Xiao Ning, Li Hongli, Shafique Laiba, Zhao Shanshan, Su Xiaoping, Zhang Yu, Cui Kuiqing, Liu Qingyou, Shi Deshun
State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi University, Nanning, China.
Front Genet. 2019 Sep 18;10:875. doi: 10.3389/fgene.2019.00875. eCollection 2019.
Coat color is of great importance in animal breed characteristics; it is not only a significant productive trait but also an indispensable economic trait, especially in the rabbit industry. In the present study, the relationship between melanocortin 1 receptor () genotypes and coat color phenotypes was observed in five rabbit breeds with popular coat colors that are present in China. These breeds comprised the Lianshan black rabbit (BR), Fujian yellow rabbit (YR), New Zealand white rabbit (WR), Gray Giant rabbit (GR), and Checkered Giant rabbit (CR), which were firstly determined, and the results showed that GR had an E allele; WR, CR, and BR had a 6-bp in-frame deletion (c.281_286del6, E allele); and YR had a 30-bp deletion (c.304_333del30 E allele). To explore the feasibility of obtaining a novel rabbit coat color through the mutation of with the CRISPR/Cas9 system, two single-guide RNAs (sgRNAs) were designed for the gene, and the editing efficiency was confirmed by injection of rabbits' zygotes. Unlike the donor rabbits whose coat color was originally black, two novel pale-yellow-coated rabbits were generated in the founders. A total of six novel gene deletions were identified in the two founder rabbits, in which the longest deletion was more than 700 bp. The histological hematoxylin-and-eosin (H&E) staining results indicated that eumelanin amounts were absent in hair follicles of -knockout (KO) rabbits, when compared with that of donor BR. In addition, the messenger RNA (mRNA) levels of some key downstream genes in the MC1R pathway were all downregulated in -KO rabbits compared with BR and YR. These results further indicate that loss-of-function MC1R contributed to blocking the synthesis of eumelanin and created a novel pale-yellow coat color in the -KO rabbits, and gene editing technology may be a useful tool to generate novel phenotypes in rabbit breeding.
毛色在动物品种特征中具有重要意义；它不仅是一个重要的生产性状，也是一个不可或缺的经济性状，尤其是在养兔业中。在本研究中，观察了中国现存的五个具有流行毛色的兔品种中黑素皮质素1受体（）基因型与毛色表型之间的关系。这些品种包括首次确定的连山黑兔（BR）、福建黄兔（YR）、新西兰白兔（WR）、灰色巨型兔（GR）和花斑巨型兔（CR），结果显示GR有一个E等位基因；WR、CR和BR有一个6碱基对的框内缺失（c.281_286del6，E等位基因）；YR有一个30碱基对的缺失（c.304_333del30 E等位基因）。为了探索通过CRISPR/Cas9系统对进行突变获得新的兔毛色的可行性，针对基因设计了两个单向导RNA（sgRNA），并通过注射兔受精卵来确认编辑效率。与毛色原本为黑色的供体兔不同，在 founders 中产生了两只新的浅黄色被毛的兔子。在两只 founder 兔中总共鉴定出六个新的基因缺失，其中最长的缺失超过700 bp。组织学苏木精-伊红（H&E）染色结果表明，与供体BR相比，基因敲除（KO）兔毛囊中真黑素含量缺失。此外，与BR和YR相比，MC1R途径中一些关键下游基因的信使RNA（mRNA）水平在KO兔中均下调。这些结果进一步表明，功能缺失的MC1R导致真黑素合成受阻，并在KO兔中产生了新的浅黄色毛色，基因编辑技术可能是在兔育种中产生新表型的有用工具。
