Structural analysis of MHC alleles in an RSV tumour regression chicken using a BAC library

The chicken major histocompatibility complex (MHC-B locus) has a strong association with resistance and susceptibility to numerous diseases. We have found a B haplotype designated WLA that associated with the regression of tumours caused by Rous sarcoma virus J strain (RSV-J). Haplotype WLA was identical to the regressive B6 haplotype when partial genotyping was performed (Poultry Science, 89, 2010, 651). We then constructed a bacterial artificial chromosome (BAC) library from a WLA homozygote chicken to evaluate the structure of this regression haplotype and compared it to those of the B6 haplotype. Comparison between WLA and B6 above 59 kb within the 167 kb, including 14 genes from BG1 to BF2, revealed 75 SNPs and 14 indels. However, several genes were identical between WLA and B6, including the BF1 and BF2 genes, which encode a class I molecule previously suggested to be related to the regression phenotype. The BLB2 gene encoding the MHC class II beta chain showed the greatest diversity, with 19 non-synonymous SNPs. A comparison of WLA and B6 haplotpyes that are associated with tumour regression and RIRa and B24 haplotypes associated with tumour progression suggests that DMA1, DMA2, BRD2, TAPBP and BLB2 genes are not involved in the intensity of RSV J tumour regression.     

用RDA技术寻找肝再生相关基因的初步研究

利用表达性差异显示分析 (RDA)技术 ,研究大鼠 2 /3肝部分切除后 1h肝组织中基因的选择性表达 ,建立了大鼠 2 /3肝部分切除术后 1h再生肝组织选择性表达基因EST库。该库约含 3× 10 4 个独立克隆 ,其中 95 %以上的克隆含有插入片段 ,长度约 2 0 0～ 70 0bp不等 ,对随机挑出的 5 2个克隆的序列分析表明其中大多数基因与肝再生调控相关 (38/5 2 )。 10株未报道序列经RNA杂交证实 ,其中 6株与肝再生相关。     

cDNA representational difference analysis of the deltamelthrin-resistant and -susceptible populations in diamondback moth (Plutella xylostella L.)

Abstract:  The improved cDNA representational difference analysis was preliminarily used to analyse the genetic differences between the deltamethrin-susceptible and -resistant strain of the diamondback moth. The driver amplicon is the cDNA from the susceptible strain while the tester amplicon is from the resistant strain. After four rounds of subtractive hybridization, we obtained one different product, the size of which was about 200 bp. Comparisons between our sequencing results and data in GenBank show that there is one sequence which has a relatively high homology to the ubiquitin gene, but there are no reports indicating that its upregulated expression is correlated with insecticide resistance. Hence further studies are warranted.     

High-resolution typing for chicken BF2 (MHC class I) alleles by automated sequencing

Sequence-based typing (SBT) was developed for major histocompatibility complex (MHC) class I and class II alleles in humans. We report here the development and application of a SBT method for alleles of the chicken BF2 locus (the more polymorphic of the two MHC class I loci in chickens). Exon 2 of the BF2 gene was selectively amplified from genomic DNA using a BF2 locus-specific PCR primer. Exon 2 sequences were sufficient to identify the 21 distinct BF2 alleles described in standard B haplotypes of Leghorns and in commercial broiler-breeder lines. Sixty-six samples from MHC typed, pedigreed chickens were tested, including 50 different heterozygous combinations. BF2 sequences from all B homozygotes were successfully amplified, and all combinations of BF2 alleles in heterozygotes were co-amplified equally. The two different BF2 alleles in heterozygotes could be identified unambiguously by distinct sequence motif patterns. In tests of samples of unknown B genotype in commercial broiler-breeder flocks, we identified expected BF2 alleles as well as an allele not previously encountered in one of the lines.     

