Research Advances on Molecular Breeding of Groupers in China

Yu H^1†, You XX^2† and Shi Q^1,2,3*

¹BGI Education Center, University of Chinese Academy of Sciences, Shenzhen 518083, China

²Shenzhen Key Lab of Marine Genomics, Guangdong Provincial Key Lab of Molecular Breeding in Marine Economic Animals, BGI, China

³Laboratory of Aquatic Genomics, College of Life Sciences and Oceanography, Shenzhen University, China †Equal contributors

*Corresponding author: Qiong Shi, Bldg 11 Room 618, Beishan Industrial Zone, BGI, Shenzhen 518083, China

Received: July 26, 2016; Accepted: August 23, 2016; Published: August 24, 2016

Abstract

As a group of economically important fish in China, groupers (Epinephelus spp.) are facing with insufficiency of high-quality gametes and germ plasm resource degradation. This situation has severely restricted the healthy development of grouper aquaculture. It is an inevitable choice to breed new varieties or species of groupers to solve these problems. Traditional hybridization and recently developed molecular breeding are effective ways to generate novel high-quality species of groupers. So far, several hybrid groupers have been cultivated in China with significant economic values. The important areas of molecular breeding include Marker-Assisted Selection (MAS) and transgenic breeding. At present, the molecular breeding of groupers in China is focusing on MAS breeding. We have constructed genetic linkage maps and carried out Quantitative Trait Loci (QTL) analysis for a couple of groupers. Furthermore, we have cooperated with scientists at Sun Yat-Sen University to sequence the whole genome of orange-spotted grouper (E. coioides). All these works will provide a valuable resource for understanding the genetic regulation of growth and establishing the foundation for MAS breeding in groupers.

Keywords: Groupers; Hybrid; Molecular breeding; Marker-assisted selection

Abbreviations

ddRAD: Double Digest Restriction-Site Associated DNA; GH/ IGF: Growth Hormone /Insulin-Like Growth Factor; GHRH: Growth Hormone-Releasing Hormone; GnRH: Gonadotropin- Releasing Hormone; MAS: Marker-Assisted Selection; MSG: Multiplexed Shotgun Genotyping; PRP-PACAP: PACAP-Related Peptide/Pituitary Adenylatecyclase Activating Polypeptide; QTL: Quantitative Trait Loci; SSR: Simple Sequence Repeat; SNP: Single- Nucleotide Polymorphism

Introduction

Groupers (Epinephelus spp.), a group of economically important marine fish species, are well-known for their rich nutrition, delicious taste and tender flesh [1,2]. Grouper industry has been developed rapidly in the past decades with wide cultivation of over 10 grouper species in China and South-East Asian countries [2]. Among them, E. coioides, E. akaara, E. awoara, and E. malabaricus are the main cultured species in China (Figure 1). In the last year, the yearly output of groupers in China is 100,006 tons, and the production of major cultured provinces, including Guangdong, Fujian and Hainan, are 42,601, 26,905, and 26,785 tons respectively [3]. At the same time, degeneration of germ plasm resources, decrease of genetic diversity and degradation of gamete quality have been threatening the development and progress of grouper industry. During the past decade, rapid development of genomic biotechnology and increasing focuses on more efficient selection programs have accelerated genetic improvement in groupers. The high throughput sequencing techniques have facilitated identification of efficient molecular markers and implementation of Marker-Assisted Selection (MAS) program.MAS breeding is going to become a prospective strategy for generation of ideal novel species of fish. Both construction of genetic linkage maps and identification of Quantitative Trait Loci (QTL) will lay the foundation for the development of MAS program in groupers. Meanwhile, traditional hybridization breeding has created several ideal grouper hybrids, which provide genetic resources for understanding the molecular mechanisms of growth superiority in the hybrid groupers.

Figure 1: The main cultured groupers in China. The pictures are downloaded from https://www.worldfishcenter.org/databases.

    
    
    Figure 1:  The main cultured groupers in China. The pictures are downloaded
from https://www.worldfishcenter.org/databases.

