Genetic Diversity in Casein Gene Cluster in a Dromedary Camel (C. dromedarius) Population from the United Arab Emirates

Genetic polymorphisms, causing variation in casein genes (CSN1S1, CSN1S2, CSN2, and CSN3), have been extensively studied in goats and cows, but there are only few studies reported in camels. Therefore, we aimed to identify alleles with functional roles in the United Arab Emirates dromedary camel (Camelus dromedarius) population to complement previous studies conducted on the same species. Using targeted next-generation sequencing, we sequenced all genes in the casein gene cluster in 93 female camels to identify and characterize novel gene variants. Most variants were found in noncoding introns and upstream sequences, but a few variants showed the possibility of functional impact. CSN2 was found to be most polymorphic, with total 91 different variants, followed by CSN1S1, CSN3 and CSN1S2. CSN1S1, CSN1S2 and CSN2 each had at least two variants while CSN3 had only one functional allele. In future research, the functional impact of these variants should be investigated further.

A hypomorphic mutation in the mouse Csn1s1 gene generated by CRISPR/Cas9 pronuclear microinjection

Caseins are major milk proteins that have an evolutionarily conserved role in nutrition. Sequence variations in the casein genes affect milk composition in livestock species. Regulatory elements of the casein genes could be used to direct the expression of desired transgenes into the milk of transgenic animals. Dozens of casein alleles have been identified for goats, cows, sheep, camels and horses, and these sequence variants are associated with altered gene expression and milk protein content. Most of the known mutations affecting casein genes’ expression are located in the promoter and 3’-untranslated regions. We performed pronuclear microinjections with Cas9 mRNA and sgRNA against the first coding exon of the mouse Csn1s1 gene to introduce random mutations in the α-casein (Csn1s1) signal peptide sequence at the beginning of the mouse gene. Sanger sequencing of the founder mice identified 40 mutations. As expected, mutations clustered around the sgRNA cut site (3 bp from PAM). Most of the mutations represented small deletions (1–10 bp), but we detected several larger deletions as well (100–300 bp). Functionally most mutations led to gene knockout due to a frameshift or a start codon loss. Some of the mutations represented in-frame indels in the first coding exon. Of these, we describe a novel hypomorphic Csn1s1 (Csn1s1c.4-5insTCC) allele. We measured Csn1s1 protein levels and confirmed that the mutation has a negative effect on milk composition, which shows a 50 % reduction in gene expression and a 40–80 % decrease in Csn1s1 protein amount, compared to the wild-type allele. We assumed that mutation affected transcript stability or splicing by an unknown mechanism. This mutation can potentially serve as a genetic marker for low Csn1s1 expression.

Comparative Genomics, Evolutionary and Gene Regulatory Regions Analysis of Casein Gene Family in Bubalus bubalis

Buffalo is a luxurious genetic resource with multiple utilities (as a dairy, draft, and meat animal) and economic significance in the tropical and subtropical regions of the globe. The excellent potential to survive and perform on marginal resources makes buffalo an important source for nutritious products, particularly milk and meat. This study was aimed to investigate the evolutionary relationship, physiochemical properties, and comparative genomic analysis of the casein gene family (CSN1S1, CSN2, CSN1S2, and CSN3) in river and swamp buffalo. Phylogenetic, gene structure, motif, and conserved domain analysis revealed the evolutionarily conserved nature of the casein genes in buffalo and other closely related species. Results indicated that casein proteins were unstable, hydrophilic, and thermostable, although αs1-CN, β-CN, and κ-CN exhibited acidic properties except for αs2-CN, which behaved slightly basic. Comparative analysis of amino acid sequences revealed greater variation in the river buffalo breeds than the swamp buffalo indicating the possible role of these variations in the regulation of milk traits in buffalo. Furthermore, we identified lower transcription activators STATs and higher repressor site YY1 distribution in swamp buffalo, revealing its association with lower expression of casein genes that might subsequently affect milk production. The role of the main motifs in controlling the expression of casein genes necessitates the need for functional studies to evaluate the effect of these elements on the regulation of casein gene function in buffalo.

