Association of restriction fragment length polymorphisms of swine leucocyte antigen class I genes with production traits of Duroc and Hampshire boars

Summary. Restriction fragment length polymorphism analyses of swine leucocyte antigen (SLA) class I genes were performed on 70 Duroc and 38 Hampshire boars from the 1986-87 national performance tests of each breed in the USA. Few boars were inbred. Southern blotting and hybridization procedures were performed on genomic DNA, isolated from white blood cells, using Pvu II endonuclease and a swine major histocompatibility complex (MHC) class I probe. Durocs had an average of 11 restriction fragments, with the most common being in 63% of the boars and the least common appearing in only one boar. Hampshire boars had an average of 12 restriction fragments, with the most common appearing in 73% of the boars and the least common appearing in only one boar. Least squares procedures and stepwise regression methods were used to examine the association between DNA restriction fragments and the selection index (INDEX), average daily gain (ADG), average backfat thickness (BF), loin muscle area (LEA), and age at 104kg (DAY104). In the Duroc breed one DNA restriction fragment was associated with decreased INDEX ( P < 0.05) and decreased ADG ( P < 0.05) whereas two other fragments were associated with increased BF ( P < 0.05). In the Hampshire breed two restriction fragments were associated with an increase in INDEX ( P < 0.05). Cluster analyses were used to group pigs of each breed on the basis of similar RFLP patterns. One cluster group in the Duroc breed was associated with lower average INDEX values ( P < 0.05), greater average DAY104 ( P < 0.05), and a larger mean LEA ( P < 0.05). In the Hampshire breed one cluster group was associated with lower INDEX ( P < 0.05). These results suggest there may be an association between swine MHC class I genes and performance traits in swine. The use of SLA class I restriction fragments, as genetic markers, may have potential in the future for improving pig performance.     

Molecular characterization of swine leucocyte antigen class I genes in outbred pig populations

The highly polymorphic swine leucocyte antigen ( SLA ) genes are one of the most important determinants in swine immune responses to infectious diseases, vaccines, and in transplantation success. Study of SLA influence requires accurate and effective typing methods. We developed a simple and rapid method to type alleles at the three classical SLA class I loci ( SLA-1 , SLA-3 and SLA-2 ) using the PCR-sequence-specific primer (PCR-SSP) strategy. This typing system relies on 47 discriminatory PCR primer pairs designed to amplify the SLA class I alleles by groups that have similar sequence motifs. We applied this low-resolution group-specific typing method to characterize the SLA class I alleles present in three outbred pig populations ( n =  202). Alleles from 24 class I allele groups corresponding to 56 class I genotypes were detected. We also identified 23 low-resolution SLA class I haplotypes in these pigs and found haplotypes Lr-1.0 ( SLA-1 *01XX- SLA-3 *01XX- SLA-2 *01XX) and Lr-4.0 ( SLA-1 *04XX- SLA-3 *04XX- SLA-2 *04XX) in all three pig populations with a high prevalence. Over 80% of the pigs examined ( n  =   162) were found to bear at least one of these haplotypes, resulting in a combined haplotype frequency of nearly 50%. This PCR-SSP-based typing system demonstrates a reliable and unambiguous detection of SLA class I alleles, and can be used to effectively investigate the SLA diversity in outbred pig populations. It will help to identify the role of SLA antigens in disease-resistant pigs and may facilitate the development of effective vaccines.     

Association analysis of lipid levels and apolipoprotein restriction fragment length polymorphisms

Summary We have analyzed the correlation between restriction site variants (RFLPs; restriction fragment length polymorphisms) of the apolipoprotein AI, AII, B, CI and CII genes and serum lipid levels in a sample of male Norwegians. We find no significant association between any of the RFLPs and lipid levels.     

Restriction maps and restriction fragment length polymorphisms of the human 21-hydroxylase genes

Restriction maps were constructed for the two human 21-hydroxylase genes (21-OHA and 21-OHB) by using DNA from subjects homozygous for a deletion of each gene. Comparing the patterns of these two genes, a KpnI restriction site occurred in the 21-OHA gene in place of a TaqI site in the 21-OHB gene about 1-kb from the 5' end of the gene, and an extra EcoRI site was located 500 bp 5' to the common EcoRI site. The DNA of fourteen unrelated normal subjects was digested with nine restriction endonucleases (AccI, BamHI, BgIII, EcoRI, HindIII, KpnI, MspI, SacI and TaqI). Restriction fragment length polymorphisms were found with EcoRI, HindIII and AccI that resulted from polymorphic endonuclease sites outside the genes.     

