Screening of Universal DNA Barcodes for Vitis amurensis

doi:10.11983/CBB16237

Abstract

Abstract: To determine the best candidate sequence that can be used as a universal DNA barcode to identify Vitis amurensis varieties, we used PCR amplification with the ideal annealing temperature and sequenced the DNA fragments of five different regions (ITS, ITS2, psbA-trnH, rbcL and matK) of Amur grape samples. The effective sequence ratios obtained by PCR amplification and sequencing, divergence of intra- and inter-varieties, DNA barcoding gap and identification by using BLAST and NJ tree methods were used to evaluate the discrimination ability of these candidate sequences. ITS2 and psbA-trnH had fairly high amplification efficiency and sequencing efficiency in 33 samples belonging to 11 V. amurensis resources; they had larger divergences of intra- and inter-varieties and better DNA barcoding gaps than the other regions tested (ITS, rbcL and matK). In addition, ITS2 could discriminate some varieties that could not be successfully discriminated by the psbA-trnH region. ITS2 and psbA-trnH may be promising DNA barcode region combinations for identifying V. amurensis resources. DNA barcoding can compensate for morphological identification and help in the exact identification of V. amurensis resources.

Key words: Vitis amurensis, DNA barcode, identification, ITS2, psbA-trnH, sequence

Song Huifang, Liu Haishuang, Yang Yiming, Fan Shutian, Li Changyu, Ai Jun. Screening of Universal DNA Barcodes for Vitis amurensis[J]. Chinese Bulletin of Botany, 2017, 52(6): 723-732.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB16237

https://www.chinbullbotany.com/EN/Y2017/V52/I6/723

Figures/Tables 9

References 33

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Number	Varieties name	Locality of origin	Parents or source	Flower type
1	Zuoyouhong	Zuojia, Jilin	Varieties	Bisexual
2	Shuanghong	Zuojia, Jilin	Varieties	Bisexual
3	Zuoshan1	Zuojia, Jilin	Wild resource	Male
4	Zuoshan2	Zuojia, Jilin	Wild resource	Male
5	4N1	Zuojia, Jilin	Genetic material	Tetraploid
6	4N2	Zuojia, Jilin	Genetic material	Tetraploid
7	Shuangqing	Zuojia, Jilin	Varieties	Bisexual
8	Shuangfeng	Zuojia, Jilin	Varieties	Bisexual
9	Shuangyou	Ji’an, Jilin	Varieties	Bisexual
10	75047	Shangzhi, Heilongjiang	Wild resource	Female
11	73061	Dunhua, Jilin	Wild resource	Female

Number	Varieties name	Locality of origin	Parents or source	Flower type
1	Zuoyouhong	Zuojia, Jilin	Varieties	Bisexual
2	Shuanghong	Zuojia, Jilin	Varieties	Bisexual
3	Zuoshan1	Zuojia, Jilin	Wild resource	Male
4	Zuoshan2	Zuojia, Jilin	Wild resource	Male
5	4N1	Zuojia, Jilin	Genetic material	Tetraploid
6	4N2	Zuojia, Jilin	Genetic material	Tetraploid
7	Shuangqing	Zuojia, Jilin	Varieties	Bisexual
8	Shuangfeng	Zuojia, Jilin	Varieties	Bisexual
9	Shuangyou	Ji’an, Jilin	Varieties	Bisexual
10	75047	Shangzhi, Heilongjiang	Wild resource	Female
11	73061	Dunhua, Jilin	Wild resource	Female

Fragment	Eight annealing temperature gradient (°C)	Annealing temperature (°C)
ITS2	54.8-55.4-56.0-56.6-57.2-57.8-58.4-59.0	56.0
psbA-trnH	52.2-52.8-53.4-54.0-54.6-55.2-55.8-56.4	55.2
matK	48.9-49.5-50.1-50.7-51.3-51.9-52.5-53.1	50.1
rbcL	52.1-52.7-53.3-53.9-54.5-55.1-55.7-56.3	54.5
ITS	49.9-50.5-51.1-51.7-52.3-52.9-53.5-54.1	52.9

Fragment	Eight annealing temperature gradient (°C)	Annealing temperature (°C)
ITS2	54.8-55.4-56.0-56.6-57.2-57.8-58.4-59.0	56.0
psbA-trnH	52.2-52.8-53.4-54.0-54.6-55.2-55.8-56.4	55.2
matK	48.9-49.5-50.1-50.7-51.3-51.9-52.5-53.1	50.1
rbcL	52.1-52.7-53.3-53.9-54.5-55.1-55.7-56.3	54.5
ITS	49.9-50.5-51.1-51.7-52.3-52.9-53.5-54.1	52.9

Marker	Amplification efficiency (%)	Sequencing success rate (%)	Effective sequence ratio (%)
ITS2	100.0	96.9	96.9
matK	100.0	96.9	96.9
psbA-trnH	100.0	100.0	100.0
rbcL	96.9	90.9	88.1
ITS	45.5	30.3	13.8