Genetic Diversity in the matK Gene of Dimocarpus longan Varieties in the Mekong Delta

MATERIALS AND METHODS

Study duration and location: The study was implemented in the Molecular Biology Laboratory of the Institute of Food and Biotechnology at Can Tho University from June, 2022 to November, 2022.

Plant sampling: Eleven different varieties of Dimocarpus longan were collected at the longan garden (Phong Nam commune, Ke Sach District, Soc Trang Province) and the longan cooperative (Thoi An ward, O Mon District, Can Tho City) in June, 2022. Dimocarpus longan cultivars collected in this study including “Thach kiet” longan, “Thanh” longan, “Idol” longan, “Long” longan, “Xuong” longan in Can Tho City, “Purple” longan, “Xuong com vang” longan, “Thach kiet” longan, “Long” longan, “Da bo” longan, “Xuong vang” longan in Soc Trang Province (Fig. 1a-k).

Amplification of DNA markers: The DNA extraction from leaves of longan fruits was performed according to the modified CTAB procedure. The obtained DNA was eluted with 50 μL of 0.1X TE solution. The solution containing DNA was stored in the freezer (-20°C) for the next procedure. The extracted DNA was treated by a Nanodrop 2000°C spectrophotometer. Then the quality of DNA was checked by 1% agarose electrophores.

To obtain the complete DNA sequence of the matK gene with a size of ~1500 bp, two pairs of primers (matK-4600/trnK-2R and tamF/tamR) were used. Which, tamF/tamR primer pair was designed in this study (Fig. 2). The tamF/tamR primer pair was synthesized at Phu Sa Biochem One Member Co., Ltd. (Vo Nguyen Giap Street, Phu Thu Ward, Cai Rang District, Can Tho City).

Each PCR reaction was performed in a volume of 50 μL containing 25 μL of ddH₂O, 20 μL of Mastermix, 3 μL of DNA template, 1 μL of forward primer (20 pmol μL^–1) and 1 μL of reverse primer (20 pmol μL^–1) (Table 1). The concentration of the DNA template was given in Table 2.

The PCR thermal cycles to amplify DNA barcodes was shown in Table 3. The quality of the PCR product was checked by 2% agarose electrophores to observe the specific bands. The selected PCR products sequenced their DNA sequences at The Institute of DNA Technology and Genetic Analysis-Genlab (Yen Hoa ward, Cau Giay District, Hanoi City).

Statistical analysis: The obtained DNA sequences were corrected by BioEdit 7.0 Software to check the peak of nucleotides. The sequences of PCR products after processing were paired by using the Denovo alignment tool. The matK sequences were analyzed by using BioEdit software, aligned with the ClustalW tool and compared on the GenBank by using the NCBI/BLAST tool integrated into the Geneious 7.0 analysis software¹⁴. The SNPs (Single nucleotide Polymorphisms) and amino acids (dN/dS) were calculated by using the MEGA7 software¹⁵. The access numbers of the matK gene having the different sequences were registered on the GenBank (NCBI).

The MEGA11 software was applied (The Molecular Evolution Genetics Analysis) with a bootstrap coefficient of 1000 to determine the distance between sequences when comparing nucleotide sequences in the matK gene of longan varieties. The phylogenetic tree was constructed by using the maximum-likelihood method in order to show the genetic relationship between different cultivars based on the sequences of the matK gene.

Fig. 1(a-k):

Some fruits and leaf morphology of 11 collected longan varieties, (a) “Thach kiet” longan (Can Tho), (b) “Thanh” longan (Can Tho), (c) “Idol” longan (Can Tho), (d) “Long” longan (Can Tho), (e) “Xuong” longan (Can Tho), (f) “Purple” longan (Soc Trang), (g) “Xuong com vang” longan (Soc Trang), (h) “Thach kiet” longan (Soc Trang), (i) “Long” longan (Soc Trang), (j) “Da bo” longan (Soc Trang) and (k)“Xuong vang” longan (Soc Trang)

Fig. 2:

