Exploration of avian gametologous genes, i.e., homologous genes located on both the Z and W chromosomes, provides a crucial information about the underlying mechanism pertaining to the evolution of these chromosomes. The domestic chicken ( Gallus gallus (Linnaeus 1758); GGA) traditionally serves as the primary reference subject of these comparative cytogenomic studies. Using bioinformatic, molecular (overgo BAC library scanning), and cytogenetic (BAC-based FISH) techniques, we have investigated in detail a pair of UBE2R2 / UBE2R2L gametologs. By screening a gridded genomic jungle fowl BAC library, CHORI-261, with a short labeled UBE2R2L gene fragment called overgo probe, we detected seven specific clones. For three of them, CH261-019I23, CH261-105E16, and CH261-114G22, we identified their precise cytogenetic location on the Gallus gallus W chromosome (GGAW). They also co-localized with the UBAP2L2 gene on the, as was shown previously, along with the CH261-053P09 BAC clone also containing the GGAW-specific UBE2R2L DNA sequence. The fine mapping of the UBE2R2 / UBE2R2L homologs in the chicken genome also shed the light on comparative cytogenetic aspects in birds. Our findings provided further evidence that bird genomes moderately changed only during evolution and are suitable for successful use of interspecies hybridization using both overgo-based BAC library screen and BAC-based FISH.

Effect of single nucleotide polymorphism (SNP) in splicing site of the LPAR1 (lysophos-phatidic acid receptor 1) gene on selected quality traits was investigated in frozen-thawed semen of Holstein-Friesian bulls. Splicing mutation A/G in the LPAR1 gene (rs43581860) was identified in 120 Holstein-Friesian bulls using PCR-RFLP technique (Hph I). Heterozygotes AG were the most frequent (37.5%) compared with AA (30.8%) and GG (31.7%) homozygotes. Observed differences in total motility (TM), sperm membrane integrity (SYBR-14/PI) and ATP content were significant between homozygotes AA or GG and heterozygotes AG. For all three traits disadvantageous effect of heterozygotes AG was detected. This means that LPAR1 splicing mutation has significant effect on semen quality and should be considered as a new marker of semen quality in Holstein-Friesian bulls.

MicroRNA-21 (miR-21) expression was quantified by real-time qRT-PCR in peripheral blood and saliva samples obtained from patients diagnosed with colorectal cancer (CRC) of varying degrees of malignancy and healthy volunteers. All patients had adenocarcinoma located in the distal colon at different stages. Significant differences were detected between the control group and the total experimental group of CRC patients (plasma, P = 0.0001; saliva, P = 5e-12). MiR-21 expression was also significantly different in certain subgroups of patients with CRC disease stages II–IV as compared to the control group. No correlation of miR-21 expression was found with regard to gender and age of patents. Also, there were no significant individual correlations and linear regression of miR-21 expression in the plasma and saliva. The estimated diagnostic sensitivity and specificity of miR-21 expression were respectively 65 and 85% in the plasma, and 97 and 91% in the saliva. Our data suggest that miR-21 in both the saliva and plasma could be a proper biomarker for CRC screening, although the saliva miR-21 expression test looks preferable due to its higher sensitivity, specificity, and technical simplicity.

Six fragments were sequenced of the regulatory and coding regions of the expressed sequence ChEST985k21 (accession number CR523443), which had been shown to affect eggshell thickness. Six sites of single nucleotide polymorphism (SNP) were found, five of them located in the regulatory region and one within the ORF. The binding sites of transcriptional factors for all obtained SNPs were revealed to be present in one allelic variant. Based on this fact they were considered as rSNPs. Genotyping of 46 Rhode Island Red birds with thick (390 +/- 13 mu m) and 45 with thin eggshell (316 +/- 21 mu m) was done. Significant difference in allelic frequency was shown of rSNPs ST2_1, ST3_1, ST3_2, ST3_3. Genotypic classes of rSNPs ST2_1, ST3_1, 5T3_2, ST3_3 and ST6_1 were shown to have significantly different shell thickness. These data could probably provide a tool for marker-assisted selection for optimization of egg shell thickness in Rhode Island layers.

