Mitochondrial DNA control region analysis of three ethnic populations in lower Northern part of Thailand

Abstract

The lower northern part of Thailand contains various genetically diverse ethnic populations. The sequences of the mitochondrial DNA hypervariable region were studied in three ethnic populations inhabiting Phitsanulok Province. One hundred and nine nucleotide sequences - 53, 29, and 27 from Hmongs (Hill tribe), Lao Songs, and Thai-Siams, respectively - were collected. The haplotypes were generated from 1130 nucleotides of the entire control region. Eighty-six haplotypes were found in the three ethnic populations, and no shared haplotypes were found between populations. Point heteroplasmy was noted at position 311 (C→Y). Haplotypes with ACAC-insertion at position 512 were observed in immigrant individuals from the Lao Song population. The Thai-Siam population showed higher genetic diversity than the other populations. The Hmong and Lao Song populations showed less genetic diversity than those living in their original area. The neutrality testing suggested that the result might be influenced by genetic drift.

Introduction

The population of Thailand is an admixture of various races and cultures. Many populations from the neighboring countries migrated into this region by forces of war or political issues; Thailand was called “Siam” until the name was changed to Thailand in 1949 A.D. (Keyes, 2002). The populations are different in physical characteristics, languages, religions, modes of living, and genetic structures. Many ethnic populations were distributed over the region, i.e., the lower northern part of Thailand, especially Phitsanulok Province (Satapanawattana et al., 2011). The native Thai (Thai-Siam) who spoke central-Thai dialect with different accents were identified and their geographical areas were mapped, according to linguistic characterization (Ngourungsi et al., 1976). Moreover, Lao Song (Thai Song Dam) and Hmong are examples of populations who migrated to Thailand a long time ago and inhabited Phitsanulok Province (Goldstein and Goldstein, 1986). Lao Song, the Tai- Kadai speaking population, is one of the minority groups living in the area near the border of Southern China, Laos, and Vietnam for more than 200 years (Schliesinger, 2001). They migrated to Thailand in the early Rattanakosin period (1778-1779 A.D.). The first wave of Lao Song colony came to the central part of Thailand - Phetchaburi, Suphan Buri, and Nakhon Prathom provinces. They then traveled upwards to Phitsanulok Province in the lower northern part of Thailand during the 1890s (Srising, 1976). Hmong (a hill tribe) is one of the Hmong- Mian ethnicities once inhabiting China. During the Indo-China conflict, they were forced to abandon their land and move to the northern part of Laos and Vietnam, where they were affected by the war going on at the time. Hmong then continued their migration to several areas in the northern part of Thailand, and some part of the population settled in Phitsanulok Province (Schliesinger, 2000).

Although languages and cultures are used as key components in categorizing ethnicity, they can be changed by environmental influence and surrounding populations. Several genetic markers, such as mitochondrial DNA (mtDNA), were then used to study the genetic structure of populations (Kivisild, 2015). The human mtDNA is an extra-chromosomal DNA. It contains 16,569 base pairs (bp), which encode 37 genes, 2 ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA) genes, and 13 polypeptide genes (Anderson et al., 1981). All are responsible for mitochondrial respiration (Asin-Cayuela and Gustafsson, 2007; Mercer et al., 2011). The genome of mtDNA is widely studied in the medicinal field because more than 250 mutations in mtDNA were identified as important causes of various diseases due to defects in oxidative phosphorylation (Tuppen et al., 2010). Several major properties of mtDNA, rather than nuclear DNA, such as maternal inheritance, high copy number, non-recombination, and higher mutation rate, are the reasons for using mtDNA as a favorable tool in evolution biology (Behar et al., 2008), anthropology (Knight et al., 2003), population history (Underhill and Kivisild, 2007), and forensic science (Irwin et al., 2011; Turchi et al., 2016). There are three high polymorphic segments called hypervariable regions (HVR) present in the non-coding control region, also called as displacement loop (D-loop). HVR-1 is at position 16,024-16,365 (342 bp), HVR-2 is at position 73-340 (268 bp), and HVR-3 is at position 438-574 (137 bp). A higher mutation rate is noted in the D-loop than in other regions in the mtDNA (Ingman et al., 2000), and can be used as a genetic marker to trace the maternal ancestors of populations that lived approximately 200,000 years ago (Behar et al., 2008). The HVRs are the key to reveal the genetic structure and relationship within and between populations (Zimmermann et al., 2009; Tillmar et al., 2010). HVR-1 contains more polymorphic sites than others do, and therefore, provides higher discrimination power for individual identification in forensic science applications (Meyer et al., 1999).