Primer location diagram

RESULTS AND DISCUSSION

DNA barcode amplification: Electrophoresis results showed that the bands were approximately 1500 bp in size. Daniell et al.¹⁶ and Steane¹⁷ found that the length of the matK gene in the chloroplasts of plants, have ~1500 bp in size. Therefore, the amplified DNA band size of ~1500 bp was relatively consistent with the theory to conduct sequencing of the amplified gene region with two pairs of primers (matK-4600/trnK-2R and tamF/tamR). According to Fig. 3a-b, the PCR products after using matK4600/trnK-2R were ~1200 and ~1000 bp, respectively. Their sequences are continuously treated by BioEdit software to remove unclear nucleotides at two ends of each sequence. Then those sequences were applied to the Denovo alignment tool to form many consensuses which had the complete matK gene of ~1500 bp in size (Table 4). Therefore, the size of the matK gene region (~1500 bp) in this research was relatively consistent with the theory presented by many previous studies.

matK gene analysis: Through Fig. 4, the quality of the obtained matK gene sequences was checked by Bioedit software and showed that most of the sequences have clear fluorescence signals and nucleotides could be identified in all samples. However, at the two ends of the sequencing results, some unclear signals appeared. Therefore, to avoid leading to inaccurate sequencing results, segments of the sequence at both ends of the obtained sequences would be removed before aligning. The analysis results after aligning consensus sequences of the complete matK gene region were shown in Table 4.

The matK gene size of Dimocarpus longan cultivars ranged from 1524-1526 bp. Sugita et al.¹⁸ and Turmel et al.¹⁹ also introduced that the matK gene size was ~1500 bp. Thus, the figures in this study were appropriate to the above previous research. Some cultivars had the highest nucleotide positions including “Thanh” longan, “Long” longan, “Xuong” longan (Can Tho), “Purple” longan, “Xuong com vang” longan, “Long” longan, “Xuong vang” longan (Soc Trang) with 3 SNPs. The similarities ranged from 99-100% when comparing all sequences in this study with Dimocarpus longan-NC_037447.1 on NCBI. Lan et al.¹⁴ showed that the similarity level when studying the matK gene sequence at 31 different longan varieties in the Dimocarpus genus varies from 99-100% so there was a correlation between the results of this study and previous studies. Significantly, the matK gene of seven cultivars was updated on NCBI including OP819669, OP819670, OP819671, OP819672, OP819673, OP819674 and OP819676 (Table 5).

The dN/dS ratio, in which, dN is non-synonymous substitutions and dS is synonymous substitutions. The result of the dN/dS ratio was recorded as 1.09. Statistically, the dN/dS ratio was higher than 1.0 could be assumed that the change of nucleotide sequences lead to the change of amino acid sequences. According to the results of amino acid sequence analysis, two positions of amino acid variations at 47 and 132 were discovered after translation in “Thanh” longan, “Long” longan, “Xuong” longan (Can Tho), “Purple” longan, “Xuong com vang” longan, “Long” longan, “Xuong vang” longan (Soc Trang) (Fig. 5).

Fig. 3(a-b):

Electrophoresis results after amplifying matK gene, (a) Amplified by matK4600/trnK-2R and (b) Amplified by tamF/tamR L: Ladder 100 bp, (+): Positive control sample, 1: “Thach kiet” longan (Can Tho), 2: “Thanh” longan (Can Tho), 3: “Idol” longan (Can Tho), 4: “Long” longan (Can Tho), 5: “Xuong” longan (Can Tho), 6: “Purple” longan (Soc Trang), 7: “Xuong com vang” longan (Soc Trang), 8: “Thach kiet” longan (Soc Trang), 9: “Long” longan (Soc Trang), 10: “Da bo” longan (Soc Trang), 11: “Xuong vang” longan (Soc Trang) and (-): Negative control sample

Fig. 4:

A fragment of sequencing results for amplified product using matK-4600 primer in purple longan (Soc Trang)

Fig. 5:

Amino acid polymorphisms after translating the nucleotide sequences of the matK gene

Fig. 6:

