Abstract
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 (MC1R) 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, ED 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 MC1R with the CRISPR/Cas9 system, two single-guide RNAs (sgRNAs) were designed for the MC1R 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 MC1R 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 MC1R-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 MC1R-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 MC1R-KO rabbits, and gene editing technology may be a useful tool to generate novel phenotypes in rabbit breeding.
Highlights
Animal coat color is one of the most important breed characteristics, which is a significant productive trait and an indispensable economic trait
There were different long-fragment deletions in every mutated blastocyst. These results indicated that a dual–single-guide RNAs (sgRNAs)-directed CRISPR/Cas9 system efficiently knocked out rabbit melanocortin 1 receptor (MC1R) in our study
We demonstrated that the MC1R/cyclic adenosine monophosphate (cAMP) pathway was disturbed and dysfunctional MC1R leads to different degrees of reduction in the expression of these genes in MC1R-KO rabbits compared with the wild yellow rabbit (Figure 6C)
Summary
Animal coat color is one of the most important breed characteristics, which is a significant productive trait and an indispensable economic trait. The activation of α-melanocyte-stimulating hormone (MSH) initiates with MC1R complex signaling that leads to the manufacturing of black and dark-brown eumelanin pigments. MC1R gene mutations linked with different coat colors have been described in humans (Valverde et al, 1995), mice (Robbins et al, 1993), pigs (Kijas et al, 1998; Kijas et al, 2001), cattle (Klungland et al, 1995; Rouzaud et al, 2000), sheep (Vage et al, 1999), goats (Fontanesi et al, 2009), dogs (Everts et al, 2000; Newton et al, 2000), chickens (Kerje et al, 2003), and foxes (Vage et al, 1997; Vage et al, 2005), in which functional mutations increased to produce black/dark coat color and lack of functions usually generated original/yellow coat color
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