SSCP analysis at the bovine CSN3 locus discriminates six alleles corresponding to known protein variants (A, B, C, E, F, G) and three new DNA polymorphisms (H, I, A1)

A high resolution SSCP protocol was developed for simultaneous discrimination of the known CSN3 alleles A, B, C, E, F and G. Furthermore, three new DNA polymorphisms were identified in different Bos taurus and Bos indicus breeds or crosses. Mendelian segregation was shown for two of these polymorphisms (named CSN3*H and 1), and the third (named CSN3*A1) was found in unrelated animals, thus indicating the presence of three additional alleles at the bovine CSN3 locus. DNA sequencing revealed single mutations that led to a Thr/Ile substitution in amino acid position 135 for CSN3*H and to a Ser/Ala substitution in position 104 of the deduced amino acid sequence of CSN3*1 (GenBank accession numbers AF105260 and AF121023) compared to CSN3*A. In CSN3*A1, a silent mutation in the third codon position of Pro150 was found (GenBank accession number AF092513).     

A Single Nucleotide Polymorphism and Sequence Analysis of CSN1S1 Gene Promoter Region in Chinese BOS Grunniens (YAK)

The aim of this study was to investigate the polymorphism of the CSN1S1 gene promoter region in 4 Chinese yak breeds, and compare the yak CSN1S1 gene promoter region sequences with other ruminants. A Polymerase Chain Reaction-Single Strand Conformation Polymorphism protocol was developed for rapid genotyping of the yak CSN1S1 gene. One hundred fifty-eight animals from 4 Chinese yak breeds were genotyped at the CSN1S1 locus using the protocol developed. A single nucleotide polymorphism of the CSN1S1 gene promoter region has been identified in all yak breeds investigated. The polymorphism consists of a single nucleotide substitution G→A at position 386 of the CSN1S1 gene promoter region, resulting in two alleles named, respectively, G₃₈₆ and A₃₈₆, based on the nucleotide at position 386. The allele G₃₈₆ was found to be more common in the animals investigated. The corresponding nucleotide sequences in GenBank of yak (having the same nucleotides as allele G₃₈₆ in this study), bovine, water buffalo, sheep, and goat had similarity of 99.68%, 99.35%, 97.42%, 95.14%, and 94.19%, respectively, with the yak allele A_386.     

Geographic distribution of haplotype diversity at the bovine casein locus

The genetic diversity of the casein locus in cattle was studied on the basis of haplotype analysis. Consideration of recently described genetic variants of the casein genes which to date have not been the subject of diversity studies, allowed the identification of new haplotypes. Genotyping of 30 cattle breeds from four continents revealed a geographically associated distribution of haplotypes, mainly defined by frequencies of alleles at CSN1S1 and CSN3. The genetic diversity within taurine breeds in Europe was found to decrease significantly from the south to the north and from the east to the west. Such geographic patterns of cattle genetic variation at the casein locus may be a result of the domestication process of modern cattle as well as geographically differentiated natural or artificial selection. The comparison of African Bos taurus and Bos indicus breeds allowed the identification of several Bos indicus specific haplotypes (CSN1S1*C-CSN2*A²-CSN3*A_I/CSN3*H) that are not found in pure taurine breeds. The occurrence of such haplotypes in southern European breeds also suggests that an introgression of indicine genes into taurine breeds could have contributed to the distribution of the genetic variation observed.     

Effect of single nucleotide polymorphisms of CAPN1 and CAST genes on meat traits in Nellore beef cattle (Bos indicus) and in their crosses with Bos taurus

The objectives of this work were to study the segregation of single nucleotide polymorphisms of the calpain 1, large subunit ( CAPN1 ) and calpastatin ( CAST ) genes in Nellore ( Bos indicus ) and Nellore × Bos taurus beef cattle, as well as to evaluate their effects on meat traits. For this, 300 animals, including 114 Nellore, 67 Angus × Nellore, 44 Rubia Gallega × Nellore, 41 Canchim, 19 Brangus three-way crosses and 15 Braunvieh three-way crosses, were genotyped for the CAPN4751 [AF_248054.2:g.6545C>T (GenBank accession AF248054.2 )] and CAST/DdeI [AF_159246.1:g.2959A>G (GenBank accession AF159246.1 )] polymorphisms and phenotyped for Ribeye Area, Backfat Thickness, Intramuscular Fat, Shear Force (SF) and Myofibrillar Fragmentation Index (MFI). In relation to the CAPN4751 polymorphism, a frequency of 10.5% was observed for the C allele in the Nellore breed. In the total sample of studied animals, a significant association was found between genotypes and meat tenderness, assessed by SF ( P  = 0.005) and MFI ( P  = 0.008), with genotype CT being more favourable than TT. For the CAST/DdeI polymorphism, a frequency of 55.7% was found for the A allele in the Nellore breed. In the total sample, a significant association was observed between genotypes and meat tenderness – SF ( P  = 0.004) and MFI ( P  = 0.006), with genotype AA being more favourable than AG. The relationship between genotypes and aged meat tenderness in confluence with the distribution of favourable alleles shows great potential for application of the CAPN4751 and CAST/DdeI polymorphisms in the genetic improvement of the Nellore breed, whilst contributing to the validation, in this breed and in its crosses with B. taurus , of the association results previously described in the literature.     

