristems were microisolated and their DNA was used as a template for polymerase chain reaction with sequence-specific primers. Four RFLP clones were assigned to cytologically defined segments of chromosome 5. This related approximately one-third of the map length of linkage group 5 to approximately one-fifth of the mitotic metaphase length of chromosome 5. The technique may substantially contribute to the connection of the RFLP-based genetic linkage maps with cytological markers of the barley chromosomes.

Mapping quantitative trait loci (QTLs) for resistance to Gibberella zeae infection in maize

The basic prerequisite for an efficient breeding program to improve levels of resistance to pathogens in plants is the identification of genes controlling the resistance character. If the response to pathogens is under the control of a multilocus system, the utilization of molecular markers becomes essential. Stalk and ear rot caused by Gibberella zeae is a widespread disease of corn: resistance to G. zeae is quantitatively inherited. Our experimental approach to understanding the genetic basis of resistance to Gibberella is to estimate the genetic linkage between available molecular markers and the character, measured as the amount of diseased tissue 40 days after inoculation of a suspension of Fusarium graminearum, the conidial form of G. zeae, into the first stalk internode. Sensitive and resistant parental inbreds were crossed to obtain F1 and F2 populations: the analysis of the segregation of 95 RFLP (restriction fragment length polymorphism) clones and 10 RAPD (random amplified polymorphic DNA) markers was performed on a population of 150 F2 individuals. Analysis of resistance was performed on the F3 families obtained by selfing the F2 plants. Quantitative trait loci (QTL) detection was based either on analysis of regression coefficients between family mean value and allele values in the F2 population, or by means of interval mapping, using MAPMAKER-QTL. A linkage map of maize was obtained, in which four to five genomic regions are shown to carry factors involved in the resistance to G. zeae.

RFLP Analysis of Genomic Regions Associated with Resistance to Preharvest Sprouting in Wheat

Resistance to preharvest sprouting (PHS) is difficult to incorporate into new varieties because heritability is low and selection is limited to one generation per year. Our objective was to identify genomic regions containing quantitative trait loci (QTL) associated with resistance to PHS in two recombinant inbred (R) populations of white wheat (Triticum aestivum L. em. Thell.) by restriction fragment length polymorphism (RFLP) markers. One population consisted of 78 RI lines from the cross of NY6432‐18 (NY18) × ‘Clark's Cream’ (CC). The second population consisted of 138 RI lines from the cross between sib lines NY18 and NY6432‐10 (NY10). The NY18/CC and NY18/10 populations were evaluated for PHS in six and seven environments, respectively, by examining physiologically mature spikes under simulated rainfall. The three parental lines were surveyed for polymorphism with 195 low‐copy RFLP clones. Individual RI lines from the NY18/CC and NY18/NY10 populations were probed with 37 and 27 polymorphic clones, respectively. Eight regions of the genome (four from each population) were significantly associated with resistance to PHS. Based on multiple regression, specific sets of markers and their interactions accounted for 44% of the genetic variance for PHS in the NY18/CC and 51% in the NY18/NY10 populations. These markers could find utility in breeding programs as indirect selection criteria for improvement of PHS resistance.