Identification of germ cells in large yellow croaker (Larimichthys crocea) and yellow drum (Nibea albiflora) using RT-PCR and in situ hybridization analyses

Yanjie Yu; Yang Yang; Huan Ye; Lei Lu; Haidong Li; Zhijin Xu; Weiye Li; Xiaolong Yin; Dongdong Xu

doi:10.1016/j.gene.2023.147280

Identification of germ cells in large yellow croaker (Larimichthys crocea) and yellow drum (Nibea albiflora) using RT-PCR and in situ hybridization analyses

Gene. 2023 May 5:863:147280. doi: 10.1016/j.gene.2023.147280. Epub 2023 Feb 18.

Authors

Yanjie Yu¹, Yang Yang², Huan Ye³, Lei Lu¹, Haidong Li⁴, Zhijin Xu⁵, Weiye Li⁵, Xiaolong Yin⁵, Dongdong Xu⁶

Affiliations

¹ School of Fisheries, Zhejiang Ocean University, Zhoushan, China; Key Lab of Mariculture and Enhancement of Zhejiang Province, Zhejiang Marine Fisheries Research Institute, Zhoushan, China.
² Key Lab of Mariculture and Enhancement of Zhejiang Province, Zhejiang Marine Fisheries Research Institute, Zhoushan, China; Ocean and Fisheries Research Institute, Zhejiang Ocean University, Zhoushan, China.
³ Key Laboratory of Freshwater Biodiversity Conservation, Ministry of Agriculture of China, Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan, China.
⁴ School of Fisheries, Zhejiang Ocean University, Zhoushan, China; Key Lab of Mariculture and Enhancement of Zhejiang Province, Zhejiang Marine Fisheries Research Institute, Zhoushan, China; Ocean and Fisheries Research Institute, Zhejiang Ocean University, Zhoushan, China.
⁵ Zhoushan Fisheries Research Institute, Zhoushan, China.
⁶ Key Lab of Mariculture and Enhancement of Zhejiang Province, Zhejiang Marine Fisheries Research Institute, Zhoushan, China; Ocean and Fisheries Research Institute, Zhejiang Ocean University, Zhoushan, China. Electronic address: xudong0580@163.com.

PMID: 36804002
DOI: 10.1016/j.gene.2023.147280

Abstract

Ocean-caught large yellow croaker (Larimichthys crocea) represents an important germplasm resource for the breeding of this species; however, these fish tend to show poor survival in captivity and would be unsuitable breeding purposes. As an alternative to the use of wild-caught croakers, germ cell transplantation has been proposed using the L. crocea specimens as donors and yellow drum (Nibea albiflora) as recipients. In this regard, the identification of L. crocea and N. albiflora germ cells is an essential prerequisite for establishing a germ cell transplantation protocol for these fish. In this study, we cloned the 3' untranslated regions (UTR) of the vasa, dnd, and nanos2 genes in N. albiflora using the rapid amplification of cDNA ends (RACE) method and then aligned and analyzed the sequences of the corresponding genes in L. crocea and N. albiflora. On the basis of gene sequence differences, we designed species-specific primers and probes for RT-PCR analysis and in situ hybridization. RT-PCR analysis revealed that these species-specific primers exclusively amplified DNA from gonads of the respective species, thus confirming that we had six specific primer pairs that could be used to distinguish the germ cells in L. crocea and N. albiflora. Using in situ hybridization analysis, we established that whereas Lcvasa and Nadnd probes showed high species specificity, the probes for Navasa and Lcdnd showed a less specificity. In situ hybridization using Lcvasa and Nadnd thus enabled us to visualize the germ cells in these two species. Using these species-specific primers and probes, we can reliably distinguish the germ cells of L. crocea and N. albiflora, thereby establishing an effective approach for the post-transplantation identification of germ cells when using L. crocea and N. albiflora as donors and recipients, respectively.

Keywords: Dnd; Germ cell transplantation; Nanos2; Species-specific primers; Species-specific probes; Vasa.

MeSH terms

Animals
Fish Proteins / genetics
Germ Cells*
Gonads
In Situ Hybridization
Perciformes* / genetics
Reverse Transcriptase Polymerase Chain Reaction

Substances

Fish Proteins