宿主基因-瘤胃微生物互作对绵羊剩余采食量调控的分子机理

Residual feed intake (RFI) is an ideal breeding index for measuring feed efficiency in livestock and its improvement is important in the genetic improvement of sheep. Recent studies have reported that RFI is co-regulated by the host gene and the rumen microorganisms, but how the interaction between the host gene and the rumen microbes can regulate sheep RFI is still unclear. This project is based on the newly established Hu sheep population with accurate residual feed intake records and further enlarges the sample size of the tested population. The sheep genotype information and rumen microbial composition and abundance were obtained using Ovine 50K SNP chip and microbial 16S rRNA sequencing. The composition and abundance of rumen microbial were defined as a complex quantitative trait - "bacterial trait", and a genome-wide association analysis is carried out to screen out the candidate genes that affect the RFI and rumen microbial flora. The population genetic effects of key genes located in important biological signaling pathways and the relative abundance of rumen microbial flora in individual with different genotypes are analyzed by KASPar SNP typing technique and real-time quantitative PCR. It is hoped that the interaction between host gene and rumen microorganisms and its regulation on sheep RFI can be analyzed from the host genome and rumen microbial metagenome.

剩余采食量是衡量家畜饲料利用效率的理想育种指标。已有研究报道，剩余采食量受宿主基因与瘤胃微生物共同调控，但有关宿主基因与瘤胃微生物互作调控绵羊剩余采食量的分子机理仍不清楚。本项目以前期建立的具有准确剩余采食量记录的湖羊群体为基础，进一步扩大群体样本含量，利用Ovine 50K SNP芯片和微生物16S rRNA测序技术，获取宿主基因型和瘤胃微生物组成和丰度，将瘤胃微生物组成和丰度标准化处理后定义为一种复杂数量性状-“细菌性状”，通过全基因组关联分析，初步筛选出影响绵羊剩余采食量和瘤胃微生物菌群的候选基因，并在验证群体中利用KASPar SNP分型技术和实时荧光定量PCR技术对重要生物学信号通路中关键基因的群体遗传学效应和具有重要功能的瘤胃微生物菌群在不同基因型个体中的相对丰度进行验证，以期从宿主基因组和瘤胃微生物宏基因组水平解析宿主基因与瘤胃微生物互作及其调控绵羊剩余采食量的分子机理。

本项目在前期工作的基础上，继续采用单栏饲养，测定了1806只湖羊80-180d阶段的采食量、日增重等性状，计算出个体的剩余采食量与饲料转化率，结果表明湖羊80d平均体重为19.62±4.08kg，180d平均体重为46.48±6.33kg，80-180d平均日增重为0.27±0.04kg/d，80-180d饲料转化率为5.96±0.65，80-180d剩余采食量为0.00±0.09。上述性状的遗传力分别为0.72±0.10、0.88±0.05、0.32±0.12、0.56±0.11、0.50±0.11。利用16S rDNA测序技术对试验群体的瘤胃内容物样品进行分析，在门水平，共鉴别出26个菌门，其中拟杆菌门和厚壁菌门为优势菌门，丰度分为别40.95%和36.36%，其后依次为纤维杆菌门和变形菌门，丰度分别为8.27%和7.28%。Observed species、ACE、Good’s coverage和Chao1指数在High-、Medium-和Low-RFI组差异显著，Negativicutes、Prevotellaceae和Selenomonadales可能是影响湖羊剩余采食量的关键微生物。基于全基因组重测序技术，采用GATK标准call SNPs流程，共检测出23409311个高质量的SNPs位点并用于后续的全基因组关联分析。利用rMVP和GEMMA软件开展剩余采食量性状的全基因组关联分析，发现NEDD9、KCND2、FER1L6等3个基因内的15个SNPs位点与湖羊80-180d剩余采食量呈显著关联。结合前期极端高、低RFI湖羊肝脏转录组数据鉴定出ME1 g.453C>T和CA1 g.199C>T两个与剩余采食量和饲料转化率相关的关键基因及遗传变异。基于大规模群体的微生物数据和基因组数据对湖羊瘤胃细菌性状的遗传力进行估计，结果发现有77个瘤胃微生物菌群是可遗传的（FDR<0.2），其中g.Christensenellaceae_R-7_group的遗传最高（0.58），表明“细菌性状”受宿主遗传控制。以标准化的瘤胃微生物丰度为“细菌性状”，开展mGWAS分析，发现Lachnospiraceae ND3007等4个微生物显著与体重关联，且基因组上9号染色体的TOX基因显著影响该菌的丰度，初步揭示了宿主基因组-瘤胃微生物-表型的分子机理。