Associations between two novel rSNPs in 5'-flanking region of the bovine casein gene cluster and milk performance traits.

Ten evolutionary conservative sequences with high identity level to homological sequences in other mammal species were revealed in 5'-flanking region of casein's genes cluster. Five novel SNPs located inside of the evolutionary conservative regions were identified. The binding sites were revealed to be present in one allelic variant of four detected SNPs. So these SNPs were considered as rSNPs. Significant differences of allelic frequencies were revealed between beef cow's group and dairy cow's group in two rSNPs (NCE4, NCE7, p<0.001). Different alleles of those two rSNPs were shown to be associated with some milk performance traits in Black-and-White Holstein dairy cows. Significant difference of protein percentage has been found between cows with G/G and A/A genotypes (P<0.05) and A/G and A/A genotypes (P<0.05) for NCE4 polymorphism. The groups of animals with genotypes G/G and A/G for NCE7 polymorphism were significantly different in milk yield at the first lactation (kg) (P<0.01), milk fat yield (kg) (P<0.05) and milk protein yield (kg) (P<0.01). For the last trait the difference was significant also between cows with genotypes G/G and A/A for rSNP NCE7 (P<0.05).

[Expression profiling of candidate genes for abdominal fat mass in domestic chicken Gallus gallus].

The quantitative traits of mass and percentage of abdominal fat in chicken and various types of obesity in mammals are homologous and functionally similar. Therefore, the genes involved in obesity development in humans and laboratory rodents as well as those responsible for pig lard thickness could be involved in abdominal fat deposition in broilers. Expression of candidate genes FABP1, FABP2, FABP3, HMGA1, MC4R, PPARG, PPARGC1A, POMC and PTPN1 was studied in fat, liver, colon, muscle, hypophysis, and brain in chicken (broilers) using real-time PCR. Significant difference in the HMGA1 gene expression in the liver of broiler chicken with high (3.5 +/- 0.18%) and low (1.9 +/- 0.56%) abdominal fat concentration has been revealed. The expression of this gene was been shown to correlate with the amount (0.7, P < or = 0.01) and mass (0.7, P < or = 0.01) of abdominal fat. The PPARG gene expression in liver in the same chicken subsets was also significantly different. Correlation coefficients of the gene expression with the abdominal fat amount and mass were respectively 0.55 (P < or = 0.05) and 0.57 (P < or = 0.01). Based on these results, we suggest that the HMGA1 and PPARG genes are involved in abdominal fat deposition. The search for single nucleotide polymorphisms (SNPs) in the HMGA and PPARG regulatory regions could facilitate identifying genetic markers for broiler breeding according to the mass and percentage of abdominal fat.

[Investigation of pseudoautosomal and bordering regions in avian Z and W chromosomes with the use of large insert genomic BAC clones].

To study pseudoautosomal and bordering regions in the avian Z and W chromosomes, we used seven BAC clones from genomic libraries as DNA probes of fragments of different gametologs of the ATP5A1 gene located close to the proximal border of the pseudoautosomal region (PAR) of sex chromosomes of domestic chicken and Japanese quail. Localization of BAC clones TAM31-b100C09, TAM31-b99N01, TAM31-b27P16, and TAM31-b95L18 in the short arm of Z chromosomes of domestic chicken and Japanese quail (region Zp23-p22) and localization of the BAC clones CHORI-261-CH46G16, CHORI-261-CH33F10, and CHORI-261-CH64F22 on W chromosomes of these species and in the short arm of Z chromosomes (region Zp23-p22) were determined by fluorescence in situ hybridization with the use of W-specific probes. The difference in the localization of the BAC clones on the Z and W chromosomes is probably explained by divergence of the nucleotide sequences of different sex chromosomes located beyond the pseudoautosomal region.

[Design of a system for genotyping of Gallus gallus based on the rSNP (regulatory single nucleotide polymorphism) alleles affecting the egg shell thickness].