While there are various ethnicities in Thailand, information about the population structure of mtDNA is still limited. Few studies were conducted in northern and northeastern part of Thailand (Fucharoen et al., 2001; Zimmermann et al., 2009; Kutanan et al., 2014). They mostly focused on hill tribes - Tai-Kadai, Thai-Isan, and Mon-Khmer (Kutanan et al., 2011; Boonsoda et al., 2013; Kutanan et al., 2014). In this research, we focused on the genetic structure of the three populations - Thai Siam, Lao Song, and Hmong - inhabiting Phitsanulok Province in the lower northern part of Thailand. The HVRs of mtDNA were used as molecular markers to trace maternal ancestors of populations. The genetic diversity and relationship within and between populations were also investigated. The genetic history and evolutionary forces which affected the population structures were discussed.

Materials and Methods

Sample collection

After the protocol was approved by Naresuan University Human Ethical Committee (COA No. 211/2015), the ethnicity of populations was explored. The dialect, culture, and population history were used to categorize the ethnicity. The volunteers were explained about the details of research and signed consent form was obtained. The family and migration history were obtained via questionnaires and personal interviews. A translator was used if the volunteers could not clearly understand our language (in case of Hmong). Three milliliters blood samples were collected from 109 unrelated individuals. They were 53 Hmongs from Hmong village in Nakhon Thai district (Lat. 16.8124112, Long. 100.9695965), 29 Lao Songs from Bang Rakam district (Lat.16.6367235, Long.100.1505811), and 27 Thai-Siams from Phrom Phiram district (Lat.17.1669996, Long. 100.0945124) (Figure 1). The collection process and data storage were done according to the human ethical protocols.

DNA analysis

Genomic DNA was extracted using PureLinkTM Genomic DNA kit (Invitrogen; Thermo Fisher Scientific, M.I., U.S.A.). The entire sequences of D-loop HVR of mtDNA were amplified using primers F15878 (5'-AAA TGG GCC TGT CCT TGT AG-3') and R649 (5'-TTT GTT TAT GGG GTG ATG TGA-3') (Brandstätter et al., 2004). The PCR mixture consisted of 2 μL DNA template, 1X OnePCRTM (GeneDirex; Keelung, Taiwan), and 200 μM each primers and the reaction was carried out in a 50-μL total volume. PCR was performed using the following conditions: 95°C for 10 min; 36 cycles of 94°C for 1 min, 56°C for 1 min, and 72°C for 2 min; and 72°C for 5 min. The PCR products were purified by PureLink® PCR Purification kit (Invitrogen) and sequenced by BigDye® Terminator v3.1 cycle sequencing kit (1st BASE, Singapore). Series of 8 overlapping PCR-amplified primers set-I - F15878, R649, F16190 (5'-CCC CAT GCT TAC AAG CAA GT-3'), R16175 (5'-TGG ATT GGG TTT TTA TGT A-3'), F16450 (5'-GCT CCG GGC CCA TAA CAC TTG-3'), R484 (5'-TGA GAT TAG TAG TAT GGG AG-3'), R285 (5'-GTT ATG ATG TCT GTG TGG AA-3'), and F314 (5'-CCG CTT CTG GCC ACA GCA CT-3') - were used for the determination of D-loop hypervariable sequences. In case of mutation or polymorphisms at the primer binding site or incomplete sequence, the primer F15971 (5'-TTA ACT CCA CCA TTA GCA CC-3') and R599 (5'-TTG AGG AGG TAA GCT ACA TA-3') were used. The PCR mixture consisting of 2 μL DNA template, 1X OnePCRTM (GeneDirex), and 200 μM each primers were carried out in a 50-μL total volume. PCR was performed using the following conditions: 95°C for 10 min; 36 cycles of 94°C for 30 s, 56°C for 30 s, and 72°C for 1 min; and 72°C for 5 min. The overlapping primer set-II - F15971, R599, R16410 (5'-GAG GAT GGT GGT CAA GGG A-3'), F34 (5'- GGG AGC TCT CCA TGC ATT TGG TA-3'), R599 (5'-TTG AGG AGG TAA GCT ACA TA-3'), and F361 (5'-ACA AAG AAC CCT AAC ACC AGC-3') - was used (Irwin et al., 2007) (Figure 2). The generated sequences of each sample were assembled using the MEGA7 software (Kumar et al., 2016). To ensure the quality of sequences, the consensus sequences were generated twice and both of consensus sequences were compared to generate the final sequences. The nucleotide positions of the mtDNA control region were numbered according to the revised Cambridge Reference Sequence (rCRS) (Andrews et al., 1999).