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).

Количественные признаки “масса и процентное содержание абдоминального жира” у домашней курицы и различные формы ожирения у млекопитающих являются гомологичными и функционально сходными. На этом основании гены, принимающие участие в формировании признаков ожирения у человека и лабораторных грызунов, толщины шпига свиньи, могут считаться функциональными генами-кандидатами депонирования абдоминального жира у бройлеров. Экспрессия генов-кандидатов FABP1, FABP2, FABP3, HMGA1, MC4R, PPARG, PPARGС1А, POMC и PTPN1 была исследована в жировой ткани, печени, толстом кишечнике, мышцах, гипофизе и головном мозге кур мясного направления (бройлеров) при помощи ПЦР в реальном времени. Выявлены достоверные различия по уровню экспрессии гена НМGA1 в печени бройлеров в группах с высоким (3.5% ± 0.18%) и низким (1.9% ± 0.56%) содержанием абдоминального жира. Показано, что уровень экспрессии этого гена коррелирует с содержанием абдоминального жира (0.70, P 0.01) и массой абдоминального жира (0.70, P 0.01). Установлено, что в тех же группах птиц достоверно различается экспрессия гена PPARG в печеночной ткани. Коэффициент корреляции экспрессии этого гена с содержанием абдоминального жира составил 0.55 (P 0.05), а с массой абдоминального жира равен 0.57 (P 0.01). Исходя из этих данных, можно сделать вывод о возможном участии генов HMGA1 и PPARG в депонировании абдоминального жира. Поиск однонуклеотидных полиморфных маркеров (SNP) в регуляторных областях генов HMGA и PPARG может позволить найти маркеры для генной селекции бройлеров по массе и процентному содержанию абдоминального жира.

The expression of nine functional candidates for QT abdominal fat weight and relative abdominal fat content was investigated by real-time polymerase chain reaction (PCR) in the liver, adipose tissue, colon, muscle, pituitary gland and brain of broilers. The high mobility group AT-hook 1 (HMG1A) gene was up-regulated in liver with a ratio of means of 2.90 (P ≤ 0.01) in the «fatty» group (relative abdominal fat content 3.5 ± 0.18%, abdominal fat weight 35.4 ± 6.09 g) relative to the «lean» group (relative abdominal fat content 1.9 ± 0.56%, abdominal fat weight 19.2 ± 5.06 g). Expression of this gene was highly correlated with the relative abdominal fat content (0.70, P ≤ 0.01) and abdominal fat weight (0.70, P ≤ 0.01). The peroxisome proliferator-activated receptor gamma (PPARG) gene was also up-regulated in the liver with a ratio of means of 3.34 (P ≤ 0.01) in the «fatty» group relative to the «lean» group. Correlation of its expression was significant with both the relative abdominal fat content (0.55, P ≤ 0.05) and the abdominal fat weight (0.57, P ≤ 0.01). These data suggest that the HMG1A and PPARG genes were candidate genes for abdominal fat deposition in chickens. Searching of rSNPs in regulatory regions of the HMG1A and PPARG genes could provide a tool for gene-assisted selection.

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.

