The giant freshwater prawn (Macrobrachium rosenbergii) exhibits sex dimorphism between the male and female individuals. To date, the molecular mechanism governing gonadal development was unclear, and limited data were available on the gonad transcriptome of M. rosenbergii. Here, we conducted comprehensive gonadal transcriptomic analysis of female (ZW), super female (WW), and male (ZZ) M. rosenbergii for gene discovery. A total of 70.33 gigabases (Gb) of sequences were generated. There were 115,338 unigenes assembled with a mean size of 1196 base pair (bp) and N50 of 2195 bp. Alignment against the National Center for Biotechnology Information (NCBI) non-redundant nucleotide/protein sequence database (NR and NT), the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, SwissProt database, Protein family (Pfam), Gene ontology (GO), and the eukaryotic orthologous group (KOG) database, 36,282 unigenes were annotated at least in one database. Comparative transcriptome analysis observed that 10,641, 16,903, and 3393 genes were significantly differentially expressed in ZW vs. ZZ, WW vs. ZZ, and WW vs. ZW samples, respectively. Enrichment analysis of differentially expressed genes (DEGs) resulted in 268, 153, and 42 significantly enriched GO terms, respectively, and a total of 56 significantly enriched KEGG pathways. Additionally, 23 putative sex-related genes, including Gtsf1, IR, HSP21, MRPINK, Mrr, and other potentially promising candidate genes were identified. Moreover, 56,241 simple sequence repeats (SSRs) were identified. Our findings provide a valuable archive for further functional analyses of sex-related genes and future discoveries of underlying molecular mechanisms of gonadal development and sex determination. delicious flesh and high value nutrition. Like other crustaceans, M. rosenbergii displays sexual dimorphic growth pattern: The male individuals grow much larger and faster than the females [1,2]. Thus, based on the economic impact, developing the monosex population culture as the efficient approach to boost production has received great attention from researchers in M. rosenbergii. Nevertheless, the premise of monosex culture is to understand the molecular mechanism of sex determination, as well as to unravel sex-related genes.Studies on sex determination have proved that many macruran species exhibit the ZW/ZZ sex determination system [3][4][5]. In M. rosenbergii, sex reversal experiments proposed that the female prawns bear the ZW sex chromosomes, and the male individuals bear the ZZ chromosomes [1,6,7]. Subsequently, isolation of sex-specific markers further deepened the insight of the ZW/ZZ sex determination mode [8,9]. Recently, Ma et al. have confirmed that M. rosenbergii has the ZW/ZZ sex determination system based on the bacterial artificial chromosome (BAC) library, and nine genes were unraveled to be the key sex-linked genes by PacBio sequencing. Amongst, three genes, including zinc knuckle domain (ZKD), reverse transcriptase, and ANCDUO, protein homologs were known genes without characteri...

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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.

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Rabbits have been domesticated for meat, wool, and fur production, and have also been cherished as a companion, artistic inspiration, and an experimental model to study many human diseases. In the present study, the muscle mass negative regulator gene myostatin (MSTN) was knocked out in rabbits at two novel sites in exon3, and the function of these mutations was determined in subsequent generations. The prominent double muscle phenotype with hyperplasia or hypertrophy of muscle fiber was observed in the MSTN-KO rabbits, and a similar phenotype was confirmed in the F1 generation. Moreover, the average weight of 80-day-old MSTN-KO rabbits (2,452 ± 63 g) was higher than that of wild-type rabbits (2,393.2 ± 106.88 g), and also the bodyweight of MSTN-KO rabbits (3,708 ± 43.06g) was significantly higher (P < 0.001) at the age of 180 days than wild-type (WT) rabbits (3,224 ± 48.64g). In MSTN-KO rabbits, fourteen rabbit pups from the F1 generation and thirteen from the F2 generation stably inherited the induced MSTN gene mutations. Totally, 194 pups were produced in the F1 generation of which 49 were MSTN-KO rabbits, while 47 pups were produced in the F2 generation of which 20 were edited rabbits, and the ratio of edited to wild-type rabbits in the F2 generation was approximately 1:1. Thus, we successfully generated a heritable double muscle buttocks rabbits via myostatin mutation with CRISPR/Cas9 system, which could be valuable in rabbit's meat production and also a useful animal model to study the development of muscles among livestock species and improve their important economic traits as well as the human muscle development-related diseases.