Single-cell transcriptome landscape of ovarian cells during primordial follicle assembly in mice.

Primordial follicle assembly in the mouse occurs during perinatal ages and largely determines the ovarian reserve that will be available to support the reproductive life span. The development of primordial follicles is controlled by a complex network of interactions between oocytes and ovarian somatic cells that remain poorly understood. In the present research, using single-cell RNA sequencing performed over a time series on murine ovaries, coupled with several bioinformatics analyses, the complete dynamic genetic programs of germ and granulosa cells from E16.5 to postnatal day (PD) 3 were reported. Along with confirming the previously reported expression of genes by germ cells and granulosa cells, our analyses identified 5 distinct cell clusters associated with germ cells and 6 with granulosa cells. Consequently, several new genes expressed at significant levels at each investigated stage were assigned. By building single-cell pseudotemporal trajectories, 3 states and 1 branch point of fate transition for the germ cells were revealed, as well as for the granulosa cells. Moreover, Gene Ontology (GO) term enrichment enabled identification of the biological process most represented in germ cells and granulosa cells or common to both cell types at each specific stage, and the interactions of germ cells and granulosa cells basing on known and novel pathway were presented. Finally, by using single-cell regulatory network inference and clustering (SCENIC) algorithm, we were able to establish a network of regulons that can be postulated as likely candidates for sustaining germ cell-specific transcription programs throughout the period of investigation. Above all, this study provides the whole transcriptome landscape of ovarian cells and unearths new insights during primordial follicle assembly in mice.

Mitochondrial genomes from modern and ancient Turano-Mongolian cattle reveal an ancient diversity of taurine maternal lineages in East Asia.

Turano-Mongolian cattle are a group of taurine cattle from Northern and Eastern Asia with distinct morphological traits, which are known for their ability to tolerate harsh environments, such as the Asian steppe and the Tibetan plateau. Through the analysis of 170 mitogenomes from ten modern breeds, two sub-lineages within T3 (T3119 and T3055) were identified as specific of Turano-Mongolian cattle. These two T3 sub-lineages, together with the previously identified T4, were also present in six Neolithic samples, dated to ~3900 years BP, which might represent the earliest domestic taurine stocks from Southwest Asia. The rare haplogroup Q, found in three Tibetan cattle, testifies for the legacy of ancient migrations from Southwest Asia and suggests that the isolated Tibetan Plateau preserved unique prehistoric genetic resources. These findings confirm the geographic substructure of Turano-Mongolian cattle breeds, which have been shaped by ancient migrations and geographic barriers.

Selection signatures of Fuzhong Buffalo based on whole-genome sequences.

BACKGROUND: Fuzhong buffalo, a native breed of Guangxi Zhuang Autonomous Region, is traditionally used as a draft animal to provide farm power in the rice cultivation. In addition, the Fuzhong buffalo also prepared for the bullfighting festival organized by the locals. The detection of the selective signatures in its genome can help in elucidating the selection mechanisms in its stamina and muscle development of a draft animal. RESULTS: In this study, we analyzed 27 whole genomes of buffalo (including 15 Fuzhong buffalo genomes and 12 published buffalo genomes from Upper Yangtze region). The ZHp, ZFst, π-Ratio, and XP-EHH statistics were used to identify the candidate signatures of positive selection in Fuzhong buffalo. Our results detected a set of candidate genes involving in the pathways and GO terms associated with the response to exercise (e.g., ALDOA, STAT3, AKT2, EIF4E2, CACNA2D2, TCF4, CDH2), immunity (e.g., PTPN22, NKX2-3, PIK3R1, ITK, TMEM173), nervous system (e.g., PTPN21, ROBO1, HOMER1, MAGI2, SLC1A3, NRG3, SNAP47, CTNNA2, ADGRL3). In addition, we also identified several genes related to production and growth traits (e.g., PHLPP1, PRKN, MACF1, UCN3, RALGAPA1, PHKB, PKD1L). Our results depicted several pathways, GO terms, and candidate genes to be associated with response to exercise, immunity, nervous system, and growth traits. CONCLUSIONS: The selective sweep analysis of the Fuzhong buffalo demonstrated positive selection pressure on potential target genes involved in behavior, immunity, and growth traits, etc. Our findings provided a valuable resource for future research on buffalo breeding and an insight into the mechanisms of artificial selection.