Statistical analysis

The mtDNA control region sequences from position 16,024-16,569 and 1-576 were analyzed. MEGA7 (Tamura et al., 2013) was used to identify variable sites (v), parsimonyinformative sites (pi), and singleton (s). The percentage of v, pi, and s was calculated by number of sites divided by range of nucleotide in each regions. The percentage of mutations - transitions (ts), transversions (tv), insertions-deletions (in/del) - were calculated by number of mutations divided by number of variable sites in each region. DnaSP ver.510.1 software (Librado and Rozas, 2009) was used to calculate the number of haplotype, haplotype diversity (H_d), and nucleotide diversity (π_i). Neutrality estimator, Fu’s Fs (Fu, 1997) and Tajima’s D (Tajima, 1989), and genetic distance within and between groups were calculated using the number of nucleotide diversity.

Results and Discussion

Variation in mtDNA control regions

One hundred and nine sequences from unrelated individuals were aligned and compared with rCRS reference sequence. The D-loop control region from position 16,024- 16,569 and from 1-576 (1130 nucleotides) was analyzed. One hundred and sixty-one variation sites (14.25%) were found (Table 1). Ninety-six parsimony-informative sites (8.5%) and 61 singletons (5.40%) were noted. The HVR-1 contained the highest number of variable and parsimony-informative sites. One hundred and sixty-two mutational sites were counted, consisting of 137 transitions and 26 transversions. The combination of transitions and transversions were found at 7 sites. This result indicated that transitions were common in the entire control region, but transversions were more frequent in HVR-3 than in other regions. The ratio of the transitions to transversions was approximately 5:1. However, the ratio of the transitions to transversions in HVR-1 was 9:1, which was lower than that reported in a previous report on Thai population (12:1) (Sangthong et al., 2015). This ratio revealed the mutation that is accumulated in isolated populations.

Table 1: Distribution of variable sites and mutations in HVR of mtDNA control regions in the three ethnic populations in Phitsanulok Province (N = 109).

A total of 1227 mutations were observed in this analysis, which contained 1108 nucleotide substitutions, of which most were transitions (Table 2). The T→C was the most frequent transition followed by A→G. Only 10.65% transversions were observed, and most of them were A→C followed by C→A. Four insertion/deletion (in/del) positions were found at position 249 (A-deletion), 512 (ACAC-insertion), and 524-525 (CC-deletion).

Table 2: Observed mutations in 1130 nucleotides of mtDNA control region in the studied populations (N = 109).

The poly-C (or C-stretch) was found around positions 16,184-16,193 and 303-309. However, they were reported as common length heteroplasmy sites in several populations (Brandstätter et al., 2004; Ramos et al., 2013). In this report, the poly-C positions were then excluded during haplotyping and statistical analysis. Point heteroplasmy was found in only one sequence at position 311(C→Y) in the Lao Song population. However, 2 individuals in Thai-Siam population were characterized with position 311 heteroplasmy, but they were not included in this result because of low quality of DNA sequences due to length heteroplasmy at position 309. There were the heteroplasmy hotspots, mostly in HVR-1 (Irwin et al., 2009), but not at position 309. The ACAC-insertion at position 512 was also reported as length heteroplasmy (Ramos et al., 2013). In this study, there were 4 individuals with ACACinsertion, but no heteroplasmy was noted.

The frequency distribution of mutation at each variation site showed a similar pattern in all populations (Figure 3). Some positions, particularly HVR-1, showed higher frequency of mutation (>0.100) in all the three ethnic populations (Table S1). The positions at which mutational frequency was over 0.3 were 16,223; 489; 523; 524; and 525. The latter four positions were in HVR-3. The distinctively high mutational frequency positions were 73 (A→G) and 263 (A→G) with frequency 1.00 and 0.991, respectively. A previous study reported that position 263A of rCRS was a rare polymorphism (Andrews et al., 1999), but the data for position 73 is still lacking.

Haplotype analysis

Within the three ethnic populations, 86 haplotypes were found, out of which 73 were unique (Table 3 and Table S2). The Thai-Siam population showed the highest percentage of unique haplotypes (85.19%). No shared haplotype was found between populations. It might indicate that each population had different maternal genetic structure. In Hmong population, 9 shared haplotypes were found with high frequencies (Table 4). The obviously high-frequency haplotypes in Hmong are Haplotype 17 (0.094) and 34 (0.075). This result indicated the close relationship of maternal lineage in Hmong population. Two shared haplotypes were found in Lao Song and Thai-Siam populations. The overall highest frequency haplotype in populations was Haplotype 37, found in the Lao Song population. This haplotype contained ACACinsertion at position 512 in HVR-3. All 4 individuals who had Haplotype 37 had maternal migration history from Nakhon Prathom Province in the central part of Thailand. This insertion was reported as heteroplasmy; as mentioned before, several reports suggested that numerous heteroplasmy sites might be eliminated from populations by genetic drift. However, this result suggested that the ACAC-insertion might be a unique pattern found in Lao Song population and might be capable of identifying this population.