Capture Sequencing to Explore and Map Rare Casein Variants in Goats

Genetic variations in the four casein genes CSN1S1, CSN2, CSN1S2, and CSN3 have obtained substantial attention since they affect the milk protein yield, milk composition, cheese processing properties, and digestibility as well as tolerance in human nutrition. Furthermore, milk protein variants are used for breed characterization, biodiversity, and phylogenetic studies. The current study aimed at the identification of casein protein variants in five domestic goat breeds from Sudan (Nubian, Desert, Nilotic, Taggar, and Saanen) and three wild goat species [Capra aegagrus aegagrus (Bezoar ibex), Capra nubiana (Nubian ibex), and Capra ibex (Alpine ibex)]. High-density capture sequencing of 33 goats identified in total 22 non-synonymous and 13 synonymous single nucleotide polymorphisms (SNPs), of which nine non-synonymous and seven synonymous SNPs are new. In the CSN1S1 gene, the new non-synonymous SNP ss7213522403 segregated in Alpine ibex. In the CSN2 gene, the new non-synonymous SNPs ss7213522526, ss7213522558, and ss7213522487 were found exclusively in Nubian and Alpine ibex. In the CSN1S2 gene, the new non-synonymous SNPs ss7213522477, ss7213522549, and ss7213522575 were found in Nubian ibex only. In the CSN3 gene, the non-synonymous SNPs ss7213522604 and ss7213522610 were found in Alpine ibex. The identified DNA sequence variants led to the detection of nine new casein protein variants. New variants were detected for alpha S1 casein in Saanen goats (CSN1S1∗C1), Bezoar ibex (CSN1S1∗J), and Alpine ibex (CSN1S1∗K), for beta and kappa caseins in Alpine ibex (CSN2∗F and CSN3∗X), and for alpha S2 casein in all domesticated and wild goats (CSN1S2∗H), in Nubian and Desert goats (CSN1S2∗I), or in Nubian ibex only (CSN1S2∗J and CSN1S2∗K). The results show that most novel SNPs and protein variants occur in the critically endangered Nubian ibex. This highlights the importance of the preservation of this endangered breed. Furthermore, we suggest validating and further characterizing the new casein protein variants.

Polymorphism of CSN1S1 (g.12164G>A) and CSN2 (g.8913C>A) genes in pure and cross dairy goats

Casein genes directly control milk protein of animals. CSN1S1 (αS1-Casein) and CSN2 (β-Casein) genes influence on milk protein fractions. Genetic polymorphisms of CSN1S1 gene at g.12164G>A locus and CSN2 gene at g.8913C>A locus were identified by PCR-RFLP technique. Animal samples were pure dairy goats providing PE (5 hds.), Saanen (8 hds.) and their crosses providing Sapera (50% Saanen, 50% PE) (51 hds.) and SaanPE (75% Saanen, 25% PE) (3 hds.) from IRIAP dairy goat station. Allele frequency, genotype frequency, heterozygosity value, and Hardy-Weinberg (H-W) equilibrium value were analyzed by Popgen32 program. CSN1S1_g.12164G>A locus resulted in two alleles, i.e. G allele (192 bp, 145 bp, and 101 bp) and A allele (337 bp and 101 bp). The G allele from the highest frequenciest was successively Saanen (0.625), Sapera (0.578), PE (0.400), and SaanPE (0.333). Most dairy goats were heterozygote (Ho>He) and in H-W equilibrium (q2 count < q2P0.05). Whereas CSN2_g.8913C>A locus was monomorphic for possesing only C allele (233 bp and 162 bp), without A allele (416 bp). The existent g.12164G>A SNP of the CSN1S1 gene of could be a potencial molecular selection marker of milk protein content in dairy goat.