Bamboo germplasm screening with nuclear restriction fragment length polymorphisms

Summary Bamboo species are difficult to identify because flowering material is seldom available and taxonomy is of necessity based on vegetative characters. To evaluate the utility of restriction fragment length polymorphism (RFLP) analysis in bamboo systematics and germplasm screening, a library of random genomic probes from a Phyllostachys nigra PstI library was constructed. Probes from the library were used to screen bamboo germplasm consisting mostly of temperate bamboos of the genus Phyllostachys. RFLP variation was abundant, and species-specific patterns were readily obtained. Chloroplast DNA showed little variation among the bamboo accessions analyzed.     

Studies on four restriction fragment length polymorphisms of the type I collagen genes in two Italian populations

Type I collagen, the most abundant of the collagen protein family, is encoded by two genes, COL1A1 and COL1A2. Two random population samples, one from central Italy and one from southern Italy, were studied for 1 restriction fragment length polymorphism (RFLP) of COL1A1 (RsaI) and 3 RFLPs of COL1A2 (EcoRI, RsaI and MspI). A considerable heterogeneity for COL1A1/RsaI was found not only between Italians and English but even among Italians. The potential usefulness of these RFLPs and haplotypes as anthropogenetic markers, particularly in distinguishing Caucasoids from Negroids, has been discussed.     

Molecular characterization of swine leucocyte antigen class II genes in outbred pig populations

The highly polymorphic swine leucocyte antigen (SLA) genes are among the most important determinants of swine immune responses to disease and vaccines. Accurate and effective SLA genotyping methods are required to understand how SLA gene polymorphisms affect immunity, especially in outbred pigs with diverse genetic backgrounds. In this study, we present a simple and rapid molecular‐based typing system for characterizing SLA class II alleles of the DRB1, DQB1 and DQA loci. This system utilizes a set of 47 sequence‐specific PCR primers developed to differentiate alleles by groups that share similar sequence motifs. We applied this typing method to investigate the SLA class II diversity in four populations of outbred pigs (n = 206) and characterized a total of 19 SLA class II haplotypes, six of which were shared by at least three of the sampled pig populations. We found that Lr‐0.1 (DRB1*01XX–DQB1*01XX–DQA*01XX) was the most prevalent haplotype with a combined frequency of 16.0%, followed by Lr‐0.2 (DRB1*02XX–DQB1*02XX–DQA*02XX) with 14.6% and Lr‐0.15b (DRB1*04XX–DQB1*0202–DQA*02XX) with 14.1%. Over 70% of the pigs (n = 147) had at least one copy of one of these three haplotypes. The PCR‐based typing system described in this study demonstrates a reliable and unambiguous detection method for SLA class II alleles. It will be a valuable tool for studying the influence of SLA diversity on various immunological, pathological and physiological traits in outbred pigs.     

Human restriction fragment length polymorphisms and cancer risk assessment

The polymorphic restriction fragments of the human Ha-ras locus, produced by the variable tandem repetition (VTR) of a short consensus sequence, fall into three classes based on allelic frequencies. Alleles of the "rare" class (individual frequencies less than 0.5%) have been detected only in white blood cell and tumor DNA of cancer patients. This phenomenon is independent of ethnic origin. No significant association of rare alleles with cancer patients has been demonstrated at an independent tandem repeat locus, VTR4.1. The results suggest that the Ha-ras restriction fragment length polymorphism is useful in cancer risk assessment.     

Genetic variability between two breeds based on restriction fragment length polymorphisms (RFLPs) of major histocompatibility complex class I genes in the pig

Summary Restriction fragment length polymorphism analyses of SLA class I genes were performed on 55 Duroc and 24 Hampshire boars from the 1986–87 national performance tests of each breed. Few boars were inbred. Southern blotting and hybridization procedures were performed on genomic DNA isolated from white blood cells by using Pvu II, Bam HI, and Eco RI endonucleases and a swine MHC class I probe. Genetic variability within and between the two breeds was estimated in terms of nucleotide diversity, by using a mathematical analysis based on the different RFLP patterns. The nucleotide diversity calculated within each breed was less than that between the two breeds. The results from the nucleotide diversity analysis suggested that genetic variability was greater in the Duroc breed than in the Hampshire breed. A relatively high level of genetic variability was shown in the class I major histocompatibility complex genes in the pig.     

Some combinatorial problems of DNA restriction fragment length polymorphisms

Recombinant DNA techniques provide a means of defining new polymorphisms at the DNA sequence level. Polymorphisms arise when individuals differ in the location and number of sites where restriction endonucleases can cleave their DNA. Each such site exhibits two possible states: one for the presence of a specific endonuclease recognition sequence, the other for its absence. The states of a system of adjacent sites can be revealed experimentally by cleaving a person's DNA into a set of fragments. For experimentally well-understood systems of sites, we consider problems of counting numbers of possible fragments, haplotypes, genotypes, and phenotypes, and the means of resolving phenotype-genotype ambiguities. The degree of polymorphism generated by such systems and the importance to gene mapping are discussed.     

