This study determined the mtGenome of the Zhijin White Goose for the first time. The size of the mtGenome was 16 739 bp. The gene combination was consistent with the typical combination of 37 genes found in the mtGenome of other goose varieties (Lin et al., 2016a, Lin et al., 2016b, Liu, Zhou, Zhang, Luo and Xu, 2013, Ren et al., 2016). The (A + T) content of the mtGenome was 52.69%. The base content of the mtGenome in Zhijin White Goose showed AT preference, which might be affected by three evolutionary forces: mutation, selection, and genetic drift(Kokate et al., 2021).
Due to its unique chemical characteristics and evolutionary status, the RNA secondary structure is closely related to its function. Therefore, RNA secondary structures have been widely used in RNA function and systematic research, and research on molecular evolution and molecular classification has mainly used rRNA. This study used the Auto Traveler software of the RNAcentral website(The, 2019)based on the swan mitochondrial 12S rRNA (b. 16. m. C. melancoryphus) and the human mitochondrial 16S rRNA (mHS_LSU_3D) files to analyse the mitochondria of Zhijin White Goose. The secondary structure of 12S rRNA and 16S rRNA in the genome has been predicted to provide basic data for further research on the structure and function of Zhijin White Goose rRNA.
As an ancient and multifunctional molecule, tRNA contain traces of early life. The secondary structure of the tRNA gene has a splicing signal that can mark certain mtGenome polycistrons (Boore, 1999). The mitochondria of Zhijin White Goose have a standard number of 22 tRNAs, ranging in length from 66 bp to 74 bp. All can form a typical clover structure, except for tRNA-Ser(AGY) due to a missing D arm. Partial mismatches of tRNA genes in the mtGenome can restore gene function through RNA self-shearing without causing amino acid transport obstacles. The mismatches of the Zhijin White Goose are all G-U mismatches, which conform to the G-U swing pairing principle, which is important for maintaining the tRNA secondary structure. Stability is also important. There is a 13-base functional DNA sequence motif (5'-TGGCAGAGCCCGG-3') on the D arm of human mitochondrial tRNA-Leu(UUR), which is the binding site of mitochondrial transcription termination factor (mTERF)(Fernandez-Silva et al., 2003) and is involved in regulating the transcription levels of two rRNA and H-chain downstream genes(Hyvarinen et al., 2007). There is an identical motif (5'- TGGCAGAGCCCGG − 3') on the D arm of the mitochondrial gene tRNA-Leu(UUR) in Zhijin White Goose., indicating that the motif is likely to have similar functions.
The secondary structure of the unique "goose hairpin" sequence(Eberhard et al., 2001) was composed of a stem consisting of seven C/G pairs and a loop containing the TCCC motif. Experiments showed that this motif and the H-chain termination are related(Dufresne et al., 1996). There was a highly similar sequence in the TAS region of Zhijin White Goose. According to the predicted stem-loop structure, the secondary structure was consistent with the characteristics of the "goose hairpin" sequence. This indicated that it might also be related to the termination of the H chain. The key sequences CSB-F, E, D, and C of the CD region of the D-loop region of the Zhijin White Goose are similar to the key sequences of the CSB region of other vertebrates and birds (Cho et al., 2009, Marshall and Baker, 1997, Quinn and Wilson, 1993, Sbisa et al., 1997), and CSB-F is a marker that distinguishes the TAS area from CD, the same as with other geese (Liu, Zhou, Zhang, Luo and Xu, 2013). In the CSB region of Zhijin White Goose, sequences corresponding to mammalian CSB-2 and CSB-3 were not recognised, research has shown that above two sequences among mammals are not universal(Saccone et al., 1991), which might be explained by species specificity (Sbisa, Tanzariello, Reyes, Pesole and Saccone, 1997).
Among the 13 PCGs of the mtGenome, CO3 and NAD4 had incomplete stop codons (T-). However, due to the presence of the PolyA tail at the 3'-end of the mRNA, the transcription process automatically completes the TAA stop codon, which does not affect transcription(Ojala et al., 1981). Codon preference refers to the unequal use of synonymous codons. The main difference between the synonymous codons is the third codon. In this study, the PCGs in Zhijin White Goose mtGenome had an AT preference, display according to RSCU calculation results, the PCGs did not have a clear AT or GC preference. This study used ENC to evaluate codon preference. Only three of out of the thirteen PCGs showed a significant codon preference. By drawing the ENC-GC3 standard curve, it was found that all PCG points were distributed below the standard curve, indicating that the PCG codon preference in Zhijin White Goose is mostly due to the influence of natural selection pressure, and artificial selection is still needed. Studies showed that codon usage preference is related to gene expression strength. Compared with the low-expression genes, the frequency of preference codons used by highly expressed genes had significantly different usage frequencies. Typically, they use a set of preferential synonymous codons. In addition, the preferentially used codons correspond to the most abundant tRNA, thereby improving the translation efficiency(Sabi and Tuller, 2014). In follow-up breeding studies, it is important to strengthen the selection and breeding of high-efficiency expression codons, promote translation efficiency, and increase gene expression to increase the expression of specific proteins and enhance the effect of molecular breeding.
Mitochondrial phylogenetic tree analysis showed that Zhijin White Goose, Swan goose and most Chinese geese belong to an evolutionary branch, in line with the notion that Chinese geese have evolved from swans(Wang et al., 2010). It also proves that the Zhijin White Goose is a local breed with a Chinese goose lineage. However, Sichuan White Goose with the GenBank accession number MK133022 is similar to geese outside China. This might be due to the integrated fragments of nuclear mitochondrial DNA. Mitochondrial DNA is first enclosed in the nucleus and then integrated into the nuclear chromosomes through non-homologous end joining after nuclear double-strand breaks(Hazkani-Covo et al., 2010), resulting in a much slower mutation rate than that of mtGenome. Therefore, this classification appears consistent with previous research results (Ren, Liang, Zhao and He, 2016).