Hybrid Breeding

Traditional hybridization has been an effective approach to improve physiological properties [4]. The first Hybrid of Epinephelus species, reported in 2014, hasobvious advantage in growth comparing with its parents [5]. It has been improved to become popular because of rapid growth rates and better disease resistance in recent years [6]. The successful hybridization of groupers are mainly composed of E. coioides♀×E. lanceolatus♂ [7,8], E. fuscoguttatus♀×E. lanceolatus♂ [9], E. coioides♀×E. fuscoguttatus♂ [10], E. marginatus♀×E. aeneus♂ [11], E. costae♀×E. marginatus♂ [12], and E. amblycephalus♀×E. akaara♂ [13]. Recently, researchers have paid more and more attention to the giant tiger groupers, Hulong (E. fuscoguttatus♀×E. lanceolatus♂) and Qinglong (E. coioides♀×E. lanceolatus♂) [14- 16]. Using microsatellite markers, researchers analyzed the level of heterozygosis and genetic structure of these grouper hybrids [6]. We also revealed the molecular mechanism of growth superiority by conducting comparative transcriptome analyses between E. fuscoguttatus & E. lanceolatus and their hybrid Hulong. Differential gene expression was observed in the brain and liver GH/IGF (growth hormone/insulin-like growth factor) axis and the downstream signaling pathways (including protein and glycogen synthesis) [17]; further transcriptome sequencing of muscles (effector tissues) proved important contribution of glycolysis in addition with calcium signaling and up-regulated troponins to the growth superiority of Hulong (Sun Y. et al., our unpublished data). In addition, the complete mitochondrial genomes of Hulong and Qinglong hybrids had been reported [18,19].

Molecular Markers and Genetic Linkage Maps

Molecular markers are fundamental for construction of genetic linkage maps, which is the key step for MAS breeding. Molecular markers provide a potential resource to revolutionize the breeding methodology by improving germ plasm characterization and costefficient marker-assisted selection [20-22]. They can also be used to evaluate genetic resources with high accuracy. Construction of genetic linkage maps, based on marker development, could make significant contribution to chromosome assembly, genome mapping, positional cloning of genes, and functional genomic studies [23,24]. In many aquatic species [25-32], genetic linkage maps have been reported with different marker types, which determined the map density. Among the reported molecular markers, microsatellite (also termed as simple sequence repeat, SSR) was the most popular marker in many previous studies. However, with the rapid development of genomic sequencing in recent years, Single-Nucleotide Polymorphism (SNP) has become the most suitable marker type for construction of high-density genetic maps [33-35] because of its abundance and stability. In addition, maturity of sequencing techniques has made it possible to identify thousands of SNPs simultaneously and perform genotyping cost-efficiently [36,37].

Genetic linkage maps of groupers have been reported, including microsatellite-based genetic maps of kelp grouper (E. bruneus) and white grouper (E. aeneus), and SNP-based genetic maps of orangespotted grouper (E. coioides) (see more details in Table 1). One sexspecific genetic linkage map of kelp grouper was constructed using 222 microsatellite markers [38]. The reported male map was 650.5cM in length while the total length of the female map reached 944.4cM; the average inter-marker distances were 5.0 cM and 6.7 cM, respectively. SSR markers (714) were employed to construct another genetic map of kelp grouper [39]. It was reported that, in male and female maps, the mean intervals were 4.0 cM and 4.1 cM respectively. With development of 222 microsatellites, a genetic linkage study of white grouper was conducted [40]. The constructed female and male maps spanned 1,053 cM and 886 cM respectively, with the average intervals of 5.8 cM and 5.0 cM. In several recent studies, we constructed two SNP-based genetic linkage maps of orange-spotted grouper [41,42]. The first one contained 4,608 SNPs, with the total length of 1,581.7 cM; the average marker interval was 0.34 cM [41]. The second one consisted of 3,029 SNPs, spanned 1,231.98 cM, with the mean distance of 0.41 cM [42]. However, the genotyping technology was different. The former was sequenced with Multiplexed Shotgun Genotyping (MSG) [41], while the latter was sequenced with recently developed Double Digest Restriction-Site Associated DNA (ddRAD) method [42]. Furthermore, we constructed a novel linkage map (Figure 2), based on 3,029 SNPs [42] corresponding to the scaffolds of the reference genome (Zhang Y. et al., our unpublished data) of orangespotted grouper.

