The Identification of the Banana Endogenous Reference Gene MaSPS1 and the Construction of Qualitative and Quantitative PCR Detection Methods

Abstract

Endogenous reference genes play a crucial role in the qualitative and quantitative PCR detection of genetically modified crops. Currently, there are no systematic studies on the banana endogenous reference gene. In this study, the MaSPS1 gene was identified as a candidate gene through bioinformatics analysis. The conservation of this gene in different genotypes of banana was tested using PCR, and its specificity in various crops and fruits was also examined. Southern blot analysis showed that there is only one copy of MaSPS1 in banana. The limit of detection (LOD) test showed that the LOD of the conventional PCR method is approximately 20 copies. The real-time fluorescence quantitative PCR (qPCR) method also exhibited high specificity, with a LOD of approximately 10 copies. The standard curve of the qPCR method met the quantitative requirements, with a limit of quantification (LOQ) of 1.14 × 10^-2 ng-about 20 copies. Also, the qPCR method demonstrated good repeatability and stability. Hence, the above results indicate that the detection method established in this study has strong specificity, a low detection limit, and good stability. It provides a reliable qualitative and quantitative detection system for banana.

Keywords: MaSPS1; banana (Musa paradisiaca); endogenous reference gene; qPCR.

Figure 1

Sequence analysis of the MaSPS1 gene. Note: The position of the forward (SPS-QF) and reverse (SPS-QR) primers and fluorescence probes (SPS-QP) of the amplified sequence are indicated separately in the sequence. Five different banana varieties with distinct genotypes, M. acuminata (AA), Guangxi hongjiao (AAA), CB5 (AAB), Musa ABB Pisang Awak (ABB), and FHIA-21 (AAAB), were selected for analysis.

Figure 2

Conventional PCR to verify MaSPS1 gene specificity. (A) Specific amplification of MaSPS1 in crops. 1~10: blank control, banana, Musa ABB Pisang Awak, rapeseed, rice, papaya, maize, soybean, cotton, and wheat, respectively. The 104 bp target band was only amplified in the banana and Musa ABB Pisang Awak reactions. (B) Specific amplification of MaSPS1 in fruits. 1~16: blank control, banana, Musa ABB Pisang Awak, mango, grapefruit, green jujube, lemon, orange, jackfruit, watermelon, wax apple, pear, grape, peach, cantaloupe, and strawberry, respectively. The 104 bp target band was only amplified in the banana and Musa ABB Pisang Awak reactions. (C) Specific amplification of MaSPS1 in the different genotypes of banana. 1~10: blank control, Malaccensis (AAA), Brazilian banana (AAA), FHIA-04 (AAAA), French Sombre (AAB), FHIA-21 (AAAB), Musa ABB Pisang Awak (ABB), M. acuminata (AAA), Guangxi hongjiao (AAA), and CB5 (AAB), respectively. All of these banana materials were able to amplify a specific target band of 104 bp.

Figure 3

Real-time fluorescence quantitative PCR (qPCR) to verify MaSPS1 specificity. 1: blank control; 2~10: Malaccensis, Brazilian banana, FHIA-04, French Sombre, FHIA-21, Musa ABB Pisang Awak, M. acuminata, Guangxi hongjiao, and CB5 had amplification signals; 11~30: rapeseed, rice, papaya, maize, soybean, cotton, wheat, mango, grapefruit, green jujube, lemon, orange, jackfruit, watermelon, wax apple, pear, grape, peach, cantaloupe, and strawberry.

Figure 4

Southern blot analysis the copy number of MaSPS1 in the banana genome. (A) Enzymatic cleavage site analysis of the MaSPS1 genomic sequence. Southern blot with BamH I (B) and EcoR I (C) digestion of banana genomic DNA. The hybridization band was about 10 kb when BamH I digestion was employed for all nine banana varieties in (B). The hybridization band was about 17 kb when EcoR I digestion was utilized (C). 1~9: Malaccensis (AA), Brazilian banana (AAA), FHIA-04 (AAAA), French Sombre (AAB), FHIA-21 (AAAB), Musa ABB Pisang Awak (ABB), M. acuminata (AAA), Guangxi hongjiao (AAA), and CB5 (AAB).

Figure 5

Conventional PCR LOD test for MaSPS1. 1~11: blank control, positive control, 5.70 × 10⁰, 5.70 × 10⁻¹, 1.14 × 10⁻¹, 5.70 × 10⁻², 2.85 × 10⁻², 1.14 × 10⁻², 5.70 × 10⁻³, 2.85 × 10⁻³, and 1.43 × 10⁻³ ng of banana genomic DNA. A clear 104 bp target band was seen when the template was 1.14 × 10⁻² ng and above.

Figure 6

qPCR LOD test for MaSPS1. 1~10: blank control, 5.70 × 10⁰, 5.70 × 10⁻¹, 1.14 × 10⁻¹, 5.70 × 10⁻², 2.85 × 10⁻², 1.14 × 10⁻², 5.70 × 10⁻³, 2.85 × 10⁻³, and 1.43 × 10⁻³ ng of banana genomic DNA. Amplification curves could be obtained when the template was 5.70 × 10⁻³ ng and above.

Figure 7

qPCR repeatability and stability test for MaSPS1. (A–C) are the amplification curves and standard curves of the qPCR test under different models of three operators; 1~6: 1.14 × 10², 1.14 × 10¹, 1.14 × 10⁰, 1.14 × 10⁻¹, 1.14 × 10⁻², and 5.70 × 10⁻³ ng of banana genomic DNA.