Detection of InDel variations within seven candidate genes and their associations with phenotypic traits in three cattle breeds.

The Xinjiang brown cattle, Red steppe cattle, and Yunling cattle are indigenous cultivated cattle breeds in Chinese frontier provinces, and they produce high-grade beef and milk products, however, their genetic diversity in many important genes related to excellent meat and milk production is still unknown. Our previous studies have found that several candidate genes (e.g., SREBP1c and PAX7) were associated with bovine economically important phenotypic traits, but none has been reported in the above-mentioned three cattle breeds. Since the InDel (insertion/deletion) marker becomes a useful tool applied in the animal molecular breeding, herein, we firstly found that the InDel variations of seven candidate genes in these cattle. Results showed that the genotypic and allelic distributions of these seven genes were remarkably different among these three cattle (p < 0.05 or p < 0.01). Furthermore, the InDel variations of SREBP1c and PAX7 genes were significantly associated with eight phenotypic traits in Xinjiang brown cattle (p < 0.05 or p < 0.01), respectively, suggesting that they can become the useful DNA markers.

Copy number variation of MYLK4 gene and its growth traits of Capra hircus (goat).

Copy number variation (CNV) is a form of genetic variation caused by genome rearrangement, with abnormal fragments ranging from 50 bp to Mb. And, CNV is closely related to disease, growth and reproductive shape of livestock. As a member of myosin light chain kinase (MYLK) family with serine/threonine specificity, MYLK4 belongs to an enzyme encoded by MYLK4 gene. Although MYLK4 is a recognized kinase, its function has yet to be revealed in subsequent studies. This study aims to analyze CNV and genetic effects of MYLK4 gene in goats. We used qPCR to detect CNV of MYLK4 gene in African Nubian goat (n = 32), Guizhou black goat (n = 196) and Guizhou white goat (n = 95), respectively, and correlated CNV data of MYLK4 gene with goat growth traits in Chinese goats. The results showed that the effect of MYLK4 gene CNV on body weight, body length and body height of goats had significantly different (p < 0.05, Q < 0.05), in which CNV showed better growth traits in type of deletion. Therefore, CNV of MYLK4 gene can be used as a molecular marker for assisted selection of goat growth traits, which provides a theoretical basis for the genetic improvement of goat breeds in China.

Exploring genotype-phenotype relationships of the CRABP2 gene on growth traits in beef cattle.

Cellular retinoic acid binding protein 2 (CRABP2) is essential to myoblast differentiation. However, there was no report about the function of CRABP2 gene in cattle. This study explored the association of CRABP2 gene polymorphisms with growth traits in cattle breeds by several methods, such as DNA sequencing, PCR, PCR-RFLP and forced PCR-RFLP. Two sequence variants were determined. There were 621 individuals in six cattle breeds from China for the experiment, and three breeds were used to test validation of polymorphisms and extent of linkage disequilibrium (LD). The results showed that both SNPs (SNP1, g.2458 G > T, SNP2, g.3878 G > A) were in intron1. Two SNPs were in low linkage disequilibrium. Association analysis suggested that SNP1 had the significant difference on growth traits with body height, height at hip cross and body slanting length (P < .05), while SNP2 showed a significant difference in growth traits with body height, height at hip cross and body slanting length(P < .05). The results of this investigation displayed that the CRABP2 gene is an available candidate gene and may be used for breed selection and conservation.

Determination of genetic effects of SERPINA3 on important growth traits in beef cattle.

Serine protease inhibitor protein 3 (serpin peptidase inhibitor, clade A, member 3, SERPINA3) is a member of the serpin superfamily, probably related to the yield and quality of muscle. This study focuses on the relationship between SERPINA3 gene polymorphism and growth traits in beef cattle. The study first uses sequencing pooled DNA samples (Pool-Seq), PCR-RFLP and Tetra-primer ARMS-PCR techniques to determine the genetic polymorphisms of SERPINA3 in 765 beef cattle. Then, the polymorphic loci were correlated with the growth characters of cattle. Five SNPs (SNP1:A-648G, SNP2:T6496A, SNP3:G2495A, SNP4:T2595A, SNP5:A2615G) were found, located in the promoter, introns 5 and SNP 3, 4, 5 were in exons 2, respectively. The observed He was from 0.44 to 0.5, Ne were approaching 2 (1.78 to 2.00). The maximum and minimum PIC (polymorphism information content) values were 0.37 and 0.34, respectively. The association analysis results showed that the SNPs had a significant height in the chest girth and body length. (p < 0.05 or p < 0.01). This will provide important information for the rapid breeding of Chinese yellow cattle and the establishment of a molecular genetic marker database.

