Background

Studying the genetic structure of population at the molecular level can not only help us understand the level of population genetic differentiation, but also be beneficial to genetic breeding and fine population genetic conservation. Compared with nuclear DNA, mitochondrial DNA (mtDNA) has a relatively simple base arrangement and stack, which reflects maternal genetic characteristics with high frequency of nucleotide transition or transversion and fast mutation rate during subculture. These unique features of mtDNA make it an important marker method widely used by many researchers in the study of fish population genetics (Oleinik et al., 2007; Teletchea, 2009). The base substitution of different regions in mtDNA genome shows different rules and characteristics. It is suitable for population genetic research of different objectives and levels. On mtDNA, D-loop is the region with the greatest variation in sequence and length of the entire mtDNA (Kocher and Lee, 1995), and the fastest-evolving region of the entire mtDNA genome, which is usually applicable for the detection and analysis of intraspecific genetic diversity among populations and the research reports on the analysis of interspecific genetic diversity (Guo et al., 2004; Jie et al., 2011). Cytochrome Cytb is a protein-coding gene, and its evolution rate is at the intermediate level compared with other regions of mtDNA. It is suitable for the detection of genetic diversity among populations and is a good method to analyze intraspecific and interspecific genetic variation at the DNA molecular level (Teletchea, 2009). However, there are few research reports on the differences and relations between the genetic variation information of Cytb and D-loop sequences, especially on the relationship between the genetic variation data obtained by the two techniques.

GIFT tilapia was introduced to China by Shanghai Fisheries University. After more than 10 years’ genetic improvement, it has become a new breeding strain with excellent production traits (Jie et al., 2011), which is of great significance to further genetic analysis of breeding population at molecular level. In this study, mtDNA Cytb and D-loop sequence variation information of GIFT tilapia breeding population was compared and analyzed in order to explore the relationship between the results of two kinds of mtDNA marker analysis, aiming to select different analysis methods for different research materials, to expand the horizontal comparison among the research results and to provide references in analysis technology, at the same time, to make the relevant research and analysis more comprehensive, and research conclusions more objective.

1 Results and Analysis

1.1 The ratio of genetic polymorphism within population based on Cytb and D-loop sequence analysis

This study was based on the ratio of genetic diversity within populations obtained by Cytb and D-loop sequence analysis, as shown in Table 1. According to the two methods, the ratio of the polymorphic loci ranged from 0.679,05 to 0.804,92 with an average of 0.776,74; the ratio of haplotype diversity ranged from 0.864,12 to 1.076,12 with an average of 0.919,45; the ratio of nucleotide diversity index ranged from 0.713,77 to 0.884,49 with an average of 0.769,77; and the ratio of average nucleotide difference ranged from 0.899,54 to 1.082,12 with an average of 0.936,19.

1.2 Base transition and transversion rate of Cytb and D-loop sequence

In this study, the comparison of the base transition rate and transversion rate in Cytb and D-loop sequence was shown in Table 2. The base transition rate of Cytb sequence ranged from 0.031,95 to 0.059,21, with an average of 0.042,67. The base transversion rate of Cytb sequence ranged from 0.002,82 to 0.005,48, with an average of 0.004,10. The base transition rate of D-loop sequence ranged from 0.027,72 to 0.047,67, with an average of 0.037,25. The base transversion rate of D-loop sequence ranged from 0.015,52 to 0.027,72, with an average of 0.022,84.

2 Discussion

In terms of inheritance pattern, mtDNA controls and affects mitochondrial function with non-Mendelian inheritance, which is inconsistent with nuclear genes. At the same time, the base mutation rate of mtDNA in fish is higher than that in nuclear DNA. There lacks effective repair mechanisms for mtDNA lesion. Specifically, the accumulation effects exist in some mtDNA mutations. The quantitative analysis of mtDNA polymorphism showed that the average difference of mtDNA base between the two unrelated individuals was 3% (Guo et al., 2004). Therefore, it is necessary to choose the appropriate mtDNA region to rigorously analyze the genetic variation characteristics of the population. There are a large number of research reports using Cytb, D-loop or other mtDNA gene regions to analyze (Gerlach and Musolf, 2000; Chen and Xiao, 2003; Yu et al., 2011). mtDNA has different evolutionary rates for different regions. The sequence genetic variation characteristics obtained based on different regions of mtDNA must be different. However, there are few research reports on the comparison and quantitative analysis of sequence variation characteristics obtained from different regions of mtDNA. Most of the current studies related to molecular marker remain in single marker analysis and lack horizontal analysis among different types of markers. To a large extent, it limits the referenced value of the results, so there is still a lot of work to be researched deeply in this field.

