LIU Yang, XU Wenteng, ZHANG Bo, 3), SHAO Changwei, WANG Xiaoxia, SUN Hejun, WANG Na, and CHEN Songlin, *



QTL Detection for Albinism-Related Loci in Chinese Tongue Sole ()

LIU Yang1), 2), XU Wenteng1), 2), ZHANG Bo1), 2), 3), SHAO Changwei1), 2), WANG Xiaoxia1), 2), SUN Hejun1), 2), WANG Na1), 2), and CHEN Songlin1), 2), *

1),,,,266071,2),,266237,3),,300457,

TheChinese tongue sole () is widely cultured in the coastal region of East Asia and has excellent economic value. However, the high albino rate of the breeding population has caused a significant loss to the aquacultureindustry. To study the molecular mechanism of albinism, the present study used an albino Chinese tongue sole family to construct three simple sequence repeat (SSR) linkage groups, and draft a preliminary linkage map related to albinism. After albinism-related loci mapping, 18 albinism-related loci were detected under two models (containing 2407 genes) compared to the Chinese tongue sole genome. One of these loci, the(), which has been reported previously as an important gene regulating both eumelanin and phaeomelanin levels, was indicated to be the possible cause of albinism. Thirty-five Gene Ontology (GO) terms and 14 Kyoto Encyclopedia of Genes and Genome pathways were annotated via bioinformatic analyses. One GO term with protein tyrosine kinase activity, which contained 10 genes, was previously suggested to affect fish albinism. These results establish a foundation for further in-depth study of albinism in Chinese tongue sole.

albinism;; locus mapping; simple sequence repeat

1 Introduction

The Chinese tongue sole () is a classic flatfish species that is widely distributed along the coast of Northeast Asia, including China, Japan, and the Korean peninsula. Due to its high nutritive value and tame habits, the Chinese tongue sole has become one of the most important flatfish species after decades of popularization(Niu, 2007). Efforts have been focused on various aspects to facilitate Chinese tongue sole breeding, including the development of sex-specific markers (Chen, 2007), construction of a genetic map (Song, 2012), genome sequencing (Chen, 2014), epigenetics research (Shao, 2014), sex-related genes study (Li, 2016), and geneediting techniques development (Cui, 2017). This progress has provided the required molecular basis for uncovering complex traits, as well as the necessary technical support to improve traits.

Albinism is a malpigmentation phenomenon that oc- curs when small doses of melanin are deposited in the eyes, skin, and hair, and is caused by a metabolic disturbance. Albino individuals can be found among mammals, birds, reptiles, and fish. Due to the lack of a protective color and the poor health, albino animals rarely survive in nature. Albinism is a complex trait, and only one type of pigment cell (melanophore) has been reported to be related to albinismin mammals (Farabee, 1903), whereas six different types of pigment cells are found in fish (Fujii, 2000). In addition, various factors, such as nutrition (Yamamoto, 1992), the environment (Seikai, 2010), and physiology (Yoo, 2000) can also affect the degree of albinism. Although the mechanism of albinism has been well studied in mammals, research on albinism is only at the initial stages in fish, such as zebrafish (Koga, 1997) and medaka (Hyodotaguchi, 1997).

The Chinese tongue sole breeding industry has been limited by many factors, andalbinism is among the leading factors. Albino individuals have an abnormal appearance rhat lead to a limited economic value; thus, further elucidation of the albino mechanism is important to improve the productivity and quality in tongue sole aquaculture. In a previous study, marker assisted selection quantitative trait locus (QTL) mapping was used to detect albinism-related loci in the Chinese tongue sole and three loci were found on previous high density simple sequence repeat (SSR) linkage map, LG15, LG17, and LG21, respectively (Ma, 2006; Song, 2012; Liu, 2014). Based on these data, this study reconstructed the three above-mentioned LGs and provided more accurate mapping of the albinism-related loci within the Chinese tongue sole genome. Furthermore, the Gene Ontology (GO) enrichment technique was used to search for genes involved in albinism, and the results will be applicable to future breeding efforts.

2 Materials and Methods

2.1 Family Establishment

In April 2012, 40 full-sib Chinese tongue sole families were chosen at the Haiyang Yellow Sea Fisheries Co. (Yantai, China). The paternal fish were selected from a wild-derived population and the maternal fish were selected from a cultured population. Some of the offspring showed varying degrees of albinism. Unlike normal individuals, albino offspring show a wide range of muciparous white skin without scales. After 16 months of culture, 233 individuals from this family were measured to calculate both body length and weight (average length, 15.91cm±2.03cm; average weight, 22.45g±8.81g) Their phe- notypes were photographed and saved as dorsal images. Whole genomic DNA was isolated from the fins of the parental fish and 94 random offspring using a tissue DNA kit (Omega-Biotek, Winooski, VT, USA).