Characteristics of the genetic structure of bulls of lebedinian breed by Beta (CSN2) - and Capa Casein genes (CSN3)

The Conservation of genetic resources in animal husbandry is a necessity arising from the possibilities and reality of the diversity of the gene pool available in the country, which determines the productive and adaptive capabilities of individual breeds of cattle. Aboriginal breeds are carriers of unique genes and gene complexes, which cannot be restored if they disappear. Marker-asissted selection is one of the current areas of improvement of dairy cattle productivity. It allows the selection of parental forms at the genetic level. An important issue in the selection of dairy cattle today is the study of the relationship between hereditary factors that determine the types of proteins in milk. Genetic variants of beta-casein significantly affect on human health, kappa-casein is associated with the quality of raw milk and cheese making properties. The aim of the research was to study the available sperm production of Lebedinian bulls by beta- (CSN2) and kappacasein (CSN3) genes. The material for research was the sperm of bulls of Lebedinian breed cattle. Determination of beta- and kappa-casein gene polymorphism was performed by PCR-RFLP method in the laboratory of the Institute of Animal Husbandry of NAAS. Analysis of the genealogical structure showed that 12 bulls-producers belong to 8 lines. Of the 12 bulls, 5 are purebred Lebedinian, 7 are crossbreeds with the swiss breed. Among those studied for the complex genotype (CSN2- (CSN3), one bull had the desired genotype A2A2BB, another bulls had genotypes A2A2AB; A1A2BB; A1A2AB; A1A1AA. Five bulls have the A1A2AA genotype and two bulls have the A1A1AB genotype. The high frequency of the A2 allele of beta-casein (46%) and the B allele of kappa-casein (33%) was observed in sires of Lebedinian breed. To create micropopulations with the desired complex A2A2 / BB genotype, sires are recommended in which the frequency of alleles of beta-casein A2 and kappacasein B satisfies the requirements. Key words: sire, beta-casein, kappa-casein, genotype, allele, frequency, haploid.

MOLECULAR GENETIC MODIFICATIONS OF κ-CASEIN

Дан анализ фракционного состава, и приведены основные модели молекулярной структуры мицелл казеина. Казеины являются фосфопротеинами и представляют коллоидный мицеллярный комплекс с фосфатом кальция и небольшим количеством других минералов. Внутренняя молекулярная структура мицелл казеина до конца не изучена, однако многие ученые согласны с моделью строения, в которой гидрофильная С-концевая часть κ -казеина находится на поверхности мицеллы, что обеспечивает стерическое и электростатическое отталкивание и предотвращает агрегацию мицелл. Фракция κ- казеина обладает широким полиморфизмом, однако на сегодняшний день наиболее распространенными являются аллельные варианты А и В, отличающиеся заменой аминокислот Thr157>Ile в аллельном варианте B. Данная генетическая модификация теряет один гликозидный центр, в результате чего повышается сыропригодность молока, но снижается его термоустойчивость. Молоко, несущее аллельный вариант В по гену CSN3, характеризуется более высоким содержанием белка, а аллельный вариант А влияет на увеличение надоев. Исследование взаимосвязи структуры мицелл казеина, полиморфных вариантов генов казеина и технологических свойств позволяет проводить селекцию коров для получения молока с требуемыми технологическими свойствами. The analysis of the fractional composition and the main models of the molecular structure of casein micelles are given. Being a phosphoprotein, casein is a colloid micellar complex with calcium phosphate and some other minerals. Inner molecule structure of casein’s micelle is not thoroughly studied. However, many scientists acknowledge one formation model, in which hydrophilic C-terminal part of kappa casein occupy micelle’s surface, thus providing steric and electrostatic repulsion and preventing micelles’ aggregation. Kappa casein fraction has a wide polymorphism, but the most common ones are A and B alleles with a distinctive replacement of amino acids Thr157>Ile in B allele. This genetic modification loses one glycoside center, resul- ting in milk’s cheeseability improvement, but also its heat resistance decrease. Moreover, milk with B allele of CSN3 has higher protein content, while A allele influences the increase in milk yield. The study of the relationship between the structure of casein micelles, polymorphic variants of casein genes and technological properties allows for the selection of cows to obtain milk with the required technological properties.