The base mutation of mtDNA sequence can be divided into two types: transition is the base substitution of the same kind, that is, the substitution processed by purine or pyrimidine between different bases of the same kind; transversion is the base substitution of different species, the substitution of a purine for a pyrimidine or vice versa, that is, the base substitution between purine and pyrimidine. In this study, Cytb transition rate averaged 0.042,67, and Cytb transversion rate averaged 0.004,10; D-loop transition rate averaged 0.037,25, and D-loop transversion rate averaged 0.022,84. The ratio of transition/transversion of Cytb region was about 10.41, and the ratio of transition/transversion of D-loop region was about 1.66. The transition rates were higher than the transversion rates. The conclusion was in agreement with other studies (Gerlach and Musolf, 2000; Zhao et al., 2010; Yang et al., 2012). The base substitution has different biological functions in different gene regions. Occurring in a gene region (such as Cytb) that encodes polypeptides, the codon information might be changed so that subsequent transcription, translation, etc. can be changed accordingly. A new amino acid structure may appear in organisms to replace the original amino acid in the corresponding position. The base substitution may also lead to the early emergence of termination codon in protein-encoding genes, and prematurely interrupt the synthesis of polypeptide chains. As a result of the base substitutions, the protein-encoding genes can not correctly form the original proteins, thus completely or partially lose some biological activities (Jia et al., 2011). In this study, the base transition rate and base transversion rate of Cytb/D-loop in different gene regions were about 1.15 and 0.18, respectively. The result was inferred to be related to the regulation of Cytb encoding protein and D-loop during mtDNA transcription and replication.

Different regions in DNA double-strand share different mutation frequencies and undertake different evolutionary pressures. Therefore, they have different evolutionary rates. This study also confirmed this conclusion. The variation rate of the D-loop region was about 5-10 times of that of the complete mtDNA molecule (Zheng et al., 2002). In this study, the results of the polymorphism ratio of Cytb/D-loop sequence in five breeding populations of Nile tilapia showed that the polymorphic loci ratio and nucleotide diversity index of Cytb and D-loop region were 0.776,74 and 0.769,77, respectively, while the haplotype diversity and average nucleotide difference ratio of Cytb/D-loop were slightly high, 0.919,45 and 0.936,19, respectively. The results and control regions were nucleotide fragments of uncoded polypeptide chains in mtDNA, without repair systems, and they were not affected by selection pressure. Therefore, more variations were accumulated (Li et al., 2011), which was related to the fairly slow evolution of the cytochrome b gene encoded protein. In general, the comparative analysis of the sequence variation information of Cytb and D-loop regions could be helpful to provide references for other similar studies to expand the scope of horizontal comparison and to better understand the results of single marker (Cytb/D-loop) analysis.

3 Materials and Methods

3.1 Experimental materials

Shanghai Ocean University introduced 5,000 Nile tilapia (base population, F0) from Philippines in 1994, and carried out breeding since 1996, producing a new generation every year. The experimental materials were selected by random sampling from 5 generation Nile tilapia breeding populations of F0 and F6~F9, 20 tilapia each population. The tail fins were cut, and 95% ethanol was used for preservation after separate numeration.

3.2 Genomic DNA extraction and PCR amplification

The DNA was extracted with the routine procedure of phenol-chloroform and the quality of extracted DNA was elementarily determined by agarose gel electrophoresis. The primer sequences of the control region referred to (Agnèse et al., 1997), and the base sequence were DL1: 5'-ACCCCTGGCTCCCAAAGC-3' and DH2: 5'-ATCTTAGCATCTTCAGTG-3'. The primers of cytochrome b gene referred to (Peng et al., 2005), and the base sequence were L14724:5'-GACTTGAAAAACCACCGTTG-3' and H15915: 5'-CTCTCTCCGGATTACAAGAC-3'. PCR amplification reaction system was 50 μL, containing 5 μL 10×Buffer, 10 mmol dNTP, 2 μL (10 pmol/μL) upstream primer and downstream primer, 200 ng template DNA, and 2.5 U Taq DNA polymerase. PCR amplified procedure: pre-degeneration at 94°C for 4 min; high-temperature denaturation at 94°C for 30 s, the template and primer were annealed at acclimated temperature for 30 s, the primer aggregation and extension at 72°C for 1 min. Each amplification program contained 35 cycles at 72°C for 10 min, stored at 4°C. The annealing temperature of control region was 50°C and the annealing temperature of cytochrome b gene was 56°C.

3.3 DNA sequencing

Amplification products were detected by 1% agarose gel electrophoresis. The well-amplified PCR products were selected to purify. Shanghai sangon Company conducted bidirectional sequencing by ABI377DNA automatic sequencer.

3.4 Data processing and analysis

The control region and cytochrome b gene sequence of Nile tilapia in GenBank were searched by BLAST (Altschul et al., 1997). The results were compared with the appropriate sequences of Nile tilapia determined by the experiment. The gene sequencing results were edited by Bioedit (Hall, 1999). The sequence was rearranged and homology was compared by CLUSTL W (Thompson et al., 1994), along with manual checking to proofread. The statistical analysis was conducted by DNASP (Rozas et al., 2003). The ratios of polymorphic loci, haplotype diversity, nucleotide diversity index and average nucleotide difference obtained by Cytb/D-loop method were calculated, respectively. The transition rate and transversion rate were calculated according to the occurrence of transition and transversion in the total number of bases in corresponding sequences.

Authors’ contributions

XXY, the executor of this study, finished experimental design, data analysis, thesis writing and revision, who was also responsible for one of the funding projects; LSF was the designer and leader of the other funding project. Both authors read and approved the final manuscript.

Acknowledgement

Thanks to Shanghai Ocean University who provided tilapia germplasm materials for this study.

References

Agnèse J.F., Adèpo-Gourène B., Abban E.K., and Fermon Y., 1997, Genetic differentiation among natural populations of the Nile tilapia Oreaochronis niloticus (Teleostei Cichlidae), Heredity, 79: 88-96