Identification of <i>Indica-Japonica</i> Subspecies Rice by Duplex Fluorescence PCR Detection for Chloroplast DNA and Endogenous <i>gos</i> Gene

Biying Shao; Tingyu Miao; Juan Peng; Bin Chen; Wenbing Chen

doi:doi:10.11648/j.ijnfs.20211005.12

| Peer-Reviewed

Identification of Indica-Japonica Subspecies Rice by Duplex Fluorescence PCR Detection for Chloroplast DNA and Endogenous gos Gene

Biying Shao, Tingyu Miao, Juan Peng, Bin Chen, Wenbing Chen

Published in International Journal of Nutrition and Food Sciences (Volume 10, Issue 5)

Received: 17 August 2021 Accepted: 6 September 2021 Published: 23 September 2021

Views: Downloads:

Download PDF

Share This Article

Twitter
Linked In
Facebook

Abstract

Asia cultivated rice are classified into two subspecies, indica and japonica rice (hsien and keng rice called in China, separately). It is necessary to establish a convenient and effective method to identify two subspecies because of the different quality characteristics between the rice products of them, which lead to different processing uses and commodity values. The identification method of PCR based on the difference of chloroplast DNA is convenient and effective, to be attempted to establish. Based on 69 bp fragment deletion of chloroplast DNA (cpDNA) found in ORF100 region nucleotides within the cpDNA Pst I-12 fragment in indica rice but not in japonica, the primer pair and a probe located on the specific cpDNA fragment in japonica rice were designed to identify indica and japonica rice. Another primer pair and probe used to detect endogenous gene gos in rice were combined with above primer pair and probe for detecting cpDNA to establish duplex fluorescence PCR to amplify cpDNA and gos gene for improving detection accuracy, to avoid the false negative results caused by DNA extraction error. The duplex fluorescence PCR detection method was established using typical japonica rice (pearl rice) and typical indica rice (Taixian 11). The accuracy of the method was validated with 547 samples including 177 samples of rice seed and eaten rice known as conventional japonica varieties and japonica type hybrid combinations and 370 samples of rice seed and eaten rice known as conventional indica rice varieties and indica type hybrid combinations. In 177 japonica samples, 170 samples with both positive results of cpDNA marker and gos gene, a coincidence rate of 96.05% agreement japonica rice, and 7 samples with positive results of gos gene and negative of cpDNA marker, not agreement japonica rice with non-coincidence rate of 3.95%, were detected. Of the 370 samples of indica rice seed and eaten rice, with positive results of gos gene and negative of cpDNA marker were detected in 340 samples, the rate of coincidence to indica rice was 91.89%. Other 30 samples with both positive results of cpDNA marker and gos gene, were detected, and the non-coincidence rate with indica rice was 8.11%. These identification results were in good agreement with known indica and japonica varieties, and the coincidence rate of japonica varieties was higher than indica. This method can be used to identify indica and japonica subspecies rice, especially suitable to identify conventional rice varieties.

Published in	International Journal of Nutrition and Food Sciences (Volume 10, Issue 5)
DOI	10.11648/j.ijnfs.20211005.12
Page(s)	101-107
Creative Commons	This is an Open Access article, distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution and reproduction in any medium or format, provided the original work is properly cited.
Copyright	Copyright © The Author(s), 2024. Published by Science Publishing Group

Keywords

Indica (hsien) Rice Seed/Eaten Rice, japonica (keng) Rice/Eaten Rice, Chloroplast DNA (cpDNA), Endogenous gos Gene, Duplex Fluorescence PCR, Identification

