Special Article - Molecular Breeding
Austin J Mol & Cell Biol. 2016; 3(1): 1008.
Research Advances on Molecular Breeding of Groupers in China
Yu H1†, You XX2† and Shi Q1,2,3*
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
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
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
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 . 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 . 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.
Traditional hybridization has been an effective approach to improve physiological properties . The first Hybrid of Epinephelus species, reported in 2014, hasobvious advantage in growth comparing with its parents . It has been improved to become popular because of rapid growth rates and better disease resistance in recent years . The successful hybridization of groupers are mainly composed of E. coioides♀×E. lanceolatus♂ [7,8], E. fuscoguttatus♀×E. lanceolatus♂ , E. coioides♀×E. fuscoguttatus♂ , E. marginatus♀×E. aeneus♂ , E. costae♀×E. marginatus♂ , and E. amblycephalus♀×E. akaara♂ . 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 . 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) ; 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 . 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 . 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 . 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 . The second one consisted of 3,029 SNPs, spanned 1,231.98 cM, with the mean distance of 0.41 cM . However, the genotyping technology was different. The former was sequenced with Multiplexed Shotgun Genotyping (MSG) , while the latter was sequenced with recently developed Double Digest Restriction-Site Associated DNA (ddRAD) method . Furthermore, we constructed a novel linkage map (Figure 2), based on 3,029 SNPs  corresponding to the scaffolds of the reference genome (Zhang Y. et al., our unpublished data) of orangespotted grouper.
Figure 2: Distribution of SNPs on the chromosomes of orange-spotted grouper. SNP markers on the scaffolds (dark green) were aligned onto the corresponding chromosomes (chr; bright green). The length unit of the upper axis (bright green, for the chromosomes) is centimorgan (cM) and the lower axis (dark green, for the sacaffolds) is millinbase (Mb)s. These data are summarized from our previous report  and unpublished results.
Length of map (cM)
Average marker interval
Male map: 202
Female map: 203
Male map: 886
Female map: 1053
Male map: 161
Female map: 173
Male map: 650.5
Female map: 944.4
Male map: 512
Female map: 509
Male map: 1,370.39
Sex-averaged map: 4,608
Sex-averaged map: 3,029
Table 1: Summary of genetic linkage maps of groupers.
QTL Mapping and Candidate Gene Identification
Genetic linkage map is essential for QTL mapping.QTL mapping can be used for qualitative and quantitative traits (such as growth related or disease resistant) that are measured quantitatively . In fishes, QTL for these two important traits have been reported [44,45]. In the examination of groupers, 3 significant QTLs affecting both body weight and total length were identified and confirmed  in the kelp grouper. In a recent report of orange-spotted grouper , we identified 27 significant growth-related QTLs and determined 17 genes corresponding to these QTLs (Table 2). Interestingly, we supposed the leptin gene to be an important candidate for controlling growth-related traits. In previous studies of leptin gene of orangespotted grouper [46,47], researchers detected 6 and 1 growth-related SNPs in leptin-a and leptin-b respectively. Moreover, Growth Hormone-Releasing Hormone (GHRH), its receptor (GHRHR) and PACAP-Related Peptide/Pituitary Adenylatecyclase Activating Polypeptide (PRP-PACAP) were verified because of their association with growth in orange-spotted grouper . These genetic linkage maps, growth-related QTLs and candidate genes could be applied together for further MAS breeding and may promote the research on genetic regulation of growth-related traits in groupers.
fasciculation and elongation protein zeta-2-like (fez2)
Dol-P-Man: Man(5) GlcNAc(2)-PP-Dol alpha-1,3-mannosyltransferase (alg3)
endothelin converting enzyme 2 (ece2)
armadillo repeat gene deleted in velocardiofacial syndrome (arvcf)
solute carrier family 27 (fatty acid transporter), member 4 (slc27a4)
tyrosine-protein kinase SgK223 (sgk223)
calcium/calmodulin-dependent protein kinase 2 (camk2)
proline-rich coiled-coil 2B (prrc2b)
melanin-concentrating hormone receptor 1 (mchr1)
pregnancy-associated plasma protein- A (papp-a)
spleen tyrosine kinase (syk)
telomerase reverse transcriptase (tert)
WD repeat-containing protein 91-like (wdrcp91)
ftz transcription factor 1 (ftz-f1)
sarcosine dehydrogenase (sardh)
multidrug and toxin extrusion protein 1-like (mate1)
multidrug and toxin extrusion protein 1-like (mate1)
neurogenic locus notch homolog protein 1-like (notch1)
*LOD: Logarithm of Odds
#a Parameter for phenotypic variation explained
Table 2: The growth-related QTLs and genes identified in the orange-spotted grouper .
Both traditional hybridization and novel MAS breeding are effective ways to generate new groupers with good properties, such as high growth rates, efficiency of food conversion and disease resistance. However, the hybridization has some disadvantages, for example, it is difficult to overcome the barrier of distant cross-incompatibility and the breeding cycle is too long. The MAS not only gets over the difficulty and shortens the breeding cycle, but also can enhance the accuracy of breeding schemes. Despite MAS is at the early development stage, it is clear that the development of sequencing techniques, genetic linkage maps, QTL analysis and availability of whole genome sequences of groupers are creating good opportunities for applications of the MAS breeding strategy. Furthermore, analyses of the hybrid grouper transcriptomes and candidate genes for growth will play an important role to facilitate the progress of MAS breeding in groupers. Meanwhile, with the rapid development of genomic sequencing and high-throughput genotyping technologies, MAS is going to be more and more cost-efficient, which supports MAS breeding to become an effective strategy for generation of novel grouper varieties or species.
This work was financially supported by Natural Science Foundation of China (No. 31370047& 31372526), Special Project on the Integration of Industry, Education and Research of Guangdong Province (No. 2013B090800017), Shenzhen Special Program for Future Industrial Development (No. JSGG20141020113728803), Special Fund for State Oceanic Administration Scientific Research in the Public Interest (No. 201305018), and Shenzhen Dapeng Special Program for Industrial Development (KY20160102).
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Citation: Yu H, You XX and Shi Q. Research Advances on Molecular Breeding of Groupers in China. Austin J Mol & Cell Biol. 2016; 3(1): 1008.