| Gymnocypris eckloni Herzenstein is a representative species of the highly specialized schizothoracine fish,which is widely distributed in the upper reaches of the Yellow River and is well adapted to the Qinghai-Tibetan plateau(QTP)aqueous environment.G.eckloni occupies an important position in the freshwater ecosystem of the plateau.The third-generation sequencing technology Pac Bio SMRT combined with Hi-C technology was used to investigate the chromosome-level genome assembly and the genome structure of embryonic and adult hemoglobin for G.eckloni.The expression and location of hemoglobin genes at different stages of embryonic development was determined by using whole embryo in situ hybridization and q RTPCR technology.The purpose of this study was to clarify the members and structural characteristics of the hemoglobin family of G.eckloni,and to explore the expression and regulation patterns of hemoglobin in the early developmental stages of G.eckloni,which will help for further research on schizothoracine fish evolution and hemoglobin switching.The main findings are as follows:(1)The chromosome-level genome of G.eckloni was assembled into 918.68 Mb sequences using Pac Bio long-read and Illumina short-read sequences,including 711 scaffolds with a contig N50 length of 4.19 Mb and a scaffold N50 length of 43.54 Mb.The assembled sequences were further anchored and orientated onto 23 pseudochromosomes.In total,24,430 protein-coding genes were predicted of which 23,157 genes were annotated,accounting for ing 94.80% of the total predicted protein-coding genes.(2)To examine G.eckloni evolution,we conducted a comparative genomics analysis using the G.eckloni genome obtained from this study and 13 other vertebrate genomes.A total of 24,619 gene families were identified among the 14 species,of which 27,39 core gene families were shared by all 14 species,856 species-specific gene families identified in G.eckloni.Analysis of the expansion and contraction of the gene families revealed that there were 464 expanded and 743 contracted gene families in G.eckloni when compared to its MRCA.The expanded and contracted gene families played important roles in the adaptation of G.eckloni to the plateau water environment.The phylogenetic analysis showed that G.eckloni was most closely related to C.carpio with an estimated divergence time of ～34.8 Mya,which coincides with the first and second tectonic uplifts of the QTP.We also found that G.eckloni underwent an additional whole-genome duplication(WGD)event(4R)at ～14.2 Ma,which had a significant effect on the evolution of chromosomes in G.eckloni.(3)The members of the embryo and adult hemoglobin gene repertoire were determined based on the genome data of G.eckloni and CDS sequences of zebrafish embryo globin genes(hbae1,hbae3,hbae4,hbae5,hbbe1,hbbe2,hbbe3)and adult globin genes(hbaa1,hbaa2,hbba1,hbba2),which have been published in the NCBI.The hemoglobin gene members of G.eckloni contains seven embryonic α-globin genes hbae1.1,hbae1.2,hbae1.4,hbae1.5,hbae1.6,hbae4 and hbae5;six embryonicβ-globin genes hbbe1.1,hbbe1.2,hbbe1.4,hbbe1.5,hbbe1.6 and hbbe3;two adult α-globin genes hbaa1 and hbaa2;two adult β-globin genes hbba1 and hbba2,and two pseudogenes hbae1.3 and hbbe1.3;it is preliminarily speculated that the hbae3 and hbbe2 genes were lost in the evolutionary process of adaptation to the plateau environment.(4)The embryo hemoglobin genes of G.eckloni includes three α-globin genes hbae1,hbae4,and hbae5.The full-length CDS sequences are 432 bp,426 bp,and 432 bp,respectively,encoding 143,141,and 143 amino acids;two β-globin genes hbbe1 and hbbe3.The CDS sequences are 444 bp and 441 bp in full length,encoding 147 and 146 amino acids,respectively.The adult hemoglobin gene of G.eckloni includes two α-globin genes: hbaa1 and hbaa2,the CDS sequences are 432 bp and 432 bp in full length,both encoding 143 amino acids;two β-globin genes,hbba1,hbba2,The CDS sequences were 447 bp and 444 bp,encoding 148 and 147 amino acids,respectively.(5)The results of whole embryo in situ hybridization and q RT-PCR showed that the expression levels of hbae1,hbbe1 and hbbe3 genes increased rapidly at 120 hpf of embryonic development,and the expression of hbae1 and hbbe1 genes increased sharply around 216 hpf after membrane rupture,and the high expression continued to432 hpf.The expression level of hbbe3 gene peaked at the membrane rupture stage,and decreased after the membrane rupture,but the hybridization signal could still be observed.The expression of hbae4 and hbae5 genes are low and there is no hybridization signal,so it is speculated that hbae4 and hbae5 genes may not play biological functions or been weakened during the embryonic development of G.eckloni.Thus,G.eckloni has unique characteristic of hemoglobin switching. |