References

[1]	Kato, S., Kosaka, H. & Hara, S. (1928). On the affinity of rice varieties as shown by the fertility of rice plants (in Japanese). Central Agriculture Institute of Kyushu Imperial University, 2: 241-276.
[2]	Ting Y. (1949). The origin of Chinese rice culture (in Chinese). Agronomy Bulletin of College of Agriculture of Chungsan University, 7: 11-24.
[3]	Ting Y. (1957). Origin and evolution of cultivated rice in China. Journal of Agriculture, 8 (3): 243-260.
[4]	Cheng, K. S. (1993). Identification of indica-japonica subspecies in Asian rice. Kunming: Yunnan Science and Technology Press, 1-23.
[5]	Chen, W. B. (1999). A Review of genetic studies on the origin and differentiation of Asian cultivated rice (Oryza sativa L.), Acta Agriculturae Shanghai, 15 (3): 42-48.
[6]	Ling, Q. H., Zhang, H. C. & Ding, Y. F. (2013). Discussion of naming for two subspecies of Oryza sativa L. Scientia Agricultura Sinica, 46 (2): 250-256.
[7]	Chen, W-B., Sato, Y. I., Nakamura, I. & Nakai, H. (1994) Indica-japonica differentiation in Chinese rice landraces. Euphytica, 74 (3): 195-201.
[8]	Tang, S. X., Jiang, Y. Z., Wei, X. H., Li, Z. C. & Yu, H. Y. (2002). Genetic Diversity of isozymes of cultivated rice in China. Acta Agronomica Sinica, 28 (2): 203-207.
[9]	Zhang, Q. F., Saghai, M. A. & Lu, T. Y. (1992). Genetic diversity and differentiation of indica and japonica rice detected by RFLP analysis. Theoretical and Applied Genetics, 83: 495-499.
[10]	Zhuang J. Y. Qian H. R., Lin H. X., Lu J., Cheng, S. H., Ying, C. S., Luo, L. J., Zhu, X. D., Dong, F. G., Min, S. K., Sun, Z. X., & Z heng K. L. (1995). RFLP based Analysis of the Origin and differentiation of Oryza sativa L. Chinese Journal of Rice Sciences, 9 (3): 135-140.
[11]	Sun, C. Q., Wang, X. K. & Yoshimura, A. (1997). RFLP Analysis of nuclear DNA in common wild rice (O. rufipogon griff.) and cultivated rice (O. sativa L.). Scientia Agricultura Sinica, 30 (4): 37-44.
[12]	Long, W. H. & Xu, M. H. (2002). RAPD-based genetic difference between indica rice and japonica rice. Journal of Yunnan Agricultural University, 17 (3): 245-247.
[13]	Yamanaka, S., Nakamura, I., Nakai, H., & Sato, Y. I. (2003). Dual origin of the cultivated rice based on molecular markers of newly collected annual and perennial strains of wild rice species, Oryza nivara and O. rufipogon. Genetic Resources and Crop Evolution, 50: 529–539.
[14]	Xu, J. X., Wang, Y. Y., & Yao C. (2012). Indica-japonica differentiation and genetic variation of upland rice varieties from Yunnan Province, China. Chinese Science Bulletin, 57 (28-29): 2705-2714.
[15]	Cai, X. X., Liu, J., Qiu, Y. Q., Zhao, W., Song, Z. P. & Lu, B. R. (2006). Differentiation of indica-japonica rice revealed by insertion/deletion fragments obtained from comparative genomic study of DNA sequences between 93-11 (indica) and nipponbare (japonica). Journal of Fudan University (Natural Science), 45 (3): 309-315.
[16]	Zhao W., Xia H. B., Zang S. J., Cai, X. X., & Lu, B. R. (2008). Differentiation of Oryza species revealed by the indica-japonica. Journal of Fudan University (Natural Science), 47 (3): 281-287.
[17]	Xiong, Z. Y., Zhang, S. J., Wang, Y. Y., Brian V. Ford-Lloyd, Tu, M., Jin, X., Wu, Y., Yan, H. X., Yang X., Liu, P. & Lu, B. R. (2010). Differentiation and distribution of indica and japonica rice varieties along the altitude gradients in Yunnan Province of China as revealed by InDel molecular markers. Genetic Resources and Crop Evolution, 57: 891–902.
[18]	Liu, P., Cai, X. X. & Lu, B. R. (2012). Single-seeded InDel fingerprints in rice: An effective tool for indica–japonica rice classification and evolutionary studies. Journal of Systematic and Evolution. 50: 1-11.