Grouper	Marker type	Marker number	Length of map (cM)	Average marker interval	References
White Grouper	microsatellite	Male map: 202 Female map: 203	Male map: 886 Female map: 1053	Male: 5.0 Female: 5.8	[40]
Kelp grouper	microsatellite	Male map: 161 Female map: 173	Male map: 650.5 Female map: 944.4	Male: 5.0 Female: 6.7	[38,39]
Kelp grouper	SSR	Male map: 512 Female map: 509	Male map: 1,370.39 Femalemap:1,475.95	Male: 4.0 Female: 4.1	[38,39]
Orange-spotted grouper	SNP	Sex-averaged map: 4,608	1,581.7	0.34	[41,42]
Orange-spotted grouper	SNP	Sex-averaged map: 3,029	1,231.98	0.41	[41,42]

QTL	Trait		LOD*		R^2#(%)		Annotation
qLG1	BW		3.3		19.6		fasciculation and elongation protein zeta-2-like (fez2)
qLG2	BL		3.3		21		Dol-P-Man: Man(5) GlcNAc(2)-PP-Dol alpha-1,3-mannosyltransferase (alg3)
			endothelin converting enzyme 2 (ece2)
qLG5_1	BW		4.7		24.3		armadillo repeat gene deleted in velocardiofacial syndrome (arvcf)
qLG5_2		BW		3.1		17.1
qLG5_3	BW		2.8		16.5		solute carrier family 27 (fatty acid transporter), member 4 (slc27a4)
			tyrosine-protein kinase SgK223 (sgk223)
qLG5_4	BW		2.9		15.8		calcium/calmodulin-dependent protein kinase 2 (camk2)
qLG5_5	BW		3.2		19.1		proline-rich coiled-coil 2B (prrc2b)
			melanin-concentrating hormone receptor 1 (mchr1)
qLG5_6		BW		4.2		24.6
qLG5_7		BW		3.5		20.8
qLG5_8	BW		3.4		20		pregnancy-associated plasma protein- A (papp-a)
qLG5_9	BW		2.6		21.5		spleen tyrosine kinase (syk)
qLG5_10		BW		2.8		24.5
qLG5_11	BW		2.8		28.3		telomerase reverse transcriptase (tert)
qLG5_12	BW		2.7		14.6		WD repeat-containing protein 91-like (wdrcp91)
			ftz transcription factor 1 (ftz-f1)
qLG5_13		BW		3		29.1
qLG5_14		BW		3.2		17.6
qLG5_15		BW		2.9		15.6
qLG5_16		BL		4.4		23.3
qLG5_17		BL		2.6		14.5
qLG5_18		BL		3.8		20.5
qLG5_19	BL		3.4		20.3		sarcosine dehydrogenase (sardh)
qLG5_20		BL		3.8		22.2
qLG5_21		BL		3.5		20.8
qLG7_1	BW		2.8		16.8		multidrug and toxin extrusion protein 1-like (mate1)
qLG7_2	BL		2.6		15.1		multidrug and toxin extrusion protein 1-like (mate1)
qLG21	BL		2.9		15.5		neurogenic locus notch homolog protein 1-like (notch1)
qLG24		BW		2.6		14.4
*LOD: Logarithm of Odds #a Parameter for phenotypic variation explained

Research Advances on Molecular Breeding of Groupers in China

Abstract

Abbreviations

Introduction

Hybrid Breeding

Molecular Markers and Genetic Linkage Maps

QTL Mapping and Candidate Gene Identification

Conclusion

Acknowledgement

References