PCR amplification of the six fragments of regulatory and coding regions of chicken ChEST985k21 gene (accession no. CR523443), substantially affecting the egg shell thickness quantitative trait, was carried out. Sequencing of these fragments in six chickens from a native Polish breed, Green-legged Partridgenous, with different manifestation of the trait of interest enabled identification of six single nucleotide polymorphism (SNP) sites within the ChEST985k21 sequence. Five of these sites were located in the regulatory region, and one site, in the coding region. For all SNPs identified, the existence of transcription factor binding sites, present in only one allelic variant, was demonstrated. This finding enables considering these sites as regulatory single nucleotide polymorphisms, rSNP. The effect of rSNP discovered on the chicken egg shell thickness was tested using PCR amplification with allele-specific primers. In the groups of chicken of Rhode Island Red breed with thick (389.9 +/- 13.09 microm) and thin (315.7 +/- 21.38 microm) egg shells statistically significant differences in the allele frequencies of the ST2_1, ST3_1, ST3_2, and ST3_3 polymorphic loci. In the same groups of birds, statistically significant differences in the shell thickness were observed in the rSNP allele genotypic classes ST2_1, ST3_1, ST3-2, ST3_3, and ST6_1. Based on these data, it was concluded that rSNPs influenced manifestation of the quantitative trait examined, and the genotyping system for marker assisted selection was constructed.

[Localization of the NotI clones from human chromosome 3 on quail microchromosomes].

For the purpose of comparative mapping of quail (Coturnix c. japonica) and human (Homo sapiens) genomes, DNA fragments from human chromosome 3 (HSA3p14-21 and HSA3q13-23) were localized on quail mitotic chromosomes. Using the method of double-color fluorescence DNA-DNA in situ hybridization, these fragments were mapped to two different microchromosomes. Earlier, similar studies were performed using chicken mitotic chromosomes. There it was demonstrated that the clones of interest were distributed among three microchromosomes (GGA12, GGA14, and GGA15). Thus, interspecific difference in the location of human chromosome 3 DNA fragments in the genomes of closely related avian species was discovered. A new confirmation of the hypothesis on the preferable localization of the gene-rich human chromosome regions on avian microchromosomes was obtained. At the same time, a suggestion on the localization of some orthologous genes in the genome of the organism under study was made: ARF4, SCN5A, PHF7, ABHD6, ZDHHC3, MAPKAPK3, ADSYNA (homolog of chicken chromosome 12), DRD2, PP2C-ETA, RAB7, CCKAR, and PKD1 (homolog of chicken chromosome 15). However, localization of the corresponding quail genes needs to be confirmed, as far as the sequences used were only the orthologs of the corresponding chicken genes.

Expression of positional candidates for shell thickness in the chicken.

Expression of 12 positional candidates for QTL affecting shell thickness at 53 wk of lay age (ST53) was investigated by real-time PCR in the distal part of chicken oviducts (uterus) with a forming eggshell. In the local chicken breed Green-legged Partridgenous, the complete cDNA CR523443 (ChEST985k21) was downregulated with ratio of means 0.49 (P < or = 0.01) in the group with low ST53 (248.6 +/- 16.62 microm) relative to the group with the highest ST53 (372.4 +/- 2.07 microm). Expression of this gene was highly correlated (0.85, P < or = 0.01) with shell thickness. No significant difference in expression between the 2 groups with thick (378.4 +/- 3.65 microm) and thin (227.8 +/- 8.99 microm) shell and no significant correlation of expression level with ST53 were detected in Rhode Island Red, which could be explained by strict selection to egg quality traits, including optimal shell thickness in this commercial layer breed. These data suggested that CR523443 was a candidate gene for QTL ST53 in the chicken.

Gene expression profiling of hereditary exencephaly in chickens.