Goat casein genotypes are associated with milk production traits in the Sarda breed

The aim of the current work was to analyze, in the Sarda breed goat, genetic polymorphism within the casein genes and to assess their influence on milk traits. Genetic variants at the CSN1S1, CSN2, CSN1S2 and CSN3 gene loci were investigated using PCR‐based methods, cloning and sequencing. Strong alleles prevailed at the CSN1S1 gene locus and defective alleles also were revealed. Null alleles were evidenced at each calcium‐sensitive gene locus. At the CSN3 gene locus, we observed a prevalence of the CSN3 A and B alleles; the occurrence of rare alleles such as CSN3 B'', C, C', D, E and M; and the CSN3 S allele (GenBank KF644565 ) described here for the first time in Capra hircus. Statistical analysis showed that all genes, except CSN3, significantly influenced milk traits. The CSN1S1 BB and AB genotypes were associated with the highest percentages of protein (4.41 and 4.40 respectively) and fat (5.26 and 5.34 respectively) (P < 0.001). A relevant finding was that CSN2 and CSN1S2 genotypes affected milk protein content and yield. The polymorphism of the CSN2 gene affected milk protein percentage with the highest values recorded in the CSN2 AA goats (4.35, at P < 0.001). The CSN1S2 AC goats provided the highest fat (51.02 g/day) and protein (41.42 g/day) (P < 0.01) production. This information can be incorporated into selection schemes for the Sarda breed goat.     

Genetic variability of MHC class II DQB exon 2 alleles in yak (Bos grunniens)

The major histocompatibility class (MHC) DQ molecules are dimeric glycoproteins revealing antigen presentation to CD⁴⁺ T cells. In the present study, the exon 2 of the MHC class II DQB gene from 32 yaks (Bos grunniens) was cloned, sequenced and compared with previously reported patterns for other bovidae. It was revealed by sequence analyses that there are 25 DQB exon 2 alleles among 32 yaks, all alleles are found to belong to DQB1 loci. These alleles exhibited a high degree of nucleotide and amino acid polymorphisms with most amino acid variations occurring at positions forming the peptide-binding sites. The DQB loci were analyzed for patterns of synonymous (d _S) and non-synonymous (d _N) substitution. The yak was observed to be under strong positive selection in the DQB exon 2 peptide-binding sites (d _N = 0.15, P < 0.001). It appears that this variability among yaks confers the ability to mount immune responses to a wide variety of peptides or pathogens.     

Distribution of <Emphasis Type="Italic">BoLA-DRB3</Emphasis> allelic frequencies and identification of a new allele in the Iranian cattle breed Sistani (<Emphasis Type="Italic">Bos indicus</Emphasis>)

The distribution of the frequencies of BoLA-DRB3 gene alleles in the Iranian cattle breed Sistani was studied by the PCR-RFLP (“hemi-nested”) assay using restriction endonucleases RsaI, HaeIII and BstYI. In the examined cattle breed (65 animals) 32 alleles have been identified one of which being described for the first time (6.15% frequency). The nucleotide sequence of the polymorphic region of exon 2 of this allele has been determined and submitted in the GenBank database under accession number DQ486519. The submitted sequence has maximum homology (92%) with the previously described sequence DRB3-mRNA from Bos indicus (AccN X79346) and differs from it by 24 nucleotide substitutions which result in 16 amino acid substitutions. The peptide (on the basis of the reconstructed amino acid sequence) has 89% identity to the sequence encoded by the BIDRBF 188 locus (Bos indicus). The results obtained permit the sequence described by us to be considered as a new allele of the BoLA-DRB3 gene (DRB3.2 ^* X). The total frequency of the main six alleles (DRB3.2*8, *10, *11, *20, *34 and *X) occurring with a frequency of over 5% is about 60% in Iranian Sistani cattle. Fifteen alleles have <1% frequency. The highest frequency was observed for DRB3.2*8 allele (21.54%) like in other previously described breeds of Bos indicus (up to 23.07%). The Iranian breed Sistani has a high level of similarity by the spectrum of BoLA-DRB3 alleles and their frequencies to other Bos indicus breeds and significantly differs by these criteria from the Bos Taurus breeds. The Iranian Sistani herd under study includes alleles associated with to resistance to leukemia (DRB3.2*11 and *23) and to different forms of mastitis (DRB3.2* 2, *7, *11, *23 and *24) although their frequencies are low (from 0.77 to 5.37%). On the whole, a high level of diversity of BoLA-DRB3 gene alleles and the availability of alleles associated with resistance to different diseases makes this breed of interest for breeding practice. The article is published in the original.     