Благовещенский И.Ю., Сазанова А.Л., Стекольникова В.А., Фомичев К.А., Баркова О.Ю., Романов М.Н., Сазанов А.А. Для изучения псевдоаутосомных и граничащих с ними районов Z- и W-хромосом птиц были использованы семь BAC-клонов, обнаруженных в геномных библиотеках с использованием в качестве ДНК-зондов фрагментов различных гаметологов гена ATP5А1, расположенного вблизи проксимальной границы псевдоаутосомного района половых хромосом домашней курицы и японского перепела. Методом флуоресцентной гибридизации in situ с использованием W-специфических зондов определены локализация BAC-клонов TAM31-b100C09, TAM31-b99N01, TAM31-b27P16 и TAM31-b95L18 в коротком плече Z-хромосом домашней курицы и японского перепела (район Zp23–p22) и локализация BAC-клонов CHORI–261-CH46G16, CHORI-261-CH33F10 и CHORI-261-CH64F22 на W-хромосомах этих видов птиц и в коротком плече Z-хромосом (район Zp23–p22). Различия в локализации BAC-клонов на Z- и W-хромосомах, возможно, связаны с наличием дивергенции в нуклеотидных последовательностях разных половых хромосом, находящихся за пределами псевдоаутосомного района. 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.

A total of 312 boars (201 Landrace and 111 Large White) were genotyped with a custom-made low throughput genotyping microarray (called SNiPORK) based on array primer extension (APEX) technology. The results were used to association studies between genotyped SNP markers and daily gains, meat content and selection index. Among the 60 SNP markers analyzed, 14 of them showed statistically significant associations between the genotype and the level of at least one trait. In order to find extremely beneficial or unfavorable intergenic diplotype combinations, 5 SNP markers were selected: CAST A499C, MYF6 T255C, PKLR C384T, SFRS1 C1146T and TNNT3 T153C, which showed statistically significant associations at P ≤ 0.01 within one of the traits and the frequency of homozygotes with a minor allele of at least 0.1. Among 10 possible permutations, statistically significant associations were found only for a combination of SNP markers in TNNT3 × SFRS1 genes and, interestingly, for all combinations with SNP located within the calpastatin ( CAST ) gene commonly known as a gene influencing pork quality traits. This study also found that CAST allele A (which is beneficial for pork tenderness) is also favorable for growth rate. This effect is clearly increased with additive alleles C of myogenic factor MYF6 and troponin T3 ( TNNT3 ) and is decreased with each allele T from serine rich splicing factor 1 ( SFRS1 ) gene. For meat content, the most favorable genotype of calpastatin gene was AC , the effect of which was generally increased with each C allele of MYF6 and TNNT3 and decreased with each allele T from SFRS1 and PKLR (puruvate kinase) genes, respectively. The calpastatin AC genotype seemed to be beneficial for selection index, although in combination with the CC genotype of troponin T3, calpastatin genotypes AA and CC reached higher values. In the case of the combination of genotypes TNNT3 × SFRS1 , the most preferable for all analyzed traits is the CC genotype of TNNT3 , especially in combination with CT or CC genotypes in the SFRS1 gene. We conclude that searching for interaction effects between candidate SNPs (even of moderate influence) may lead to interesting and valuable findings enabled better understanding and applications of SNPs in pork yield and quality improvement programmes.

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 μm) and thin (315.7 ± 21.38 μm) egg shells statistically significant differences in the allele frequencies of the ST2_1, ST3_1, ST3_2, and ST3_3 polymorphic sites. 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.

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, pituitary gland, 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 ≤ 0.01) and mass (0.7, P ≤ 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 ≤ 0.05) and 0.57 ( P ≤ 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.

Identification of genes determining the expression of economically important traits of plants and animals is a main research focus in agricultural genomics. Most of these traits are characterized by a wide variability of the expression of genes at certain loci called quantitative trait loci (QTL). Characterization of the chromosomal regions carrying QTL can be applied in marker-assisted selection (MAS) to improve breeding efficiency. Molecular linkage maps in combination with powerful statistical methods facilitate the genetic dissection of complex traits, and the chicken is ideally suited for this task due to a relatively short life cycle and large number of progeny. Two major approaches are employed in understanding the genomic architecture of economically important traits: QTL mapping and, more recently, functional genomics.

Six primer-pairs satisfying the requirements of sequencing were obtained. PCR-amplification allowed to get per 6 fragments from 6 biological samples in 3 technical repeats. Thirty six DNA-sample were sequenced. Six SNPs were revealed (5 ones in regulatory region and one within ORF). Based on these data the system for MAS of layers on shell thickness was developed.