代表性差异分析比较两株来自不同地区的猪源Lactobacillus菌株

[目的]对分离自猪肠道的乳酸杆菌S1菌株进行鉴定,并比较该菌株与同种的001T菌株的基因差异.[方法]对S1菌株进行16S rRNA基因序列分析和种特异PCR检测,并且对S1菌株和Lactobacillus sobrius 001T进行代表性差异分析(Representational difference analysis,RDA).[结果]16S rRNA基因序列分析表明,与S1菌株最相似的已知菌为L.sobrius.变性梯度凝胶电泳分析显示,仔猪空、回肠细菌图谱中有一与S1菌株有相同迁移位置的优势条带,克隆、测序鉴定表明,与该条带相匹配的16S rRNA基因克隆(Clone S)的最相似已知菌也为L.sobrius.16S rRNA基因系统进化分析表明,S1菌株与Clone S和L.sobrius 16S rRNA基因序列同源性分别为99.8%和99.6%.L.sobrius特异性引物也可以扩增S1株菌的16S rRNA基因的特定片段.因此S1菌株可被确定为Lsobrius.RDA对菌株S1和同种的猪源L.sobrius 001T菌株的基因差异进行分析,未发现这两株菌的基因组差异.[结论]猪肠道乳杆酸菌S1菌株属于L.sobrius,其与猪源L sobrius 001T菌株为相似菌株.     

A radiation hybrid map of chicken chromosome 15

We have constructed a radiation hybrid (RH) map of chicken chromosome (GGA) 15. This map can be used as a resource to efficiently map genes to this chromosome. The map has been developed using a 6000 rad chicken-hamster whole-genome radiation hybrid panel (ChickRH6). In total, six microsatellite loci, 18 sequence tagged sites (STSs) from BAC end sequences and 11 genes were typed on the panel. The initial framework map comprised eight markers, and an additional 23 markers were then added to generate the final map. The total map length was 334 centiRay6000 (cR6000). The estimated retention frequency for the data set was 18%. Using an estimated physical length of 21 Mb, the ratio between cR6000 and physical distance over GGA15 was estimated to be 0.063 Mb/cR6000. The present map increases the marker density and the marker resolution on GGA15 and enables fast mapping of new chicken genes homologous to genes from human chromosomes 12 and 22.     

Labile DNA sequences in flax identified by combined sample representational difference analysis (csRDA)

Flax (Linum usitatissimum) has a genome in which changes have been associated with environmental factors. The inbred flax variety, Stormont Cirrus (Pl), served as the parent, and several lines (termed genotrophs) were derived from this parent. The phenotypes of the genotrophs were stable in a number of different growth environments, unlike the original Pl line in which changes associated with environmental factors continued to occur. These genotrophs differed from the original line in a number of characteristics, but the only known phenotypic characteristic that is shared by all the genotrophs and different from the parental, Pl, line is the lack of changes associated with the original environmental factors. However, some of these genotrophs have changed in both phenotype and nuclear DNA subsequent to their original growth and differentiation from Pl. Representational difference analysis (RDA) has been used to identify differences between Pl and all the genotrophs in an attempt to identify the loci controlling these aspects of plasticity. Subtractions between Pl DNA as a tester (target) and one of the genotrophs (individual RDA) or a mixture of different types of genotroph (L6, S6, C2, and LH) DNAs as a driver were done (combined sample RDA; csRDA). In addition, contrary RDA, where of the genotroph DNA was used as a tester and Pl DNA as a driver, was also executed. Three difference clones (163-4-2, 123-5-2, and 163-13), from 74 primary clones obtained after three rounds of subtractions with Pl DNA as tester were further characterized. In addition, 2 difference products (213-r1 and 213-r9) were characterized from contrary RDA. The clones 163-4-2 and 163-13 from the csRDA showed polymorphisms between Pl and all the genotrophs when PCR was done with primers derived from sequences of the clones, but only the clone 163-13 polymorphism was confirmed by Southern blot analysis. Four of 5 clones (163-4-2, 123-5-2, 163-13 and 213-r9) that have been characterized appear to be associated with structural changes in the DNA. From the contrary csRDA, it was observed that no clones could be recovered from subtractions between a mixture of genotrophs as a tester and Pl as a driver, and several possible explanations have been proposed.     