[19]	Chen, W. B., Nakamura, I., SATO, Y.-I. & Nakai, H. (1993). Distribution of deletion type in cpDNA of cultivated and wild rice [J]. Japanese Journal of Genetics, 68 (6): 579-603.
[20]	Yang, J. Wang, J., Can, Q., Chen, Z. D., Tang L. H., Wang Y. P., Fang X. W., Wang C. L. & Zhong, W. G. (2009). Indica-japonica differentiation of chloroplast DNA of weedy rice in the Changjiang and Huaihe River Valley of China. Chinese Journal of Rice Sciences, 23 (4): 391-397.
[21]	Wang, R. S., Wei, X., Cao, L. R., Qiao, W. H., Zhang, W. X., & Yang, Q. W. (2011). Origin and evolution of cultivated rice (O. sativa L.) in China based on gene diversity of chloroplast genome. Journal of Plant Genetic Resources, 12 (5): 686-693.
[22]	Cao, L. R., Wei, X., Huang, J., Qiao, W. H., Zang, W. X. & Yang, Q. E. (2013). Study on the origin and evolution of Asian cultivated rice based on gene fragment nucleotides diversity of mitochondrial genome. Journal of Plant Genetic Resources, 14 (1): 18-24.
[23]	Kanno, A., Watanabe, N., Nakamura, I. & Hirai, A. (1993). Variations in chloroplast DNA from rice (Oryza sativa): differences between deletions mediated by short direct-repeat sequences within a single species. Theoretical and Applied Genetics, 86: 579-584.
[24]	Rong, Q. Y., Chen, X. X., Liu, Y. N., Xu, Z., Ding, Y. F., Wu, Y. L., Ji, X. F., Zhang, Y. & Li, B. Q. (2018). Establishment of a duplex fluorescence quantitative PCR technique for detection of Helicobacter pylori with TaqMan MGB probes. Journal of Pathogen Biology, 6: 572-574, 579.
[25]	Ma, L., Yao, G. Z., Wang, Z., Meng, Q. F., Li, Y., Xiao, F., Liu, J. H., & Wang, W. L. (2020). Establishment of duplex fluorescence RT-PCR method for detection of H5N8 subtype avian influenza virus HA and NA genes. ProgressinVeterinaryMedicine, 41 (6): 32-37.
[26]	Shao M. L., Dong X., Zhao Y. L., Kong, B. H. & Liu, S. G.. (2013). A duplex fluorescence quantitative PCR assay for detecting Listeriamonocytogenes and Staphylococcus aureus. Food Science, 34 (16): 169-172.
[27]	Yang, L. L., Sun, L. X., Ruan, X. L., Qiu, D. Y., Chen, D. H., Cai, X. Q., & Li, H. P. (2015). Development of a single-tube duplex real-time fluorescence method for the rapid quantitative detection of Fusarium oxysporum f. sp. cubense race 1 (FOC1) and race 4 (FOC4) using TaqMan probes, Crop Protection, 68 (2): 27-35.
[28]	Hou, D. J., Han, H. J., Hao, Z. H. & Jiang, Y. B. (2016). The method of duplex fluorescence PCR for rapid detection of cattle-and sheep-derived ingredients in feedstuff. Heilongjiang Animal Science and Veterinary Medicine, 2: 247-250.
[29]	Wang F.-J., Ye S.-D., Chen G.-W. & Bao, Y. H. (2018). Establishment and verified of a duplex fluorescence quantitative PCR for screening of genetically modified soybeans and products. Science and Technology of Food Industry, 8: 236-239, 271.
[30]	Yuan, J. J., Wei, S., Long, Y., Wu, X. Y., Li, X. & Fu, W. (2020). Development and application of duplex real-time PCR assay for the detection of genetically modified soybean (Glycine max) MON87701 and MON87708. Journal of Agricultural Biotechnology, 28 (2): 342-348.
[31]	Chen, W. B., Shao, B. Y., Lin, Y. W., Miao, T. Y., Peng, J., Guo, J., Chen, B. & Peng, H. Y. (2020). Identification method of hsien (indica) and keng (japonica) rice by SYBR-Green fluorescence-PCR detection for chloroplast DNA. China Port Science and Technology, 10: 17-25.
[32]	Chen, H. Y., Chen, X. M., Chen, S. Y., Wang, Z. H., Li, L, Huang, W. S., Hu, X. Z., Zhu, S. F. & Chen, H. J. (2010). Protocol of real-time polymerase chain reaction for detecting genetically modified components in rice and its derived products. Commodity Inspection Standards of the People's Republic of China, SN/T2584-2010.