Table 3. Numbers of haplotypes, as well as unique and shared haplotypes, observed in the three ethnic populations.

Table 4: Frequencies of haplotypes in the three ethnic populations. Number of samples of Hmong, Lao Song, and Thai-Siam populations were 53, 29, and 27, respectively.

The diversity parameters of mtDNA control region haplotype in the three ethnic populations are presented in Table 5. The insertion and deletion sites were ignored (Tillmar et al., 2010; Jankova-Ajanovska et al., 2014). There were 1119 positions in the final dataset. The average number of nucleotide differences (k) was approximately 13. The Thai-Siam population showed the highest haplotype diversity (H_d). The nucleotide diversity (π_i) varied from 0.01130 to 0.01174.

Table 5: Diversity and neutrality testing parameters of mtDNA control region haplotypes in the three ethnic populations.

Several lines of evidence indicated decrease in genetic diversity in migrated populations. A previous report on the Hmong population in China, the original habitat of Hmong, revealed that the H_d varied from 0.937 to 1.000, which was consistent with the Hmong population H_d observed in this study. Meanwhile, π_i of Hmong population in this report was relatively low when compared to that in a similar report, which varied from 0.00174 to 0.00218 (Wen et al., 2005). Lao Song population in this study revealed lower percentage of unique haplotype as compared to that in Suphan Buri (92.0%, N = 25), where their first wave colonized (Fucharoen et al., 2001). The evidence of migration of these 2 populations resulted in a decrease in genetic diversity by founder effect. Additionally, there were higher values of genetic diversity in the Thai-Siam population who had no history of migration.

The Tajima’s D and Fu’s Fs were estimated to prove the null hypothesis of neutrality of genetic effect. The negative values of both parameters in all populations suggested that they were evolved without selective effect and growth in constant population size. The evolution of population was influenced by the founder effect of population expansion.

Most of the previous reports revealed only HVR-1 data because of the forensic tradition of mtDNA control region study. The HVR-1 H_d and π_i of the three ethnic populations were calculated by the sequences from position 16,024-16,365. The HVR-1 H_d in Hmong, Lao Song, and Thai-Siam populations was 0.957, 0.975, and 0.989, respectively. The HVR- 1 π_i in Hmong, Lao Song, and Thai-Siam populations was 0.02365, 0.02112, and 0.02110, respectively. Several studies on several populations in Thailand reported the range of the HVR- 1 H_d, which varied from 0.931 to 1.000, and that of π_i varied from 0.0169 to 0.0235 (Fucharoen et al., 2001; Lertrit et al., 2008; Bodner et al., 2011; Boonsoda et al., 2013; Kutanan et al., 2014). These values indicated that the genetic diversity of the three ethnic populations in this study were relatively high. Moreover, the genetic distance, referred to as the differentiation of genetic structure between populations, between the three populations (Table 6) was relatively high as compared to that revealed in previous reports (Fucharoen et al., 2001; Kutanan et al., 2014). The previous study on Tai-Kadai populations - Yuan, Leu, and Yong in the northern part of Thailand - reported that the range of percentage of unique haplotypes was 26.3-67.7 and H_d range was 0.878-0.992 (N = 39-62). The negative value of Fs also suggested the demographic expansion of populations (Kampuansai et al., 2007).

Table 6: The genetic differentiation by nucleotide diversity between groups of the three ethnic populations.

Conclusion

The present study demonstrated the genetic structure and diversity of Thai-Siam, Hmong, and Lao Song populations living in Phitsanulok Province, Thailand. The haplotype study of mtDNA control region revealed no admixture between populations. The unique patterns of mutation in mtDNA control region were found at position 73 (A→G) and 512 (ACAC-insertion), which can be used as population markers. Thai-Siam population represented the highest diversity in all the three populations, while the genetic diversities of Hmong and Lao Song populations, who had migration history, decreased as compared to their original populations. These results confirmed the history of long-term isolation of Hmong and Lao Song populations.

Conflicts of interest

The authors declare no conflicts of interest.

Acknowledgments

Research supported by funding from the National Research Council of Thailand. The authors greatly appreciate the helpful comments and suggestions from anonymous reviewers. The authors would like to thank all individuals who were helpful in specimen collection.