Identification of sulI allele of dihydropteroate synthase by representational difference analysis in Haemophilus parasuis serovar 2

AIMS: Identification of genes differentially present in Haemophilus parasuis serovar 2 by representational difference analysis (RDA). METHODS AND RESULTS: Bacterial genomic DNA was extracted, cleaved with Sau3AI and ligated to oligonucleotide adapter pair. The optimal tester (H. parasuis serovar 2)/driver ratio (H. parasuis serovars 1, 3 and 5) for the hybridization was established and the mixture was hybridized, and amplified by PCR. The products were cloned and transformed into Escherichia coli TOP10 cells and checked for specificity by Southern blotting analysis. The RDA subtractive technique yielded six bands ranging from 1500 to 200 bp, which were cloned into pCR II-TOPO vector and 40 clones were analysed. A fragment of 369 bp was specific for H. parasuis serovar 2, and showed 99% homology to sulI gene encoding for dihydropteroate synthase (dhps). The dhps gene conferring sulfonamide resistance was detected in H. parasuis serovar 2 but was absent in serovars 1, 3, 5 and in most of the Actinobacillus pleuropneumoniae serotypes (except serotype 7). CONCLUSION: sulI allele of dihydropteroate synthase has been identified in H. parasuis serovar 2 by RDA technique. SIGNIFICANCE AND IMPACT OF THE STUDY: The RDA technique seems to be an useful method for the identification of genes that are differentially present in H. parasuis, a respiratory pathogen of veterinary interest.     

Isolation and characterization of cDNA clones for chicken major histocompatibility complex class II molecules

The chicken major histocompatibility complex (MHC), the B complex, is being intensively analysed at the DNA level. To further probe the molecular structure of chicken MHC class II genes, cDNA clones coding for chicken MHC class II (B-L) p chain molecules were isolated from an inbred G-B2 Leghorn chicken spleen and liver. Twenty-nine cDNA clones were isolated from the spleen and eight cDNA clones were isolated from the liver. Based on restriction maps, most clones could be clustered into one family of genes. Four cDNA clones were sequenced (S7, S10 and S19 from the spleen and L1, which was identical to S19, from the liver). Complete amino acid sequences of B-Lβ chain molecules were predicted from the nucleotide sequences of the cDNA clones. Although both the nature and the location of the conserved residues were similar in chicken and mammalian sequences, some species-specific differences were found, suggesting that the structures of the B-L molecules of this haplotype are similar, but not identical, to their mammalian counterparts.     

Closing a gap in the physical map of the ovine major histocompatibility complex

A 184 kb gap in an ovine MHC physical map was successfully closed by identification of two overlapping clones (304C7 and 222G18) from a Chinese fine wool merino sheep BAC library. The location and tiling path of the two clones were confirmed by BAC‐end sequencing and PCR amplification of loci in overlapping regions. Full‐length sequencing of the clones identified 13 novel ovine genes in the gap between loci Notch4 and Btnl2, and eight of them belonging to the Butyrophilin‐like (Btn‐like or Btnl) gene family. The scattered distribution of the Btnl gene cluster at the gap provided a clue to explain the difficulties previously experienced in closing the gap. Completed BAC contigs of the ovine MHC will facilitate sequencing of the entire ovine leukocyte antigen (OLA) region, providing detailed information for comparative studies of MHC evolution.     

Maize DNA enrichment by representational difference analysis in a maize chromosome addition line of oat