2.2 Phenotyping

Two phenotype models were used for albinism-related loci mapping (model 1, albinism rate; model 2, albinism/ normal). Ninety-four individuals were randomly chosen from the 233 offspring, and 83 had albinism at different levels, resulting in a percentage of 86.27%, which was consistent with the family albino proportion (201/233, 88.30%). Dorsal image photographs were used to calculate the albino rate in model 1. The photographs were divided into squares, and the squares with normal and albino skin were designated S1 and S2, respectively. The albinism rate was calculated as a percentage: S2/(S1+S2), and the albino rate of normal individuals was metered to 0. All albino samples were metered to 1, and normal individuals were metered to 0 in model 2.

2.3 Genotyping

Using the genomic DNA of 94 selected offspring and their parents, all 126 SSR markers in the above-men- tioned three LGs (LG15, 17, and 21) were restructured based on a high density SSR linkage map (Song, 2012). A polymerase chain reaction (PCR) analysis was performed, and the products were examined by 6% polyacrylamide gel electrophoresis (Wang, 2014).

2.4 Linkage Group Structure

The LGs were structuredJoinmap 4.0 with the cross pollinator model,which considers F1 outbreeding population data containing various genotypic configurations (Van, 2006). Pairwise analyses were performed to sort markers into LGs at a minimum logarithm of the odds (LOD) score of 3.0. Linkage distances were estimated for each LG using the Kosambi mapping function.

2.5 Albinism-Related Loci Mapping

Albinism-related loci mapping was conducted using WinQTL Cart 2.5 software and the composite interval mapping (CIM) method (Wang, 2007). The CIM analysis was conducted using model 6 with four parameters for forward and backward stepwise regression, a 10- cM window size, five control markers, and a step size of 1-cM. The location of each QTL was determined according to its LOD peak location and the surrounding region. Furthermore, the correlations between phenotype and every single marker were analyzed using the single marker analysis method with WinQTL Cart 2.5. Loci were trans- formed into the physical area by scanning the whole genome of the Chinese tongue sole based on the nearest boundary markers at both ends of the loci.

2.6 Albinism-Related Gene Prediction

All genes in the loci were used to detect the albinism- related genes via bioinformatics methods. GO terms were predictedDAVID (https://david.ncifcrf.gov/). DAVID and the Kyoto Encyclopedia of Genes and Genomes (KEGG, http://www.kegg.jp/) were used to detect the can- didate pathway related to albinism.

3 Results

3.1 Linkage Groups

LG15, LG17, and LG21, as well as their male and female LGs, were structured (Figs.1–3). In general, the marker that constituted the LG ranged from 15 to 26, the observed length ranged from 69.2 to 139.2cM, and the average interval of a single linkage remained <7cM, all of which can provide a reliable basis for loci mapping (Table 1).

3.2 Albinism-Related Loci

Eighteen albinism-related loci (15 from model 1 and three from model 2) were detected in nine LGs (Table 2 and Figs.1–3). The interval size of 18 loci ranged from 0.2 to 7.2cM (most of them were <3cM), which was relatively accurate (Figs.1–3). All 70 markers that had been mapped on the LGs in this study were followed by single marker analysis with the phenotype from the two different models. Three markers were significantly corre- lated with albinism (Table 3). All loci were mapped on the Chinese tongue sole genome (Genbank accession number: PRJNA73987) using the physicallocation of the boundary markers. After merging intervals from male and female LGs, 2407 genes were detected at albinism-related loci.

Fig.1 Albinism-related loci on the consensus linkage groups. (A) Consensus linkage groups and the location of albinism- related loci. (B) Logarithm of the odds (LOD) value of the albinism-related loci.

Fig.2 Albinism-related loci on the female linkage groups. (A) Female linkage groups and the location of albinism-related loci. (B) Logarithm of the odds (LOD) value of the albinism-related loci.

Fig.3 Albinism-related loci on the male linkage groups. (A) Male linkage groups and the location of the albinism-related loci. (B) Logarithm of the odds (LOD) value of the albinism-related loci.

