Construction of a High-Density Genetic Map and Identification of Quantitative Trait Loci for Nitrite Tolerance in the Pacific White Shrimp (Litopenaeus vannamei)

Abstract

Nitrite is a major environmental toxin in aquaculture systems that disrupts multiple physiological functions in aquatic animals. Although nitrite tolerance in shrimp is closely related to successful industrial production, few genetic studies of this trait are available. In this study, we constructed a high-density genetic map of Litopenaeus vannamei with 17,242 single nucleotide polymorphism markers spanning 6,828.06 centimorgans (cM), with an average distance of 0.4 cM between adjacent markers on 44 linkage groups (LGs). Using this genetic map, we identified two markers associated with nitrite tolerance. We then sequenced the transcriptomes of the most nitrite-tolerant and nitrite-sensitive individuals from each of four genetically distinct L. vannamei families (LV-I-4). We found 2,002, 1,983, 1,954, and 1,867 differentially expressed genes in families LV-1, LV-2, LV-3, and LV-4, respectively. By integrating QTL and transcriptomics analyses, we identified a candidate gene associated with nitrite tolerance. This gene was annotated as solute carrier family 26 member 6 (SLC26A6). RNA interference (RNAi) analysis demonstrated that SLC26A6 was critical for nitrite tolerance in L. vannamei. The present study increases our understanding of the molecular mechanisms underlying nitrite tolerance in shrimp and provides a basis for molecular-marker-assisted shrimp breeding.

Keywords: Litopenaeus vannamei; QTL; genetic map; nitrite tolerance; transcriptomic.

FIGURE 1

High-density linkage sex-average map for Litopenaeus vannamei, showing genetic distances among specific length amplified fragment (SLAF) markers (shown as black bars).

FIGURE 2

Quantitative trait loci (QTL) for nitrite tolerance in Litopenaeus vannamei, showing the logarithm of odds (LOD) values of the linkage groups. The gray line indicates the LOD threshold (3.00; P = 0.05).

FIGURE 3

The Gene Ontology (GO) terms overrepresented in the differentially expressed genes (DEGs) across four genetically distinct Litopenaeus vannamei families (LV-1, LV-2, LV-3, and LV-4).

FIGURE 4

The KEGG terms overrepresented in the in the DEGs across four genetically distinct Litopenaeus vannamei families (LV-1, LV-2, LV-3, and LV-4).

FIGURE 5

Expression of SLC26A6 from the transcriptomic analysis validated by qRT-PCR. Expression of SLC26A6 was detected in nitrite-sensitive and nitrite-tolerant shrimp from genetically distinct Litopenaeus vannamei families (LV-1, LV-2, LV-3, and LV-4). Data were normalized to 18s rRNA expression, and presented as a relative log2-fold change to validate the transcriptomic analysis results. Error bars show the standard deviation of three technical replicates.

FIGURE 6

qRT-PCR quantification of relative SLC26A6 expression in shrimp from Litopenaeus vannamei family LV-1 at 36, 48, and 72 h after injection with 0.9% normal saline (blue bars; buffer control), dsRNA-egfp (green bars; negative control), or dsRNA-SLC26A6 (orange bars; experimental group). L. vannamei 18S RNA was used as the internal reference gene. Asterisks (*) above bars indicate significant differences (P < 0.01) in relative gene expression as compared to both controls. Error bars show the standard deviation of three biological replicates.

FIGURE 7

Median lethal time of Litopenaeus vannamei after injection with 0.9% normal saline (buffer control), dsRNA-egfp (negative control), or dsRNA-SLC26A6 (experimental group). Asterisks (*) above bars indicate significant differences (P < 0.01) in median lethal time as compared to both controls. Error bars show the standard deviation of three biological replicates.