Single nucleotide polymorphism (SNP) in growth hormone gene of Jakhrana,a prominent milk goat breed in India

PCR-SSCP patterns on non-degenerative PAGE revealed 6 amplicons in caprine GH exon-4 and 3 alleles A₄, B₄ and C₄ were identified. In exon-5, six SSCP variants revealed three alleles A₅, B₅, and C₅. Out of 54 AA sites of GH-4 coding region, six codons were polymorphic. At codon-6, nucleotide substitution of G/A resulted in to genotypes 6_RR, 6_HH and G/C into genotypes 6_PP, 6_RP. At codon-36, A/G nucleotide substitution resulted in to newer genotypes 36_GG from that of 36_DD in reference Genbank sample. At codon-54, C/T nucleotide substitution caused change of amino acid (AA) from arginine (R) to tryptophan (W) resulted into a new genotype of 54_WW in comparison to 54_RR of Genbank reference sample. In exon-5, out of 67 AA sites 8 codons were polymorphic, but the codons 14 and 60 were preponderant. At codon-14, A/G substitution resulted into 3 genotypes 14_KK, 14_EE and 14_KE with frequency of 0.52, 0.38 and 0.10, respectively. At codon-60, G/C and G/A substitutions resulted in to 3 genotypes 60_GG, 60_RR and 60_GR with frequencies of 0.48, 0.42 and 0.10, respectively. Synonymous mutations as compared to Genbank accession D00476.1 were present at codons 25, 31 and 62 in all the animals of Jakhrana goats. The high genetic variability in GH gene exon-4 and exon-5 may be useful in exploring their associations with milk and growth traits in goat for further genetic improvement.     

Isoelectric focusing reveals additional casein variants in German sheep breeds

Isoelectric focusing (IEF) was applied for screening milk protein variants in milk samples from altogether 1078 sheep of different breeds, in detail Black Faced Mutton sheep (SKF; n = 57), East Friesian Milk sheep (OMS; n = 254), Gray Horned Heath (GGH; n = 190), Merinoland sheep (MLS; n = 363), Merino Mutton sheep (MMS; n = 88), and Rhön sheep (RHO; n = 126). Besides the known genetic variants of α_s1-casein (CSN1S1) (A, C, D), α_s2-casein (CSN1S2) (A, B), and β-lactoglobulin (LGB) (A, B, C) additional variants could be demonstrated in CSN1S1 (H, I) and CSN1S2 (C, D) and their genetic control confirmed by segregation analyses. CSN1S1*H corresponds to a previously mentioned phenotype “X” occurring in OMS, whereas CSN1S1*I was identified for the first time in GGH. CSN1S2*C appeared in OMS, GGH, MLS, and RHO in low frequencies and CSN1S2*D in MLS. Within LGB all three alleles occurred in Merino breeds while α-lactalbumin (LAA) and κ-CN (CSN3) were monomorph at protein level. The haplotype CSN1S1*C–CSN1S2*A was predominant in five out of six breeds with frequencies between 0.325 and 0.919.     

A microsatellite within the bovine K-casein gene reveals a polymorphism correlating strongly with polymorphisms previously described at the protein as well as the DNA level

The polymorphism of a (TA)n(CA)n repeat microsatellite present in the third intaron of the bovine K-casein gene (CASK) has been investigated. The existence of six alleles differing only in the number of dinucleotide repeats has been established. A total of 330 animals belonging to nine different pure bred Bos Taurus French breeds or to a cross-bred Bos taurus*Bos indicus population (Créole) were genotyped. The distribution of the microsatellite alleles was examined and clear breed differences were noted. Genotyping of animals by isoelectric focusing (IEF) or restriction fragment length polymorphism (RFLP) (TaqI) was performed, in order to examine the relationship of the microsatellite polymorphism to other previously described CASK polymorphisms, at the protein and DNA levels. Strong correlation was seen, indicating that evolution of the various polymorphisms was not independent, and nine CASK haplotypes were observed.     