Благовещенский И.Ю.; Сазанова А.Л.; Романов М.Н.; Фомичев К.А.; Стекольникова В.А.; Сазанов А.А.

In all studied neognathous birds, the Z–W pair of sex chromosomes shows strictly localized recombination in a very short pseudoautosomal region. There are five published W-linked genes in this region that have homologs on the Z, reflecting their common origin from an ancestral homologous chromosome pairs: CHD1, HINT, SPIN, UBAP2, and ATP5A1. Using BAC-based FISH, we have investigated in detail the genes located on the chicken Z and W chromosomes, especially those in the pseudoautosomal region. For one of them, UBAP2Z, we identified its precise cytogenetic location on the Z chromosome and mapped its W-linked counterpart, UBAP2W. The fine FISH mapping of two more homologs, ATPA5A1Z and ATPA5A1W, has been completed. By screening two gridded genomic jungle fowl BAC libraries, TAMU 031-JF256-BI and CHORI-261, with labeled amplified gene fragments or overgos, we detected four and five specific clones, respectively. BAC clones TAM31-100C09, TAM31-099N01, TAM31-027P16 and TAM31-095L18 were assigned to GGAZ at Zp23-p22 site (Flpter, 0.12 ± 0.034 to 0.15 ± 0.033). Clones CH261-046G16, CH261-033F10 and CH261-064F22 were detected on GGAZ (Flpter, 0.12 ± 0.033 to 0.14 ± 0.033). They also co-localized on GGAW, along with the CH261-114G22 BAC clone containing the GGAW-specific UBAP2W DNA sequence. We cytogenetically mapped in the chicken genome the sixth, previously unknown pair of Z- and W-linked homologs, UBE2R2Z and UBE2R2W. Using a chicken UBE2R2W-specific overgo, a positive clone was identified in the California condor BAC library and is being used for FISH in this endangered species and other birds.

MilkProtChip is oligonucleotide microarray allowing bovine genotyping based on single nucleotide polymorphisms (SNPs) in genes influencing milk protein biosynthesis. A total of 71 SNPs in 42 genes were selected as associated with milk protein biosynthesis. Genotyping of about 300 animals of Polish Black-and-White cattle showed that SNPs in acyl-CoA:1,2-diacylglycerol O-transferase (DGAT1), lactoferrin (LTF), casein kappa (CSN3) and growth hormone receptor (GHR) genes were associated with several milk performance traits. Analysis of correlations between SNPs and milk production traits showed that SNPs in single genes rarely affect the investigated traits. Only 4 of 42 investigated single SNPs had impact on milk production traits while 22 combinations of paired SNPs in these genes had impact. Positive effect SNP combinations in two genes can be a result of additive effect on these SNPs on the same traits or effect of genes interaction. The MilkBovExp chip representing 90 genes encoding transcription factors expressed in the bovine mammary gland and/or involved in mammary gland signaling pathways was designed for further investigation of impact of gene expression and/or its encoded products on milk traits performance.

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 ≤ 0.01) in the group with low ST53 (248.6 ± 16.62 μm) relative to the group with the highest ST53 (372.4 ± 2.07 μm). Expression of this gene was highly correlated (0.85, P ≤ 0.01) with shell thickness. No significant difference in expression between the 2 groups with thick (378.4 ± 3.65 μm) and thin (227.8 ± 8.99 μm) 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.

Nineteen BAC clones were identified by hybridization of the bovine genomic BAC library CHORI-240 with mixedCSN1S1- andCSN3-specific probes. Two of the clones were shown to contain the genesCSN1S1, CSN1S2, CSN2, STATH andCSN3, and five were proved to include the genesCSN2, STATH, CSN1S2 andCSN3. These data showed that the BAC contig was established for the whole casein cluster, including all known five genes.