Integrative analysis of APOL3 gene CNV for adult cattle stature.

Copy number variations (CNVs) have been identified as another important structural variation of genome. In recent years, a large amount of CNVRs have been identified in humans and animals. However, association and dosage effects studies of CNVs are very limited. Apolipoprotein L3 (APOL3) gene plays a central role in modulating gene transcription and is located within a CNVR that encompasses quantitative trait locis (QTLs) for economic traits like meat quality. Herein, we analyzed the CNV polymorphism of APOL3 in 421 individuals from five distinct cattle breeds, and then correlated their genotypes with growth traits. Association analysis revealed that the APOL3 CNV was significantly associated with hip height and cannon circumference of Xianan (XN) cattle (P < .01), and visibly associated with body slanting length and hucklebone width of Pinan (PN) cattle (P < .05). Overall, the data provide evidence for the functional role of APOL3 CNV and a basis for future applications in cattle breeding.

Genetic Variants in ADD1 Gene and their Associations with Growth Traits in Cattle.

The α-adducin (ADD1) is a subunit of adducin which is a cytoskeleton heterodimeric protein. Adducin participates in oocytes chromosome meiosis of mice, prompting adducin has an effect on embryonic development. Adducin gene mutation has significantly functional change. So the present study was to identify and characterize polymorphisms within the coding region of the bovine ADD1 gene among different cattle breeds. Here, 11 novel single nucleotide polymorphisms (SNPs 1-11) were identified by DNA sequencing and polymerase chain reaction-single stranded conformational polymorphism, there were one synonymous mutation in exon 1 (SNP1); four missense mutations in exons 4, 7, and 8 (SNPs 3-6); and six mutations in introns 4, 12, 13, and 14 (SNPs 2, 7-10). The statistical analyses indicated that the some SNPs are associated with the growth traits (body length, body height, chest circumference, and hucklebone width) in Chinese Jiaxian cattle population. Our results provide evidence that polymorphisms in the ADD1 gene are associated with growth traits, and may be used for marker-assisted selection in beef cattle breeding program.

Genetic Variant of MYLK4 Gene and its Association with Growth Traits in Chinese Cattle.

As a member of MYLK family, MYLK4 gene may play a vital role in muscle development. In this study, one novel single-nucleotide polymorphism (SNP) was identified the bovine MYLK4 by sequencing pooled DNA samples (pool-Seq) and forced polymerase chain reaction-restriction fragment length polymorphism (forced PCR-RFLP) methods. Overall, we reported one mutation (SNP1) in the intron 10 region within the bovine MYLK4 gene in 559 individuals representing five main cattle breeds from China (Nanyang, NY; Qinchuan; Jiaxian, JX; Pinan cattle; and Caidamu cattle, CDM). Genotype AA and allele A were predominant in the QC, PN, and XN populations. Association analysis with growth traits in the QC breed showed that the animals with genotype GG had significantly greater chest breadth and hip width (P < 0.05). Meanwhile, the genotype GG was strongly associated with withers height and body length than those with genotype AA (P < 0.01 or P < 0.05) at 12 months in the NY breed. These statistical results exhibited that the MYLK4 gene might be a potential candidate gene to improve cattle's growth traits, and the SNP could be used as molecular markers in early marker-assisted selection (MAS) in beef cattle breeding program.

Worldwide patterns of ancestry, divergence, and admixture in domesticated cattle.