Table 1 Linkage group information

Table 2 Parameter values for the loci and estimation of the genetic effects

Table 3 Results of the single marker analysis

Notes: *<0.05, **<0.01.

3.3 GO and KEGG Enrichment Analysis

After GO enrichment, 35 albinism-related terms, containing 880 genes in total, were found. Most of them belonged to basic biological processes, molecular functions, and energy metabolism (Table 4). The term encoding protein tyrosine kinase activity contained 10 genes in the mapped loci and was related to tyrosine, which has been reported as the key element of albinism. A pathway en- richment analysis helped to further understand the genes’ biological functions. In this study, 139 of 2407 genes in mapped loci were annotated to 14 KEGG signaling pathways (Table 5).

Table 4 GO term enriched via DAVID

Table 5 Pathways related to albinism

4 Discussion

4.1 Phenotype Selection

In this study, we employed two models to represent the albino level of a mapped population, in which models 1 and 2 considered quantitative and qualitative traits, respectively. WinQTL Cart 2.5 was used as the QTL mapping software to detect albinism-related loci. A previous study reported that using a qualitative trait as a phenotype for QTL mapping was a feasible method;., Song(2012) mapped a sex-related locus on the sex chromosome, which led to a more reliable method. Wang(2014) mapped a coincidentresistance locus in Japanese flounder with two types of phenotypes, supporting the viability of this method. In the present study, albinism-related loci were detected in LG17 using model 1 and model 2, and sharing the same boundary mar- kers makes these loci credible.

4.2 Albinism-Related Loci

In a previous study, a preliminary SSR linkage map was constructed and albinism-related loci of the Chinese tongue sole were mapped on LG15, LG17, and LG21 (Liu, 2014). Although low genomic coverage limited its application for albino research, the map provided a foundation for a subsequent study. In this study, we reconstructed three LGs in a more efficient and economical way and mapped new loci on these LGs. The loci were relatively accurate and exhibited more than 85% genomic coverage, which made it possible to transform the loci to the genome and explore related genes more accurately.

4.3 Albinism-Related Genes

Because 2407 genes were located in the albinism-re- lated loci, GO and KEGG analyses were further used to narrow the list of genes, and more reliable candidates were found. Based on these 2407 genes and the inbuilt model species background (using the online bioinformation database DAVID), 35 GO terms were enriched and possibly involved in the albino process. All GO terms belonged to basic biological processes, molecular function, and energy metabolism. As albinism is a complex biological process, we propose that the basic biological function may play a role in the process. Consistent with this hypothesis, a study about the Japanese flounder albinism transcriptome also obtained GO terms regarding gly- cometabolism (Wang, 2017). A GO term for protein tyrosine kinase activity was identified in this study. As tyrosine is the most important amino acid in mammalian albinism, this term may play an important role in fish albinism (Bilandžija, 2013). Moreover, some qRT- PCR-verifiedupregulated genes in the Japanese flounder albinism transcriptome, such as()and(), were also contained in our albinism-related loci (Wang, 2017). The albinism-related gene(), also known as L-dopachrome tautomerase (which has been intensively studied in humans and reported in a study on the Japanese flounder albinism transcriptome), has also been found on albinism loci (Fang., 2001; Wang, 2017). MAM and LDL-receptor classes, a domain-containing protein (), and a G-protein coupled receptor 143 () cause oculocutaneous albinism and ocular albinism, respectively, in humans, and they have also been found in albinism-related loci (Gronskov, 2013; Bassi, 1995). All genes detected in this study provide potential candidate genes for future albinism studies on the Chinese tongue sole.

In conclusion, we mapped albinism-related loci based on three reconstructed LGs in Chinese tongue sole. After a comparison with the genomic data, 2407 genes were identified, and a GO analysis revealed that 880 genes were annotated in 35 GO terms. KEGG enrichment distributed 135 genes into 14 pathways. Five genes that have been previously reported to cause various forms of albinism were found at albinism-related loci. These data provide a preliminary understanding for the molecular basis of albinism. However, it remains difficult to find a major gene for fish albinism among abundant genes due to the relatively low density of the SSR LGs. A high density single nucleotide polymorphisms (SNP) linkage map would be an ideal option to improve the studies in this field.

Acknowledgements

This study was financially supported by the National Natural Science Foundation of China (Nos. 31570078, 31472273), the China Agriculture Research System (No. CARS-47-G03), and the Taishan Scholar Climbing Program of Shandong Province, China.

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