 The recent recovery of maize (Zea mays L.) single-chromosome addition lines of oat (Avena sativa L.) from oat x maize crosses has provided novel source materials for the potential isolation of maize chromosome-specific sequences for use in genetic mapping and gene cloning. We report here the application of a technique, known as representational difference analysis (RDA), to selectively isolate maize sequences from a maize chromosome-3 addition line of oat. DNA fragments from the addition line and from the oat parent were prepared using BamHI digestion and primer ligation followed by PCR amplification. A subtractive hybridization technique using an excess of the oat parental DNA was employed to reduce the availability for amplification of DNA fragments from the addition lines that were in common with the ones from the oat parental line. After three rounds of hybridization and amplification, the resulting DNA fragments were cloned into a plasmid vector. A DNA library containing 400 clones was constructed by this method. In a test of 18 clones selected at random from this library, four (22%) detected maize-specific repetitive DNA sequences and nine (50%) showed strong hybridization to genomic DNA of maize but weak hybridization to genomic DNA of oat. Among these latter nine clones, three detected low-copy DNA sequences and two of them detected DNA sequences specific to chromosome 3 of maize, the chromosome retained in the source maize addition line of oat. The other eight out of the 13 clones that had strong hybridization to maize DNA detected repetitive DNA sequences or high-copy number sequences present on most or all maize chromosomes. We estimate that the maize DNA sequences were enriched from about 1.8% to over 72% of the total DNA by this procedure. Most of the isolated DNA fragments detected multiple or repeated DNA sequences in maize, indicating that the major part of the maize genome consists of repetitive DNA sequences that do not cross-hybridize to oat genomic sequences. Received: 18 November 1997 / Accepted: 12 March 1998     

Identification of imprinted loci by methylation-sensitive representational difference analysis: application to mouse distal chromosome 2

Imprinted genes are distinguished by different patterns of methylation on their parental alleles, a property by which imprinted loci could be identified systematically. Here, representational difference analysis (RDA) is used to clone HpaII fragments with methylation differences on the maternal and paternal copies of distal chromosome (Chr) 2 in the mouse. Uniparental inheritance for this region causes imprinting phenotypes whose molecular basis is only partially understood. RDA led to the recovery of multiple differentially methylated HpaII fragments at two major sites of imprinted methylation: paternal-specific methylation at the Nesp locus and maternal-specific methylation at the Gnasxl locus. Nesp and Gnasxl represent oppositely imprinted promoters of the Gnas gene, which encodes the G-protein subunit, Gsalpha. The organization of the Nesp-Gnasxl-Gnas region was determined: Nesp and Gnasxl were found to be 15 kb apart, and Gnasxl was found to be 30 kb upstream of Gnas. Sites of imprinted methylation were also detected at the loci for neuronatin on Chr 2 and for M-cadherin on Chr 8. RDA was highly effective at identifying imprinted methylation, and its potential applications to imprinting studies are discussed.     

Erythrocyte alloantigen loci Ea-D and Ea-I map to chromosome 1 in the chicken

A test cross was conducted to analyse some linkage relationships in the chicken. Pea comb (P), naked neck (Na), tardy feathering (t), four erythrocyte alloantigen loci (Ea-C, -D, -I, -P), and the rearrangement break point (RB) of the NM 7092 t(Z;1) chromosome translocation were tested. Significant linkages were found between P and Ea-I (32.9 +/- 4.2), the RB and Ea-D (30.7 +/- 4.3), and t and Ea-D (38 +/- 4.8). The data suggest the linear order of t, Ea-D, and the RB, with t closest to the centromere. Significant linkage was also found between Na and Ea-P (32.4 +/- 4.9), confirming earlier reports.     

Identification of Brachyspira hyodysenteriae-specific DNA fragments using representational difference analysis

Two novel Brachyspira hyodysenteriae-specific DNA fragments, designated as Bh100 and Bh400, were identified using representational difference analysis. To isolate the fragments the combined DNA of the Brachyspira pilosicoli, Brachyspira intermedia, Brachyspira murdochii and Brachyspira innocens reference strains was subtracted from the genome of B. hyodysenteriae strain B204. Both fragments were present in a single copy and mapped to different positions on the genome of B. hyodysenteriae B78(T). Larger fragments encompassing the continuous open reading frames (ORF) of Bh100 and Bh400 were cloned and analysed. Whereas the ORF of 2130 bp encompassing Bh100 did not show homology to any known bacterial protein, Bh400 was part of a putative operon with significant homology to the phosphotransferase system of Bacillus subtilis.     

Chromosomal assignment of chicken clone contigs by extending the consensus linkage map

The bacterial artificial clone-based physical map for chicken plays an important role in the integration of the consensus linkage map and the whole-genome shotgun sequence. It also provides a valuable resource for clone selection within applications such as fluorescent in situ hybridization and positional cloning. However, a substantial number of clone contigs have not yet been assigned to a chromosomal location or have an ambiguous chromosome assignment. In this study, 86 single nucleotide polymorphism markers derived from 86 clones were mapped on the genetic map. These markers added anchoring information for 56 clone contigs and 13 individual clones, covering a total of 57,145 clones.     