Linkage of restriction fragment length polymorphisms and isozymes in Citrus

Summary Genetic linkage analysis was performed using two segregating populations of citrus. One population arose from an intergeneric backcross of Citrus grandis (L.) Osb. cv Thong Dee and Poncirus trifoliata (L.) Raf. cv Pomeroy, using the former as the recurrent (female) parent. The other population came from an interspecific backcross of C. reticulata Blanco cv Clementine and C. x paradisi Macf. cv Duncan, using the former as the recurrent (male) parent. A total of 11 isozyme and 58 restriction fragment length polymorphisms were found to segregate in a monogenic fashion in one or both populations. Linkage analysis revealed that 62 of the loci examined mapped to 11 linkage groups, while 7 loci segregated independently from all other markers. Gene order was highly conserved between the maps generated from the two divergent segregating populations. Possible applications of the use of such maps in tree fruit breeding are discussed.     

Type I interferon genes in cattle: restriction fragment length polymorphisms,gene numbers and physical organization on bovine chromosome 8

Multiple, superimposed Type I interferon (IFN) restriction fragments were resolved following 72–92 h of horizontal electrophoresis. Restriction fragment length polymorphisms (RFLPs) for α IFN (IFNA), β IFN (IFNB), ωIFN (IFNW) and trophoblast IFN (IFNT) genes were identified in Hin dill, Eco RI and Taql digestions from 313 cattle. RFLPs with codominant segregation in cattle pedigrees were considered alleles, and 19 distinct polymorphic Type I IFN loci (5 IFNA, 4 IFNB, 8 IFNW and 2 IFNT) were identified. Allele frequencies and observed heterozygosity values were calculated for each locus and several loci were considered highly informative for linkage analysis. Bovine IFN gene numbers (10 IFNA, 6 IFNB, 20 IFNW and 6 IFNT) were estimated from the number of polymorphic loci plus additional monomorphic hybridizing bands present in Eco RI and Hindlll digestions. Physical linkage of the Type I IFN gene families on bovine chromosome 8 was demonstrated by pulsed field gel electrophoresis (PFGE). Hybridization of two or more IFN probes to similarly sized PFGE fragments suggested the tentative gene family order: IFNA/IFNW-IFNT-IFNB. These studies provide a basis for the development of more detailed genetic and physical maps of the bovine Type I IFNs.     

DNA restriction fragment length polymorphisms and heterozygosity in the human genome

Summary A list is presented of published reports of DNA polymorphisms found in the human genome by restriction enzyme analysis. While the list indicates the large number of restriction fragment length polymorphisms (RFLPs) detected to date, the information collated is insufficient to permit an estimate of heterozygosity for the genome as a whole. Data from our laboratory are therefore also presented on RFLPs detected using a random sample of cloned DNA segments. Such an analysis has permitted a first unbiassed estimate of heterozygosity for the human genome. Since this figure is an order of magnitude higher than previous estimates derived from protein data, the majority of polymorphic variation present in the human genome must, by implication, occur in noncoding sequences. In addition it was confirmed that enzymes containing the dinucleotide CpG in their recognition sequences detect more polymorphic variation than those that do not contain a CpG. Also presented are the clinical applications of DNA polymorphisms in the diagnosis of human genetic disease.Supported by the Deutsche Forschungsgemeinschaft     

Identification of restriction fragment length polymorphisms linked to genes controlling soluble solids content in tomato fruit

Summary Gene(s) conferring high soluble solids (SS) in tomato fruit had been backcrossed previously from a wild tomato species, Lycopersicon chmielewskii LA1028 ( 10% SS), into a L. esculentum cultivar, VF36 ( 5% SS), to derive a BC₅S₅ line, LA1563, similar to VF 36 but with 7–8% SS. DNAs from these lines and a tomato breeding line, H2038, were screened for restriction fragment length polymorphisms (RFLPs) using four restriction endonucleases and sixty clones chosen at random from a tomato cDNA library. Most of the cDNA clones (56) identified the same RFLP in VF 36 and LA1563 and a different RFLP in LA1028. However, two cDNA clones identified the same RFLP in LA1563 and LA1028 and a different RFLP in VF36. To determine whether RFLPs identified by these two cDNA clones were linked to SS genes, a H2038 x LA1563 F₂ population was screened for segregation of the RFLPs and for SS content. The segregation ratios of these RFLPs were consistent with ratios expected for codominant alleles at unlinked loci. Analysis of variance of SS content for different RFLP genotypic classes indicated that RFLP alleles at one of the loci were linked to genes controlling SS content. The RFLP allele from the high SS tomato line, LA1563, was associated with significantly higher SS content and, therefore, could be useful in selecting for high SS gene(s) in a tomato breeding program.     