Characterisation of novel S-alleles from cherry (Prunus avium L.)

In plant populations exhibiting gametophytic self-incompatibility, individuals harbouring rare S alleles are likely to have a reproductive advantage over individuals having more common alleles. Consequently, determination of the self-incompatibility haplotype of individuals is essential for genetic studies and the development of informed management strategies. This study characterises six new S alleles identified in wild cherry (Prunus avium L.). Investigations to determine the S genotype of individuals in recently planted woodland through length polymorphisms of introns associated with the stylar S-RNase gene and the pollen SFB gene revealed six S intron profiles which did not correspond to those of known S alleles. These are now attributed to S ₂₇ to S ₃₂ . Consensus primers, annealing in the S-RNase sequence coding for the signal peptide and C5 regions, were used to isolate the S-RNase alleles associated with the novel S intron profiles. The proteins corresponding to the new alleles were separated by isoelectric focusing from stylar extracts and their pI values determined. Similarities between the deduced amino acid sequence for the new alleles isolated and other cherry S-RNase sequences available on the databases ranged from 40% to 86%. Amplification products for SFB introns ranged from 172 to 208bp. New sequence regions exposed to positive selection were identified and the significance of the PS3 region reinforced. A phylogenetic relationship between P. avium S-RNases for S ₁₀ and S ₁₃ and between corresponding SFB alleles may indicate co-evolution of allele specificities of these two genes. The nucleotide sequences reported in this paper have been submitted to the EMBL/GenBank database under the following accession numbers: S ₂₇ (DQ266439), S ₂₈ (DQ266440), S ₂₉ (DQ266441), S ₃₀ (DQ266442), S ₃₁ (DQ266443), S ₃₂ (DQ266444).     

Cloning, Function, and Expression of sanS: A Gene Essential for Nikkomycin Biosynthesis of Streptomyces ansochromogenes

A 2-kb SmaI DNA fragment was cloned from the cosmid library of Streptomyces ansochromogenes. This DNA fragment contains a complete open reading frame which is 1275 bp in length, designated sanS (GenBank accession no. AF322179). The deduced SanS protein consists of 424 amino acids and belongs to a superfamily of enzymes with an unusual ATP-grasp fold. The disruption and complementation of sanS indicated that sanS is essential for nikkomycin biosynthesis in Streptomyces ansochromogenes. The sanS gene was subcloned into expression vector pET23b and overexpressed in E. coli BL21 (DE3). The protein was then purified and showed ATPase activity.     

Hypervariability of intronic simple (gt)n(ga)m repeats in HLA-DRB genes

We have investigated the extent of DNA variability in intronic simple (gt)_n(ga)_m repeat sequences and correlated this to sequence polymorphisms in the flanking exon 2 of HLA-DRB genes. The polymerase chain reaction (PCR) was used to amplify a DNA fragment containing exon 2 and the repeat region of intron 2. The PCR products were separated on sequencing gels in order to demonstrate length hypervariability of the (gt)_n(ga)_m repeats. In a parallel experiment, the PCR products were cloned and sequenced (each exon 2 plus adjacent simple repeats) to characterize the simple repeats in relation to the HLA-DRB sequences. In a panel of 25 DRB1, DRB4, and DRB5 alleles new sequences were not detected. Restriction fragment length polymorphism (RFLP) subtyping of serologically defined haplotypes corresponds to translated DNA sequences in 85% of the cases, the exceptions involving unusual DR/DQ combinations. Many identical DRB1 alleles can be distinguished on the basis of their adjacent simple repeats. We found group-specific organization of the repeats: the DRw52 supergroup repeats differ from those of DRB1*0101, DRB4*0101, and DRB5*0101 alleles and from those of pseudogenes. Finally, we amplified baboon DNA and found a DRB allele with extensive similarity to DRB1 sequences of the DRw52 supergroup. The simple repeat of the baboon gene, however, resembles that of human pseudogenes. In addition to further subtyping, the parallel study of polymorphic protein and hypervariable DNA alleles may allow conclusions to be drawn on the relationships between the DRB genes and perhaps also on the theory of trans-species evolution.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M 34258.     