In this preliminary study, differentially expressed genes were investigated in cranial tissues from chickens with hereditary exencephaly using cDNA microarrays containing 1,152 genes and expressed sequence tags (ESTs). Genes showing twofold or greater differences at P < 0.05 between affected and normal cranial cells were considered to be candidates for hereditary exencephaly in chicken. Eighteen ESTs (11 known genes/homologues) were upregulated and 108 ESTs (51 known genes/homologues) were downregulated. The EST AL584231 (ROS006C9), orthologous to human MTHFD1, a known candidate gene for human neural tube defects (NTDs), was expressed at the same level both in normal and affected chicken cranial tissues. ESTs AL584253 (ROS006F7, thioredoxin reductase 1) and AL585511 (ROS024H9, thioredoxin), both involved in NTD pathogenic pathways in mice, were downregulated and had mean ratios of 0.41 and 0.04 for expression in affected vs. normal cells respectively. Expression differences of these two ESTs were confirmed by quantitative real-time polymerase chain reaction. These data indicate that ESTs AL584253 and AL585511 are candidates for hereditary exencephaly in chickens.

Chromosomal localization of seven HSA3q13-->q23 NotI linking clones on chicken microchromosomes: orthology of GGA14 and GGA15 to a gene-rich region of HSA3.

Double-color fluorescence in situ hybridization was performed on chicken chromosomes using seven unique clones from the human chromosome 3-specific NotI linking libraries. Six of them (NL1-097, NL2-092, NL2-230, NLM-007, NLM-118, and NLM-196) were located on the same chicken microchromosome and NL1-290 on another. Two chicken microchromosome GGA15-specific BAC clones, JE024F14 containing the IGVPS gene and JE020G17 containing the ALDH1A1 gene, were cytogenetically mapped to the same microchromosome that carried the six NotI linking clones, allowing identification of this chromosome as GGA15. Two GGA14-specific clones, JE027C23 and JE014E08 containing the HBA gene cluster, were co-localized on the same microchromosome as NL1-290, suggesting that this chromosome was GGA14. The results indicated that the human chromosomal region HSA3q13-->q23 is likely to be orthologous to GGA15 and GGA14. The breakpoint of evolutionary conservation of human and chicken chromosomes was detected on HSA3q13.3-->q23 between NL1-290, on the one hand, and six other NotI clones, on the other hand. Considering the available chicken-human comparative mapping data, another breakpoint appears to exist between the above NotI loci and four other genes, TFRC, EIF4A2, SKIL and DHX36 located on HSA3q24-->qter and GGA9. Based on human sequences within the NotI clones, localization of the six new chicken coding sequences orthologous to the human/rodent genes was suggested to be on GGA15 and one on GGA14. Microchromosomal location of seven NotI clones from the HSA3q21 T-band region can be considered as evidence in support of our hypothesis about the functional analogy of mammalian T-bands and avian microchromosomes.

[Comparative compositional mapping of chicken and quail chromosomes]. / Sravnitel'noe kompozitsionnoe kartirovanie khromosom kuritsy i perepela.

The distribution of various isochore families on mitotic chromosomes of domestic chicken and Japanese quail was studied by the method of fluorescence in situ DNA--DNA hybridization (FISH). DNA of various isochore families was shown to be distributed irregularly and similarly on chromosomes of domestic chicken and Japanese quail. The GC-rich isochore families (H2, H3, and H4) hybridized mainly to microchromosomes and a majority of macrochromosome telomeric regions. In chicken, an intense fluorescence was also in a structural heterochromatin region of the Z chromosome long arm. In some regions of the quail macrochromosome arms, hybridization was also with isochore families H3 and H4. On macrochromosomes of both species, the pattern of hybridization with isochores of the H2 and H3 families resembled R-banding. The light isochores (L1 and L2 families) are mostly detected within macrochromosome internal regions corresponding to G bands, whereas microchromosomes lack light isochores. Although mammalian and avian karyotypes differ significantly in organization, the isochore distribution in genomes of these two lineages of the warm-blooded animals is similar in principle. On macrochromosomes of the two avian species studied, a pattern of isochore distribution resembled that of mammalian chromosomes. The main specific feature of the avian genome, a great number of microchromosomes (about 30% of the genome), determines a compositional specialization of the latter. This suggests the existence of not only structural but also functional compartmentalization of the avian genome.