Cite This Article

Plain Text BibTeX RIS

APA Style

Biying Shao, Tingyu Miao, Juan Peng, Bin Chen, Wenbing Chen. (2021). Identification of Indica-Japonica Subspecies Rice by Duplex Fluorescence PCR Detection for Chloroplast DNA and Endogenous gos Gene. International Journal of Nutrition and Food Sciences, 10(5), 101-107. https://doi.org/10.11648/j.ijnfs.20211005.12

Copy | Download

ACS Style

Biying Shao; Tingyu Miao; Juan Peng; Bin Chen; Wenbing Chen. Identification of Indica-Japonica Subspecies Rice by Duplex Fluorescence PCR Detection for Chloroplast DNA and Endogenous gos Gene. Int. J. Nutr. Food Sci. 2021, 10(5), 101-107. doi: 10.11648/j.ijnfs.20211005.12

Copy | Download

AMA Style

Biying Shao, Tingyu Miao, Juan Peng, Bin Chen, Wenbing Chen. Identification of Indica-Japonica Subspecies Rice by Duplex Fluorescence PCR Detection for Chloroplast DNA and Endogenous gos Gene. Int J Nutr Food Sci. 2021;10(5):101-107. doi: 10.11648/j.ijnfs.20211005.12

Copy | Download

@article{10.11648/j.ijnfs.20211005.12,
  author = {Biying Shao and Tingyu Miao and Juan Peng and Bin Chen and Wenbing Chen},
  title = {Identification of Indica-Japonica Subspecies Rice by Duplex Fluorescence PCR Detection for Chloroplast DNA and Endogenous gos Gene},
  journal = {International Journal of Nutrition and Food Sciences},
  volume = {10},
  number = {5},
  pages = {101-107},
  doi = {10.11648/j.ijnfs.20211005.12},
  url = {https://doi.org/10.11648/j.ijnfs.20211005.12},
  eprint = {https://article.sciencepublishinggroup.com/pdf/10.11648.j.ijnfs.20211005.12},
  abstract = {Asia cultivated rice are classified into two subspecies, indica and japonica rice (hsien and keng rice called in China, separately). It is necessary to establish a convenient and effective method to identify two subspecies because of the different quality characteristics between the rice products of them, which lead to different processing uses and commodity values. The identification method of PCR based on the difference of chloroplast DNA is convenient and effective, to be attempted to establish. Based on 69 bp fragment deletion of chloroplast DNA (cpDNA) found in ORF100 region nucleotides within the cpDNA Pst I-12 fragment in indica rice but not in japonica, the primer pair and a probe located on the specific cpDNA fragment in japonica rice were designed to identify indica and japonica rice. Another primer pair and probe used to detect endogenous gene gos in rice were combined with above primer pair and probe for detecting cpDNA to establish duplex fluorescence PCR to amplify cpDNA and gos gene for improving detection accuracy, to avoid the false negative results caused by DNA extraction error. The duplex fluorescence PCR detection method was established using typical japonica rice (pearl rice) and typical indica rice (Taixian 11). The accuracy of the method was validated with 547 samples including 177 samples of rice seed and eaten rice known as conventional japonica varieties and japonica type hybrid combinations and 370 samples of rice seed and eaten rice known as conventional indica rice varieties and indica type hybrid combinations. In 177 japonica samples, 170 samples with both positive results of cpDNA marker and gos gene, a coincidence rate of 96.05% agreement japonica rice, and 7 samples with positive results of gos gene and negative of cpDNA marker, not agreement japonica rice with non-coincidence rate of 3.95%, were detected. Of the 370 samples of indica rice seed and eaten rice, with positive results of gos gene and negative of cpDNA marker were detected in 340 samples, the rate of coincidence to indica rice was 91.89%. Other 30 samples with both positive results of cpDNA marker and gos gene, were detected, and the non-coincidence rate with indica rice was 8.11%. These identification results were in good agreement with known indica and japonica varieties, and the coincidence rate of japonica varieties was higher than indica. This method can be used to identify indica and japonica subspecies rice, especially suitable to identify conventional rice varieties.},
 year = {2021}
}