The domestication and development of cattle has considerably impacted human societies, but the histories of cattle breeds and populations have been poorly understood especially for African, Asian, and American breeds. Using genotypes from 43,043 autosomal single nucleotide polymorphism markers scored in 1,543 animals, we evaluate the population structure of 134 domesticated bovid breeds. Regardless of the analytical method or sample subset, the three major groups of Asian indicine, Eurasian taurine, and African taurine were consistently observed. Patterns of geographic dispersal resulting from co-migration with humans and exportation are recognizable in phylogenetic networks. All analytical methods reveal patterns of hybridization which occurred after divergence. Using 19 breeds, we map the cline of indicine introgression into Africa. We infer that African taurine possess a large portion of wild African auroch ancestry, causing their divergence from Eurasian taurine. We detect exportation patterns in Asia and identify a cline of Eurasian taurine/indicine hybridization in Asia. We also identify the influence of species other than Bos taurus taurus and B. t. indicus in the formation of Asian breeds. We detect the pronounced influence of Shorthorn cattle in the formation of European breeds. Iberian and Italian cattle possess introgression from African taurine. American Criollo cattle originate from Iberia, and not directly from Africa with African ancestry inherited via Iberian ancestors. Indicine introgression into American cattle occurred in the Americas, and not Europe. We argue that cattle migration, movement and trading followed by admixture have been important forces in shaping modern bovine genomic variation.

Correlation between ZBED6 Gene Upstream CpG Island methylation and mRNA expression in cattle.

DNA methylation is essential for the regulation of gene expression and important roles in muscle development. To assess the extent of epigenetic modifications and gene expression on the differentially methylated region (DMR) in ZBED6, we simultaneously examined DNA methylation and expression in six tissues from two different developmental stages (fetal bovine and adult bovine). The DNA methylation pattern was compared using bisulfite sequencing polymerase chain reaction (BSP) and combined bisulfite restriction analysis (COBRA). The result of quantitative real-time PCR (qPCR) analysis showed that ZBED6 has a broad tissue distribution and is highly expressed in adult bovine (P < 0.05 or P < 0.01). The DNA methylation level was significantly different in liver, lung and spleen between the two cattle groups (P < 0.05 or P < 0.01). The adult bovine group exhibited a significantly higher mRNA level and lower DNA methylation level than the fetal bovine group in liver, lung, and spleen. No significant association was detected between DNA methylation level and muscle, heart, and kidney at two different stages. In this study, the statistical analyses indicated that DNA methylation patterns are associated with mRNA level in some tissues, these results may be a useful parameter to investigate muscle developmental in cattle and as a model for studies in other species, potentially contributing to an improvement of growth performance selection in beef cattle breeding program.

miR-30-5p Regulates Muscle Differentiation and Alternative Splicing of Muscle-Related Genes by Targeting MBNL.

MicroRNAs (miRNAs), a class of single stranded, small (~22 nucleotides), non-coding RNAs, play an important role in muscle development. We focused on the role of the miR-30-5p family during bovine muscle development from previous high-throughput sequencing results and analyzed their expression profiles. MHC and MyoG mRNAs expression as well as their proteins were suppressed in differentiated C2C12 cells, suggesting the importance of miR-30-5p in muscle development. MBNL, the candidate target of miR-30-5p, is an alternative splicing regulation factor. MBNL1 and MBNL3 have opposite effects on muscle differentiation. Our results confirmed that miR-30a-5p and miR-30e-5p repress the expression of MBNL1, MBNL2 and MBNL3, whereas miR-30b-5p inhibits MBNL1 and MBNL2 expression. This provides direct evidence that MBNL expression can be flexibly regulated by miR-30-5p. Previous studies showed that MBNL1 promotes exon inclusion of two muscle-related genes (Trim55 and INSR). Through RNA splicing studies, we found that miR-30-5p had an effect on their alternative splicing, which means miR-30-5p via MBNL1 could be integrated into muscle signaling pathways in which INSR or Trim55 are located. In conclusion, miR-30-5p could inhibit muscle cell differentiation and regulate the alternative splicing of Trim55 and INSR by targeting MBNL. These results promote the understanding of the function of miRNAs in muscle development.

Copy number variations of the extensively amplified Y-linked genes, HSFY and ZNF280BY, in cattle and their association with male reproductive traits in Holstein bulls.