Compositional mapping of chicken chromosomes and identification of the gene-richest regions.

'Compositional chromosomal mapping', namely the assessment of the GC level of chromosomal bands, led to the identification, in the human chromosomes, of the GC-richest H3+ bands and of the GC-poorest L1+ bands, which were so called on the basis of the isochore family predominantly present in the bands. The isochore organization of the avian genome is very similar to those of most mammals, the only difference being the presence of an additional, GC-richest, H4 isochore family. In contrast, the avian karyotypes are very different from those of mammals, being characterized, in most species, by few macrochromosomes and by a large number of microchromosomes. The 'compositional mapping' of chicken mitotic and meiotic chromosomes by in-situ hybridization of isochore families showed that the chicken GC-richest isochores are localized not only on a large number of microchromosomes but also on almost all telomeric bands of macrochromosomes. On the other hand, the GC-poorest isochores are generally localized on the internal regions of macrochromosomes and are almost absent in microchromosomes. Thus, the distinct localization of the GC-richest and the GC-poorest bands observed on human chromosomes appears to be a general feature of chromosomes from warm-blooded vertebrates.

[Mapping the chicken genome: problems and perspectives]. / Kartirovanie genoma kuritsy: problemy i perspektivy.

Various molecular methods are now used to map the chicken genome, including chromosome scraping, flow cytofluorimetry, zonal centrifugation, construction of chromosome-specific libraries, genetic analysis with polymorphic DNA markers, and in situ hybridization. Two main drawbacks are characteristic of existing maps of chicken chromosomes. First, classic genetic maps (i.e., linkage groups of genes for morphological, physiological, and biochemical characters), physical maps of chromosomes, and new genetic maps constructed on the basis of polymorphic DNA markers (RFLP, RAPD, VNTR, SSR, and CR1-PCR) do not coordinate with one another. Second, a relatively low number of genes is present in classic genetic maps and physical chromosome maps. Application of cytogenetic methods to chromosome mapping in birds is limited because of some specific features characteristic of the organization of avian genomes. For the same reason, studying the location and expression of avian genes is very important. Since mammalian and avian genomes differ in structure, revealing their possible common functional characteristics will provide for a better understanding of the general mechanisms that control biologically important characters in higher animals.

[Use of the pGB725 probe containing poly-TG-repeats for detecting polymorphic microsatellite loci in the genome of the chicken Gallus gallus domesticus]. / Ispol'zovanie proby pGB725, soderzhashchei poli-TG-povtory, dlia vyiavleniia polimorfnykh mikrosatellitnykh lokusov v genome kuritsy Gallus gallus domesticus.

Using [32P]-labeled hybridization probe of plasmid pGB725 enriched with poly-TG-pairs of nucleotides, chicken genome fingerprints were obtained. High genetic informativity of hybridization profiles and the possibility of polymorphic loci in the telomeric macrochromosomes' regions was revealed.

[Localization of various DNA sequences on the mitotic chromosomes of the chicken]. / Lokalizatsiia nekotorykh posledovatel'nostei DNK na mitoticheskikh khromosomakh kuritsy.

The chromosomal localization of the chicken transferrin receptor gene, as well as sequences that were homologous to the viral oncogene v-fos and the human gene families ZFY and SRY were determined by the method of nonisotopic DNA-DNA in situ hybridization. A correspondence was revealed between the Comptonian linkage group 10 and chromosome 1. A common origin of avian chromosome Z and mammalian chromosome Y is hypothesized.