Phylogenetic tree of Dimocarpus longan variations based on the matK gene by maximum-likelihood (ML) method Data at each branch showed the bootstrap values, Dimocarpus longan-NC_037447.1: Reference sequence, Acer negundo-NC_051957.1: Outgroup sequence and Xanthoceras sorbifolium-NC_037448.1: Outgroup sequence

The matK sequences in some other subjects previously studied on NCBI were used to combine the construction of the phylogenetic tree for this research. Specifically, the matK gene sequences in plants of Acer genus (Acer negundo-NC_051957.1), Xanthoceras genus (Xanthoceras sorbifolium-NC_037448.1) of the family Sapindaceae were selected as outgroup sequences.

By the method of maximum-likelihood (ML), the matK gene sequences of longan varieties in the Dimocarpus genus were grouped separately from cultivars in the Acer genus and Xanthoceras genus in the Sapindaceae family (Fig. 6). For eleven studied longan sequences, some longan varieties such as “Thach kiet” longan (Can Tho), “Idol” longan (Can Tho) and “Thach kiet” longan (Soc Trang) had the most correlation when they were located in the same clade with matK reference sequence on NCBI (Dimocarpus longan-NC_037447.1).

The above varieties were grouped with “Long” longan (Can Tho) and “Xuong com vang” longan (Soc Trang). “Xuong” longan (Can Tho) and “Xuong vang” longan (Soc Trang) belonged to the same group and had a relatively close relationship with the above longan varieties. Then the varieties of “Purple” longan (Soc Trang) and “Da bo” longan (Soc Trang) were grouped, while “Thanh” longan (Can Tho) and “Long” longan (Soc Trang) were analyzed that they had a more different genetic relationship. Thus, the results of the phylogenetic tree based on the nucleotide sequences of the matK gene had taxonomic significance to the species level in many different longan varieties.

Schmitz-Linneweber et al.²⁰ evaluated that the matK gene is one of the genes with a high evolutionary rate in the chloroplast genome. Therefore, this gene has been widely applied to genealogical studies at the species level. Yesson et al.²¹ carried out that the species recognition efficiency of the matK gene has also been evaluated in 528 species of the Cactaceae family, of which 75% were native succulents. This group of authors concluded that the matK gene sequencing was able to correctly identify 77% of the collected species, these studies have shown that the matK gene has been used to differentiate many species effectively. In this study, by the maximum-likelihood (ML) method, the phylogenetic tree was built with the phylogenetic branches and the bootstrap values recorded in all branches were higher than 75%.

The results of this study was reliable to conclude about phylogenetic resources based on the matK gene of longan varieties at the species level. It can be applied to marking varieties of longan through barcodes on labels, helping to understand the origin of longan varieties and improving their commercial value when exporting. The result was not clear in nucleotide sequence differences of the longan varieties in this study. However, it can be promising in the future if some further research is carried out in other genomic regions to determine the differences in gene sequences of longan varieties.

CONCLUSION

The genetic diversity of eleven D. longan varieties was detected based on molecular biology in Mekong Delta. The results of this study contributed to distinguishing longan varieties at the species level based on nucleotide sequences after analyzing the DNA sequencing results. However, these findings did not clearly show the significant differences in genetic diversity between the longan varieties. Therefore, the study could be extended by further research in other genomic regions of the chloroplast genomes of several longan varieties.

SIGNIFICANCE STATEMENT

The objective of this study was to investigate the genetic diversity of the matK gene of the longan varieties in the Mekong Delta. It is one of the first molecular biology studies including “purple” longan. Based on the results of this study, the nucleotide sequences in the matK gene were different in many longan varieties. Therefore, this study has made several important contributions to the research of the matK’s genetic diversity to improve the commercial value of the longan varieties. Furthermore, the study was as a premise for further applied studies to build DNA barcodes to help distinguish different cultivars.

ACKNOWLEDGMENTS

The fund for this study was invested in the Scientific Research Project of Can Tho University (T2022-131). We thank the Institute of Food and Biotechnology at Can Tho University for its instrumental support.