Copy | Download

TY  - JOUR
T1  - Identification of Indica-Japonica Subspecies Rice by Duplex Fluorescence PCR Detection for Chloroplast DNA and Endogenous gos Gene
AU  - Biying Shao
AU  - Tingyu Miao
AU  - Juan Peng
AU  - Bin Chen
AU  - Wenbing Chen
Y1  - 2021/09/23
PY  - 2021
N1  - https://doi.org/10.11648/j.ijnfs.20211005.12
DO  - 10.11648/j.ijnfs.20211005.12
T2  - International Journal of Nutrition and Food Sciences
JF  - International Journal of Nutrition and Food Sciences
JO  - International Journal of Nutrition and Food Sciences
SP  - 101
EP  - 107
PB  - Science Publishing Group
SN  - 2327-2716
UR  - https://doi.org/10.11648/j.ijnfs.20211005.12
AB  - Asia cultivated rice are classified into two subspecies, indica and japonica rice (hsien and keng rice called in China, separately). It is necessary to establish a convenient and effective method to identify two subspecies because of the different quality characteristics between the rice products of them, which lead to different processing uses and commodity values. The identification method of PCR based on the difference of chloroplast DNA is convenient and effective, to be attempted to establish. Based on 69 bp fragment deletion of chloroplast DNA (cpDNA) found in ORF100 region nucleotides within the cpDNA Pst I-12 fragment in indica rice but not in japonica, the primer pair and a probe located on the specific cpDNA fragment in japonica rice were designed to identify indica and japonica rice. Another primer pair and probe used to detect endogenous gene gos in rice were combined with above primer pair and probe for detecting cpDNA to establish duplex fluorescence PCR to amplify cpDNA and gos gene for improving detection accuracy, to avoid the false negative results caused by DNA extraction error. The duplex fluorescence PCR detection method was established using typical japonica rice (pearl rice) and typical indica rice (Taixian 11). The accuracy of the method was validated with 547 samples including 177 samples of rice seed and eaten rice known as conventional japonica varieties and japonica type hybrid combinations and 370 samples of rice seed and eaten rice known as conventional indica rice varieties and indica type hybrid combinations. In 177 japonica samples, 170 samples with both positive results of cpDNA marker and gos gene, a coincidence rate of 96.05% agreement japonica rice, and 7 samples with positive results of gos gene and negative of cpDNA marker, not agreement japonica rice with non-coincidence rate of 3.95%, were detected. Of the 370 samples of indica rice seed and eaten rice, with positive results of gos gene and negative of cpDNA marker were detected in 340 samples, the rate of coincidence to indica rice was 91.89%. Other 30 samples with both positive results of cpDNA marker and gos gene, were detected, and the non-coincidence rate with indica rice was 8.11%. These identification results were in good agreement with known indica and japonica varieties, and the coincidence rate of japonica varieties was higher than indica. This method can be used to identify indica and japonica subspecies rice, especially suitable to identify conventional rice varieties.
VL  - 10
IS  - 5
ER  -