Genomic regions associated with antibody response to sheep red blood cells in the chicken

F(1) and F(2) populations were generated by crossing two lines of chickens divergently selected from a common founder population for 32 generations for either high or low antibody response 5 days post-injection of a non-pathogenic antigen, sheep red blood cells (SRBCs). The number of loci with major effects on day 5 SRBC titers was estimated to be more than 7 in this population. There was a significant association between MHC haplotype and day 5 antibody titers as well as body weight at sexual maturity. A significant difference between reciprocal F(2) crosses for both 5- and 12-day antibody titers suggests that sex chromosome and/or parent of origin effects on autosomal loci have an important role in immune response. A single marker-trait association analysis on 1024 genetic markers and 128 F(2) individuals detected 11 genomic regions associated with antibody response traits and 17 regions associated with body weight gain. Several of the genomic regions identified as being associated with antibody response have been described previously, while novel regions associated with antibody response were identified on chromosomes 11 and 24. Based on the lack of overlap of the regions associated with body weight and antibody response, we conclude that while these phenotypes are inversely correlated in the selected lines, they are controlled by distinct genetic loci and may be reflective of intense selection pressure on loci affecting the partitioning of nutrients between the immune system and growth pathways.     

Use of representational difference analysis to identify Escherichia coli O157-specific DNA sequences

Whereas several important virulence factors in Escherichia coli O157 have been identified, studies suggest they are not always essential and are probably insufficient to account for the severe clinical manifestation of E. coli O157 infection. Identification of putative virulence determinants is crucial to the understanding of bacterial pathogenesis and genomic comparison analysis may aid the characterisation of unidentified virulence attributes. In this study, representational difference analysis (RDA) was used for genomic comparison of E. coli O157 with the proposed ancestral strain, E. coli O55. Unique E. coli O157 gene sequences were isolated and one, termed RDA-1, taken forward for further analysis. Southern blotting with labelled RDA-1 as a probe showed it to be present in 77% of E. coli O157 isolates and absent in all non-E. coli O157 screened. Sequence flanking RDA-1 was obtained from a genomic clone identified by hybridisation, and contained an open reading frame predicted to encode a novel iron-regulated outer membrane protein.     

Assigning alleles to different loci in amplifications of duplicated loci

Duplicated loci, for example those associated with major histocompatibility complex (MHC) genes, often have similar DNA sequences that can be coamplified with a pair of primers. This results in genotyping difficulties and inaccurate analyses. Here, we present a method to assign alleles to different loci in amplifications of duplicated loci. This method simultaneously considers several factors that may each affect correct allele assignment. These are the sharing of identical alleles among loci, null alleles, copy number variation, negative amplification, heterozygote excess or heterozygote deficiency, and linkage disequilibrium. The possible multilocus genotypes are extracted from the alleles for each individual and weighted to estimate the allele frequencies. The likelihood of an allele configuration is calculated and is optimized with a heuristic algorithm. Monte‐Carlo simulations and three empirical MHC data sets are used as examples to evaluate the efficacy of our method under different conditions. Our new software, mhc‐typer V1.1, is freely available at https://github.com/huangkang1987/mhc-typer .     

利用鸡F2资源群体构建1号染色体遗传连锁图谱

在鸡1号染色体上选取23个微卫星标记,利用东北农业大学鸡F2资源群体构建了遗传连锁图谱。选用369只F2个体用于基因型测定。结果表明23个微卫星位点除MCW0058为低度多态,其他位点均为中高度多态。构建的连锁图谱覆盖1号染色体全长,总共637.9 cM。MCW0115和ROS0025标记顺序与EL图谱不同,但与WAU图谱一致。其他标记顺序与3大参考家系标记顺序一致,图谱总长和标记间距离大于3大参考家系。此连锁图谱的构建为数量性状位点（QTL）定位奠定了良好的基础。