BACKGROUND: Recent transcriptomic analysis of the bovine Y chromosome revealed at least six multi-copy protein coding gene families, including TSPY, HSFY and ZNF280BY, on the male-specific region (MSY). Previous studies indicated that the copy number variations (CNVs) of the human and bovine TSPY were associated with male fertility in men and cattle. However, the relationship between CNVs of the bovine Y-linked HSFY and ZNF280BY gene families and bull fertility has not been investigated. RESULTS: We investigated the copy number (CN) of the bovine HSFY and ZNF280BY in a total of 460 bulls from 15 breeds using a quantitative PCR approach. We observed CNVs for both gene families within and between cattle breeds. The median copy number (MCN) of HSFY among all bulls was 197, ranging from 21 to 308. The MCN of ZNF280BY was 236, varying from 28 to 380. Furthermore, bulls in the Bos taurus (BTA) lineage had a significantly higher MCN (202) of HSFY than bulls in the Bos indicus (BIN) lineage (178), while taurine bulls had a significantly lower MCN (231) of ZNF280BY than indicine bulls (284). In addition, the CN of ZNF280BY was positively correlated to that of HSFY on the BTAY. Association analysis revealed that the CNVs of both HSFY and ZNF280BY were correlated negatively with testis size, while positively with sire conception rate. CONCLUSION: The bovine HSFY and ZNF280BY gene families have extensively expanded on the Y chromosome during evolution. The CN of both gene families varies significantly among individuals and cattle breeds. These variations were associated with testis size and bull fertility in Holstein, suggesting that the CNVs of HSFY and ZNF280BY may serve as valuable makers for male fertility selection in cattle.

SNP and haplotype analysis reveal IGF2 variants associated with growth traits in Chinese Qinchuan cattle.

Insulin-like growth factor 2 (IGF2) is a potent cell growth and differentiation factor and is implicated in mammals' growth and development. The objective of this study was to evaluate the effects of the mutations in the bovine IGF2 with growth traits in Chinese Qinchuan cattle. Four single nucleotide polymorphisms (SNPs) were detected of the bovine IGF2 by DNA pool sequencing and forced polymerase chain reaction-restriction fragment length polymorphism (forced PCR-RFLP) methods. We also investigated haplotype structure and linkage disequilibrium (LD) coefficients for four SNPs in 817 individuals representing two main cattle breeds from China. The result of haplotype analysis showed eight different haplotypes and 27 combined genotypes within the study population. The statistical analyses indicated that the four SNPs, combined genotypes and haplotypes are associated with the withers height, body length, chest breadth, chest depth and body weight in Qinchuan cattle population (P < 0.05 or <0.01). The mutant-type variants and mutant haplotype (Hap 8: ATGG; likely to be the beneficial QTN allele) was superior for growth traits; the heterozygote diplotype was associated with higher growth traits compared to wild-type homozygote. Our results provide evidence that polymorphisms in the IGF2 gene are associated with growth traits, and may be used for marker-assisted selection in beef cattle breeding program.

Expression, SNP identification, linkage disequilibrium, and haplotype association analysis of the growth suppressor gene ZBED6 in Qinchuan beef cattle.

Zinc finger, BED-type containing 6 (ZBED6) is a novel transcription factor that was identified and shown to act as a repressor of IGF2 transcription in skeletal muscle myogenesis and development. The aims of this study were to determine ZBED6 expression level and examine the association of the ZBED6 polymorphism with growth traits in Qinchuan beef cattle. The bovine ZBED6 mRNA was detected in eight tissues by quantitative real-time PCR (qPCR), being highly expressed in skeletal muscle. Three single nucleotide polymorphisms (SNPs) were identified the bovine ZBED6 by sequencing pooled DNA samples (Pool-Seq) and forced polymerase chain reaction-restriction fragment length polymorphism (forced PCR-RFLP) methods. In this study, we reported one mutation in the promoter and two missense mutations in the coding regions within the bovine ZBED6 gene, and the haplotype variability and extent of linkage disequilibrium (LD) in 817 individuals from the Qinchuan (QC) and Chinese Holstein (CH). We also investigated haplotype structure and linkage disequilibrium coefficients for three SNPs of ZBED6 in the study populations. The result of haplotype analysis of three SNPs showed that eight different haplotypes were identified in two breeds. The wild-type haplotype (Hap 1: GCA) and mutant-type haplotype (Hap 8: AGG) shared by two populations accounted for 29.8%, 57.5%, and 8.6%, 0% of all haplotypes observed in QC and CH, respectively. The statistical analyses indicated that three SNPs, 23 combined genotypes, and 8 haplotypes were significantly associated with different growth traits in the QC cattle population (P < 0.05 or P < 0.01). The mutant-type variants and mutant haplotype were superior for growth traits; the heterozygote diplotype was associated with higher growth traits compared to the wild-type homozygote. The results of this study suggest that the ZBED6 gene possibly is a strong candidate gene that affects growth traits in QC beef cattle breeding program.