Detection of linkage between restriction fragment length polymorphism markers and quantitative traits

Summary Methodologies commonly used to detect linkage of marker loci to loci affecting quantitative traits are discussed. It is shown that variances for the quantitative trait differ among marker genotypes when using F₂ or pooled backcross data if linkage exists. Hence, to analyze this type of data by single factor ANOVA or other statistical techniques that assume a common variance is inadequate. Restriction fragment length polymorphism (RFLP) markers are a powerful tool in plant breeding but cost is an important drawback; hence, a methodology is suggested to obtain the minimum number of plants in F₂ populations to detect such linkage.     

Phylogenetic analysis of pines using ribosomal DNA restriction fragment length polymorphisms

Phylogenetic relationships among 30 species of the genusPinus were studied using restriction site polymorphism in the large subunit of nuclear rDNA. Of the 58 restriction sites scored, 48 were phylogenetically informative, and the 30 species reduced to ten taxa when species with identical restriction site patterns were combined. These ten taxa corresponded to the currently recognized subsections of the genus, with the sole exception ofP. leiophylla, which was identical in its pattern of restriction sites to all three species included from subsect.Oocarpae despite its being in a different section of subg.Pinus (Pinea instead ofPinus). A measure of the proportion of phylogenetic information contained within the data set (Homoplasy Excess Ratio, or HER) revealed that the character states were significantly non-randomly distributed among the ten taxa (HER = 0.71, p < 0.01). Branchand-bound searches using either Wagner or Dollo parsimony as the optimization criterion were carried out using PAUP in order to estimate phylogenetic relationships among the ten taxa. Three taxa (Picea pungens, Tsuga canadensis, andLarix decidua) were used independently as outgroups for purposes of rooting the trees. Despite the extreme differences in the assumptions underlying the Wagner and Dollo parsimony, the two gave surprisingly similar estimates of phylogeny, with both analyses supporting the monophyly of the two major subgeneraPinus andStrobus and differing in topology only in the placement of subsect.Ponderosae within subg.Pinus. The likelihood for the Wagner tree was only slightly higher than that computed for the Dollo tree.     

The use of restriction fragment length polymorphisms in paternity analysis.

This paper examines the utility of restriction fragment length polymorphisms (RFLPs) for paternity analysis. While, on the average, 99% of falsely accused males can be excluded with the standard battery of blood group antigens, red cell enzymes, serum proteins, and HLA antigens, there are still mother-child pairs for whom the exclusion probability is not high. It has been suggested that additional resolution would be available with RFLPs. We have examined the strategic aspects of using RFLPs for paternity analysis, comparing the efficacy and cost of a multimarker haplotypic set with those of a comparable set of unlinked RFLPs, using published frequencies for the beta-globin complex, the serum albumin region, and the growth hormone region. There are four major findings. (1) Greater resolution is obtained with a carefully chosen set of tightly linked RFLPs producing chromosomal haplotypes than with a comparable set (same allele frequencies) of unlinked markers, but only if it is possible to establish linkage phase unambiguously. (2) Assay of linked sets is cheaper than is the assay of unlinked markers, but the cost advantage is optimized with sets of no more than two or three linked markers. (3) Also, with more than two or three tightly linked markers, the haplotypic frequencies are too poorly estimated to provide a reliable measure of the probability of paternity for unexcluded males, given the sample sizes likely to be available in the near future. (4) Optimal resolution, minimal cost, and acceptable accuracy are obtained with several independent sets of no more than two or three tightly linked RFLP markers each. With current technology, RFLP analysis is more expensive for the same level of genetic resolution than is the standard battery, but gradual replacement of the latter can be anticipated as economies of scale reduce the cost of the DNA technology.     

Comparison of restriction fragment length polymorphisms in wild and cultivated barley.

This study was undertaken to assess the relative level of molecular diversity between cultivated barley, Hordeum vulgare ssp. vulgare (HV), and one of its wild relatives, H. vulgare ssp. spontaneum (HS), and to identify possible restriction fragment length polymorphism (RFLP) patterns that may provide information concerning the phylogenetic relationship between these two barley groups. A total of 363 barley accessions were assayed, including 95 entries of HV collected from 36 major barley growing countries of the world and 268 entries of HS from 25 natural populations in Israel and Iran. The 26 RFLP marker loci used in the survey represent single-copy, low-copy, and repetitive DNA sequences and mark all of the chromosome arms. A randomization test, on the basis of equal sample sizes, showed that HS is more polymorphic than HV, as evaluated by the number of alleles and diversity indices. The analysis also indicated extensive RFLP differentiation between these two barley groups; highly significant differences of allele frequencies were detected at the majority of the loci. The HV sample can be subdivided according to winter or spring growth habits, and two- or six-rowed spikes. Analysis of genetic polymorphisms in these subgroups showed that levels of diversity were about equal in spring and winter groups and also in the groups with two- and six-rowed spikes. However, significant differences of allelic frequencies were detected between subgroups of the two divisions.