Copy | Download

Author Information

Biying Shao

Technology Center of Fuzhou Customs District, Fuzhou, China
Tingyu Miao

Technology Center of Fuzhou Customs District, Fuzhou, China
Juan Peng

Technology Center of Fuzhou Customs District, Fuzhou, China
Bin Chen

Technology Center of Fuzhou Customs District, Fuzhou, China
Wenbing Chen

Technology Center of Fuzhou Customs District, Fuzhou, China

Download PDF

Sections

Plain Text BibTeX RIS

APA Style

Biying Shao, Tingyu Miao, Juan Peng, Bin Chen, Wenbing Chen. (2021). Identification of Indica-Japonica Subspecies Rice by Duplex Fluorescence PCR Detection for Chloroplast DNA and Endogenous gos Gene. International Journal of Nutrition and Food Sciences, 10(5), 101-107. https://doi.org/10.11648/j.ijnfs.20211005.12

Copy | Download

ACS Style

Biying Shao; Tingyu Miao; Juan Peng; Bin Chen; Wenbing Chen. Identification of Indica-Japonica Subspecies Rice by Duplex Fluorescence PCR Detection for Chloroplast DNA and Endogenous gos Gene. Int. J. Nutr. Food Sci. 2021, 10(5), 101-107. doi: 10.11648/j.ijnfs.20211005.12

Copy | Download

AMA Style

Biying Shao, Tingyu Miao, Juan Peng, Bin Chen, Wenbing Chen. Identification of Indica-Japonica Subspecies Rice by Duplex Fluorescence PCR Detection for Chloroplast DNA and Endogenous gos Gene. Int J Nutr Food Sci. 2021;10(5):101-107. doi: 10.11648/j.ijnfs.20211005.12

Copy | Download

@article{10.11648/j.ijnfs.20211005.12,
  author = {Biying Shao and Tingyu Miao and Juan Peng and Bin Chen and Wenbing Chen},
  title = {Identification of Indica-Japonica Subspecies Rice by Duplex Fluorescence PCR Detection for Chloroplast DNA and Endogenous gos Gene},
  journal = {International Journal of Nutrition and Food Sciences},
  volume = {10},
  number = {5},
  pages = {101-107},
  doi = {10.11648/j.ijnfs.20211005.12},
  url = {https://doi.org/10.11648/j.ijnfs.20211005.12},
  eprint = {https://article.sciencepublishinggroup.com/pdf/10.11648.j.ijnfs.20211005.12},
  abstract = {Asia cultivated rice are classified into two subspecies, indica and japonica rice (hsien and keng rice called in China, separately). It is necessary to establish a convenient and effective method to identify two subspecies because of the different quality characteristics between the rice products of them, which lead to different processing uses and commodity values. The identification method of PCR based on the difference of chloroplast DNA is convenient and effective, to be attempted to establish. Based on 69 bp fragment deletion of chloroplast DNA (cpDNA) found in ORF100 region nucleotides within the cpDNA Pst I-12 fragment in indica rice but not in japonica, the primer pair and a probe located on the specific cpDNA fragment in japonica rice were designed to identify indica and japonica rice. Another primer pair and probe used to detect endogenous gene gos in rice were combined with above primer pair and probe for detecting cpDNA to establish duplex fluorescence PCR to amplify cpDNA and gos gene for improving detection accuracy, to avoid the false negative results caused by DNA extraction error. The duplex fluorescence PCR detection method was established using typical japonica rice (pearl rice) and typical indica rice (Taixian 11). The accuracy of the method was validated with 547 samples including 177 samples of rice seed and eaten rice known as conventional japonica varieties and japonica type hybrid combinations and 370 samples of rice seed and eaten rice known as conventional indica rice varieties and indica type hybrid combinations. In 177 japonica samples, 170 samples with both positive results of cpDNA marker and gos gene, a coincidence rate of 96.05% agreement japonica rice, and 7 samples with positive results of gos gene and negative of cpDNA marker, not agreement japonica rice with non-coincidence rate of 3.95%, were detected. Of the 370 samples of indica rice seed and eaten rice, with positive results of gos gene and negative of cpDNA marker were detected in 340 samples, the rate of coincidence to indica rice was 91.89%. Other 30 samples with both positive results of cpDNA marker and gos gene, were detected, and the non-coincidence rate with indica rice was 8.11%. These identification results were in good agreement with known indica and japonica varieties, and the coincidence rate of japonica varieties was higher than indica. This method can be used to identify indica and japonica subspecies rice, especially suitable to identify conventional rice varieties.},
 year = {2021}
}