Comparative analysis of the IGF2 and ZBED6 gene variants and haplotypes reveals significant effect of growth traits in cattle.

Muscle growth is a complex phenomenon regulated by many factors, whereby net growth results from the combined action of synthesis and turnover. Insulin-like growth factor 2 (IGF2) is a fetal growth and differentiation factor that plays an important role in muscle growth and in myoblast proliferation and differentiation; Zinc finger, BED-type containing 6 (ZBED6) is a novel transcription factor that was identified and shown to act as a repressor of IGF2 transcription in skeletal muscle. In this study, a total of seven single nucleotide polymorphisms (SNPs) were identified, four SNPs in intron 8 of IGF2 and one promoter SNP and two missense mutations in the coding region of ZBED6, two of which were in complete linkage disequilibrium (LD) in the bovine IGF2. The 58 haplotypes were inferred in 1522 individuals representing four purebred cattle breeds from China. The seven SNPs, 79 and 66 combined diplotypes were revealed for association with body mass in Nanyang and Jiaxian cattle populations at five different ages (P < 0.05 or 0.01). The mutant-type variants and haplotype 58 (likely in LD with the beneficial quantitative trait nucleotide allele) was superior for body mass; the heterozygote diplotype of the most common haplotypes 58 was associated with higher body mass compared to either heterozygote or homozygote. The statistical analyses indicated that the mutant-type variants and haplotypes are significantly associated with body mass in study cattle populations at different ages. These data demonstrate that variants and haplotypes are associated with growth traits, and these results may provide important biological insights into the phenotypic differentiation that is associated with adaptation and specialization of cattle breeds.

The effect of haplotype variation in the bovine PAX6 gene.

The transcription factor paired box 6 gene (PAX6) plays a key role during the early development of various organs. In this study, three single nucleotide polymorphisms (SNPs) have been identified in the introns 2, 8 and 11 of the bovine PAX6 gene. 8 haplotypes and 13 diplotypes were revealed and the linkage disequilibrium (LD) was assessed in 817 cows representing four main cattle breeds from China (Nanyang, NY; Qinchuan, QC; Jiaxian, JX; Chinese Holstein, CH). The LD analysis revealed that the additional three SNPs were not in strong LD, the wild haplotype (Hap 1: CTT) and mutant haplotype (Hap 8: TCC) have a frequency of 9.36, 35.61, 18.32, 57.15 % and 15.60, 14.19, 0.83, 0 % in the four cattle populations. The statistical analyses indicated that the SNP2 and 3 are associated with the body weight at birth, 6 and 12 months in NY and JX cattle population (P < 0.01 or P < 0.05); and significant association was detected between 19 (NY) or 13 (JX) diplotype (combined genotypes) and body weight at different ages (birth, 6 and 12 months). Our results provide evidence that some polymorphisms in PAX6 are associated with growth traits at some ages, and may be used as candidates for marker-assisted selection in beef cattle breeding program.

Y chromosome diversity and paternal origin of Chinese cattle.

To determine the Y chromosome genetic diversity and paternal origin of Chinese cattle, 369 bulls from 17 Chinese native cattle breeds and 30 bulls from Holstein and four bulls from Burma were analyzed using a recently discovered USP9Y marker that could distinguish between taurine and indicine cattle more efficiently. In total, the taurine Y1, Y2 haplogroup and indicine Y3 haplogroup were detected in 7 (1.9 %), 193 (52.3 %) and 169 (45.8 %) individuals of 17 Chinese native breeds, respectively, although these frequencies varied amongst the Chinese native cattle breeds examined. Y2 dominates in northern China (91.4 %), while Y3 dominates in southern China (81.2 %). Central China is an admixture zone with Y2 predominating overall (72.0 %). Our results demonstrate that Chinese cattle have two paternal origins, one from B. taurus (Y2) and the other from B. indicus (Y3). The Y1 haplogroup may originate from the imported beef cattle breeds in western countries. The geographical distributions of the Y2 and Y3 haplogroup frequencies reveal a pattern of male indicine introgression from south to north China, and male taurine introgression from north to south China.