ABSTRACT. The detection method based on the mathematical expectation (ME) strategy is fast and accuracy for low frequency mutation screening in large samples. Previous studies have found that the 14-bp insertion/deletion (indel) variants of the 3 0 untranslated region (3 0 UTR) within bovine PRNP gene have been characterized with low frequency (5%) in global breeds outside China, which has not been determined in Chinese cattle breeds yet. Therefore, this study aimed to identify the 14-bp indel within PRNP gene in 5 major Chinese indigenous cattle breeds and to evaluate its associations with phenotypic traits. It was the first time to use ME strategy to detect low frequency indel polymorphisms and found that minor allele frequency was 0.038 (Qinchuan), 0.033 (Xianan), 0.013 (Nanyang), 0.003 (Jiaxian), and zero (Ji'an), respectively. Compared to the traditional detection method by which the sample was screened one by one, the reaction time by using the ME method was decreased 62.5%, 64.9%, 77.6%, 88.9% and 66.4%, respectively. In addition, the 14-bp indel was significantly associated with the growth traits in 2 cattle breeds, with the body length of Qinchuan cattle as well as the body weight and waistline of Xianan cattle. Our results have uncovered that the method based on ME strategy is rapid, reliable, and cost-effective for detecting the low frequency mutation as well as our findings provide a potential valuable theoretical basis for the marker-assisted selection (MAS) in beef cattle.

The growth differentiation factor 9 (GDF9) gene, a candidate gene for high prolificacy in livestock, regulates several key granulosa cell enzymes involved in cumulus expansion in female reproduction. 1 In this study, we found a novel 12bp insertion/deletion (indel) in the 3 0 regulatory region of the goat GDF9 gene and studied its association with firstborn litter size and growth traits.Genomic DNA was extracted from ear tissues of 1816 Shaanbei white cashmere (SBWC) ewes and from blood samples of 133 Guanzhong dairy goats (GZ). For the SBWC ewes, 1422 individuals had growth traits data records and 1328 individuals had first-born litter size data, and all GZ individuals had related growth traits data. A novel 12-bp indel polymorphism (NC_022299.1:g.66028950insTACTTT CAACAA; rs670709574; Fig. S1) was detected by amplication and sequencing of a GDF9 gene fragment with primers (forward: 5 0 -ACCCATCTAGTGTTCTCGCC-3 0 ; reverse: 5 0 -GCCATCTAAAGCGCAGAGTT-3 0 ) in randomly pooled DNA samples from 50 SBWC goats. This polymorphism was genotyped in all studied individuals by 3.0% agarose gel electrophoresis. Association of the 12-bp indel with firstborn litter size and growth traits in all individuals with data was determined using a mixed linear model with R version 3.2.0 software. A fixed effects model was used to establish the influence of different parameters on first-born litter size. Ewe and lambing year factors were considered random, and least squares means were used for multiple comparisons in litter size among different genotypes: Y ijklm = l + S i + HYS j + P k + G l + e ijklm , where Y ijklm is the phenotypic value of litter size, l is the overall population mean, S i is the effect of lambing year, HYS j is the mean of the population, P k is the fixed effect of the parity, G l is the fixed effect of the genotype and e ijklm is the random error. Allele and genotype distributions of single-kid and multi-kid goats were compared using the chi-square test. 2 For growth traits, analysis of variance was applied to the general linear model with a reduced linear model:where Y ijk is the observation of the growth trait (body height, etc.) evaluated on the i th level of the fixed factor age (a i ) and the j th level of the fixed factor genotype (b j ), l is the overall mean for each trait and e ijk is the random error for the ijk th individual. 3,4 For the 12-bp indel locus, genotypes II, ID and DD were detected (Fig. S2) in SBWC goats with frequencies of the I and D alleles of 0.455 and 0.545 respectively (Table S1). Importantly, first-born litter size in ewes with the II and DD genotypes was significantly larger than in ewes with the ID genotype (P = 0.033) ( Table 1) with genotype distributions significantly different between mothers of singlelamb and multi-lamb litters (v 2 test, P = 4.5 9 10 -7 ) (Table S2). Moreover, a significant relationship was observed between this indel locus and body height (P = 0.006) in the SBWC breed, again II-and DD-genotype individuals being superior. Interestingly, we found a significan...

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Skeletal muscle is one of the three major muscle types in an organism and has key roles in the motor system, metabolism, and homeostasis. RNA-Seq analysis showed that novel lncRNA, lncFAM200B, was differentially expressed in embryonic, neonatal, and adult cattle skeletal muscles. The main aim of this study was to investigate the molecular and expression characteristics of lncFAM200B along with its crucial genetic variations. Our results showed that bovine lncFAM200B was a 472 nucleotide (nt) non-coding RNA containing two exons. The transcription factor binding site prediction analysis found that lncFAM200B promoter region was enriched with SP1 transcription factor, which promotes the binding of myogenic regulatory factor MyoD and DNA sequence. The mRNA expression analysis showed that lncFAM200B was differentially expressed in embryonic, neonatal, adult bovine muscle tissues, and the lncFAM200B expression trend positively correlated with that of MyoG and Myf5 in myoblast proliferation and differential stages. To identify the promoter active region of lncFAM200B, we constructed promoter luciferase reporter gene vector pGL3-Basic plasmids containing lncFAM200B promoter sequences and transfected them into 293T, C2C12, and 3T3-L1 cells. Our results suggested that lncFAM200B promoter active region was from −403 to −139 (264 nt) of its transcription start site, covering 6 SP1 potential binding sites. Furthermore, we found a novel C-T variation, named as SNP2 (ERZ990081 in European Variation Archive) in the promoter active region, which was linked to the nearby SNP1 (rs456951291 in Ensembl database). The genotypes of SNP1 and combined genotypes of SNP1 and SNP2 were significantly associated with Jinnan cattle hip height. The luciferase activity analysis found that the SNP1-SNP2 haplotype CC had the highest luciferase activity, which was consistent with the association analysis result that the combined genotype CC-CC carriers had the highest hip height in Jinnan cattle. In conclusion, our data showed that lncFAM200B is a positive regulator of muscle development and that SNP1 and SNP2 could be used as genetic markers for marker-assisted selection (MAS) breeding of beef cattle.