Copy | Download

TY  - JOUR
T1  - Identification of Indica-Japonica Subspecies Rice by Duplex Fluorescence PCR Detection for Chloroplast DNA and Endogenous gos Gene
AU  - Biying Shao
AU  - Tingyu Miao
AU  - Juan Peng
AU  - Bin Chen
AU  - Wenbing Chen
Y1  - 2021/09/23
PY  - 2021
N1  - https://doi.org/10.11648/j.ijnfs.20211005.12
DO  - 10.11648/j.ijnfs.20211005.12
T2  - International Journal of Nutrition and Food Sciences
JF  - International Journal of Nutrition and Food Sciences
JO  - International Journal of Nutrition and Food Sciences
SP  - 101
EP  - 107
PB  - Science Publishing Group
SN  - 2327-2716
UR  - https://doi.org/10.11648/j.ijnfs.20211005.12
AB  - Asia cultivated rice are classified into two subspecies, indica and japonica rice (hsien and keng rice called in China, separately). It is necessary to establish a convenient and effective method to identify two subspecies because of the different quality characteristics between the rice products of them, which lead to different processing uses and commodity values. The identification method of PCR based on the difference of chloroplast DNA is convenient and effective, to be attempted to establish. Based on 69 bp fragment deletion of chloroplast DNA (cpDNA) found in ORF100 region nucleotides within the cpDNA Pst I-12 fragment in indica rice but not in japonica, the primer pair and a probe located on the specific cpDNA fragment in japonica rice were designed to identify indica and japonica rice. Another primer pair and probe used to detect endogenous gene gos in rice were combined with above primer pair and probe for detecting cpDNA to establish duplex fluorescence PCR to amplify cpDNA and gos gene for improving detection accuracy, to avoid the false negative results caused by DNA extraction error. The duplex fluorescence PCR detection method was established using typical japonica rice (pearl rice) and typical indica rice (Taixian 11). The accuracy of the method was validated with 547 samples including 177 samples of rice seed and eaten rice known as conventional japonica varieties and japonica type hybrid combinations and 370 samples of rice seed and eaten rice known as conventional indica rice varieties and indica type hybrid combinations. In 177 japonica samples, 170 samples with both positive results of cpDNA marker and gos gene, a coincidence rate of 96.05% agreement japonica rice, and 7 samples with positive results of gos gene and negative of cpDNA marker, not agreement japonica rice with non-coincidence rate of 3.95%, were detected. Of the 370 samples of indica rice seed and eaten rice, with positive results of gos gene and negative of cpDNA marker were detected in 340 samples, the rate of coincidence to indica rice was 91.89%. Other 30 samples with both positive results of cpDNA marker and gos gene, were detected, and the non-coincidence rate with indica rice was 8.11%. These identification results were in good agreement with known indica and japonica varieties, and the coincidence rate of japonica varieties was higher than indica. This method can be used to identify indica and japonica subspecies rice, especially suitable to identify conventional rice varieties.
VL  - 10
IS  - 5
ER  -

Copy | Download