Long-term persistence of both functional and non-functional alleles at the leukocyte immunoglobulin-like receptor A3 (LILRA3) locus suggests balancing selection

The leukocyte immunoglobulin-like receptor (LILR) family consists of 13 loci, and a number of variations have been identified in these genes. Some polymorphisms of the LILR genes are reported to be associated with susceptibility to diseases such as rheumatoid arthritis and multiple sclerosis. LILRA3, one of the LILR genes, exhibits a presence or absence variation due to a 6.7-kb deletion in various populations. In this study, variation screening of the LILRA3 gene revealed high allele frequency of the 6.7-kb LILRA3 deletion (71%) in Japanese, in contrast to the frequency reported for the other populations. In addition, we identified a splice acceptor mutation in intron 1 with allele frequency of 19%, resulting in three alternatively spliced isoforms. Surprisingly, all of these isoforms were found to contain premature termination codons (PTCs) in the exon 3. Taken together, approximately 80% of Japanese lack functional LILRA3 alleles. The maximum likelihood coalescent analysis suggested that two major lineages, functional alleles and PTC-containing alleles, have been maintained for 2.75 million years in humans. These results prompted us to hypothesize that balancing selection had maintained both the functional and non-functional alleles at the LILRA3 locus. This hypothesis is consistent with the observation that the 6.7-kb LILRA3 deletion is detected worldwide in the presence of functional LILRA3 alleles.Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB236941, AB236942, AB236943, AB236944, AB236945, AB236946, AB236947, AB236948, AB236949, AB236950.     

云南莲瓣兰主栽品种SSR指纹图谱的构建和遗传差异分析

为了解云南莲瓣兰(Cymbidium tortisepalum)的遗传多样性,利用SSR技术对32个莲瓣兰主栽品种进行遗传变异分析,并构建莲瓣兰栽培品种的指纹图谱。结果表明,筛选出的12对多态性高、稳定性好的引物共检测到95个等位基因,每对引物检测到4~18个等位基因,有效等位基因数(N E)为61.489,平均有效等位基因数(NA)为5.124,Shannon信息指数(I)和多态性信息含量(PIC)分别为0.806~2.624和0.789~0.953。12对引物中,以引物SSR03的等位基因数、NE、观测杂合度、I和PIC最高。32个品种在12对引物上都具有不同的特异性条带,可以彼此区别。从12对引物中筛选出3对核心引物SSR02、SSR03和SSR12构建了莲瓣兰主栽品种SSR分子指纹图谱,这3对核心引物组合即可鉴定32个莲瓣兰栽培品种。这为莲瓣兰的品种鉴定、遗传多样性分析和分子育种研究提供理论基础和技术支持。     

Molecular evolution of bovine Toll-like receptor 2 suggests substitutions of functional relevance

Background  

There is accumulating evidence that polymorphism in Toll-like receptor (TLR) genes might be associated with disease resistance or susceptibility traits in livestock. Polymorphic sites affecting TLR function should exhibit signatures of positive selection, identified as a high ratio of non-synonymous to synonymous nucleotide substitutions (ω). Phylogeny based models of codon substitution based on estimates of ω for each amino acid position can therefore offer a valuable tool to predict sites of functional relevance. We have used this approach to identify such polymorphic sites within the bovine TLR2 genes from ten Bos indicus and Bos taurus cattle breeds. By analysing TLR2 gene phylogeny in a set of mammalian species and a subset of ruminant species we have estimated the selective pressure on individual sites and domains and identified polymorphisms at sites of putative functional importance.     

A Study of Conservation Genetics in Cupressus chengiana,an Endangered Endemic of China,Using ISSR Markers

ISSR markers were used to analyze the genetic diversity and genetic structure of eight natural populations of Cupressus chengiana in China. ISSR analysis using 10 primers was carried out on 92 different samples. At the species level, 136 polymorphic loci were detected. The percentage of polymorphic bands (PPB) was 99%. Genetic diversity (H _e) was 0.3120, effective number of alleles (A _e) was 1.5236, and Shannon’s information index (I) was 0.4740. At the population level, PPB = 48%, A _e=1.2774, H _e=0.1631, and I=0.2452. Genetic differentiation (G _st) detected by Nei’s genetic diversity analysis suggested 48% occurred among populations. The partitioning of molecular variance by AMOVA analysis indicated significant genetic differentiation within populations (54%) and among populations (46%; P < 0.0003). The average number of individuals exchanged between populations per generation (N _m ) was 0.5436. Samples from the same population clustered in the same population-specific cluster, and two groups of Sichuan and Gansu populations were distinguishable. A significantly positive correlation between genetic and geographic distance was detected (r=0.6701). Human impacts were considered one of the main factors to cause the rarity of C. chengiana, and conservation strategies are suggested based on the genetic characters and field investigation, e.g., protection of wild populations, reestablishment of germplasm bank, and reintroduction of more genetic diversity.