Rice is one of the most important food crops in China. More than 60% of the population in China rely on rice as the staple food, and rice plays an important role in national food security (Xu et al., 2010, China Rice, 16(S1): 26-31). In recent years, because of its special rice fragrance, the demand for fragrant rice is increasing, and it is widely recognized and sought after in the market. Its price is more than 2 times that of non-fragrant rice, and most of the high-grade rice on the market is fragrant rice. With the development of people's dietary demand towards high quality, nutrition and function and the intensification of international trade competition, fragrant rice breeding has become a hot topic in rice research nowadays.

Fine mapping and map-based cloning of important agronomic traits genes in rice were carried out by molecular means, and development of functional markers for target genes can enhance the purpose and efficiency of breeding and greatly reduce the breeding years (Moose and Mumm, 2008). Guangxi's unique geographical location and climate resources breed and contain a number of fragrant rice germplasm resources, which have a long cultivation history and are widely distributed, mainly in the west of Guangxi, central Guangxi and some areas of South Guangxi. Local fragrant rice in Guangxi is mostly waxy rice, with large grains, rich fragrance, soft and sticky, fragrant and high nutritional value. Compared with ordinary fragrant rice, local fragrant rice has incomparable advantages. It is very valuable local rice seed resources, and its aroma gene resources need to be further explored and applied in breeding research.

Studies have shown that more than 200 compounds are involved in the aroma of fragrant rice (Mahattanatawee and Rouseff, 2014; He et al., 2015), in which 2-acetyl-1-pyrroline (2AP) is the main aromatic compound in fragrant rice, which has the aroma of popcorn and a low odor threshold (Routray and Rayaguru, 2018; Hashemi et al., 2015). The genetic basis of aroma is complex, but it is generally believed that the deletion of Badh2 on chromosome 8 of rice promotes the accumulation of 2AP, and Badh2 is the only aroma gene cloned at present (Bradbury et al., 2005; Shao et al., 2013; Hashemi et al., 2015). Further studies showed that Badh2 was a dominant allele encoding BADH2 protein (betaine aldehyde dehydrogenase) and inhibited aroma, and its non-functional recessive allele was the source of aroma in rice. Any mutation that disables the Badh2 gene results in a new functional badh2 allele; badh2 encodes a substantially truncated BADH2 enzyme that is responsible for elevated 2AP levels (Bradbury et al., 2008; Chen et al., 2008; Fitzgerald et al., 2008; Okpala et al., 2019). The formation of 2AP in aromatic cucumber, sorghum and other plants has also been confirmed to be caused by mutations in different locations of badh2 gene (Yundaeng et al., 2013; Pramnoi et al., 2013). The mutation of Badh2 gene is directly related to the aroma of rice. At least 18 alleles of aroma have been reported in the Badh2 loci that produce aroma. However, relevant scholars have only developed molecular markers for seven mutation sites such as G/A mutation at the junction between exon 1-intron 1 (badh2-E2.1), 7 bp deletion in exon 2 (badh2-E2.1), 803 bp deletion in exon 4 and 5 (badh2-E4-5.2), 8 bp deletion in exon 7 and 3 bp mutation (badh2-E7), 3 bp deletion in exon 12 (badh2-E12), 3 bp insertion in exon 13 (badh2-E13.1), and 1 bp insertion in exon 14 (badh2-E14.1). However, no corresponding molecular markers have been developed for 11 mutation types, including 806 bp deletion of exon 4 and 5 (badh2-E4-5.1) (He et al., 2015). The development of efficient molecular markers for different markers is of great practical significance for the identification, screening and breeding of new varieties of fragrant rice using these markers. Guo et al. (2017) used badh2-E2.1 and badh2-E7 molecular markers to select 15 germplasm materials containing aroma genes from 208 japonica rice germplasm resources in regions alongside Huanghe Rive of Henan. Chen et al. (2020) used the molecular marker of badh2-E7 to assist in breeding the fragrant rice variety "Jijing 816".

In the previous study, we used the reported four alleles badh2-E2.1, badh2-E4-5.2, badh2-E7 and badh2-E13.1 to identify 179 local fragrant rice materials in Guangxi. It was found that only 71 fragrant rice materials were identified with aroma genotypes, and the remaining 108 fragrant rice materials were not identified with genotypes. Therefore, it is speculated that there may be fragrance alleles other than the four reported alleles (Zeng et al., 2017). In this study, 40 fragrant rice materials without genotypes were selected to detect SNP loci of their Badh2 genes by direct sequencing method. The main genotypes of fragrant rice in Guangxi region were excavated and a set of fluorescence molecular markers badh2-E4-5 and badh2-E7 were developed based on real-time fluorescence PCR. These results could be helpful for the application of this gene in molecular marker-assisted breeding of fragrant rice.

1 Results and Analysis

1.1 There were two mutation types in Guangxi fragrant rice 

Ten pairs of overlapping primers were used to amplify the Badh2 gene of 40 Guangxi fragrant rice samples, and the sequence of each fragment was obtained by Sanger sequencing. Sequence alignment with non-fragrant rice Nipponbare revealed that there were two main mutation types of Badh2 gene in these fragrant rice from Guangxi, among which 37 fragrant rice had 806 bp deletion between exon 4 and exon 5 (29 homozygous deletion and 8 heterozygous deletion). In addition, 8 cultivars had 8 bp deletion and 3 bp mutation (badh2-E7) in exon 7 (2 of which were heterozygous) (Table 1). Six of the 40 fragrant rice cultivars had both badh-E4-5.1 and badh2-E7 deletions. The results indicated that two deletion types, badh-E4-5.1 and badh2-E7, were mainly found in the Guangxi fragrant rice materials collected in this study.

1.2 Design of primers and probes for Real-time PCR detection

Compared with conventional PCR detection, real-time fluorescence PCR with probe method can detect double or multiple mutations in one reaction without electrophoresis, which greatly reduces the workload and facilitates the popularization of the technology with simple judgment method. In this study, a set of primers and probes for real-time fluorescence PCR detection were designed for mutation types of Badh2 gene badh2-E4-5.1, badh2-E4-5.2 and badh-E7 (Table 2). In the detection of badh2-E4-5 type, the binding site of the wild-type primer and probe was on the intron missing from the mutant type, and the binding site of the probe in the deletion type was between exon 4 and exon 5 linked together after the deletion of 806 bp fragment or 803 bp fragment, and the primers were corresponding to the upstream and downstream of the probe. The designed primers can simultaneously detect badh2-E4-5.1 and badh2-E4-5.2 mutants (Figure 1A). In the detection of badh-E7, the positive and negative primers of the wild-type and mutant were the same, and the probes were the wild-type sequence of the mutant site and the sequence after the mutation respectively (Figure 1B). In order to enable simultaneous double detection of the same site, the wild-type probes were labeled with FAM and the mutant probes were labeled with VIC.

To verify the effectiveness of the developed molecular markers, 40 samples of fragrant rice tested by Sanger sequencing were detected by real-time PCR. By comparison, of the 40 samples, the detection of badh2-E4-5 was invalid in 2 samples, the results of 2 samples were inconsistent with Sanger sequencing, and the results of the remaining 36 samples were consistent with sequencing (Figure 2A). In the detection of badh-E7 locus, 39 samples were consistent with Sanger sequencing results, and the detection data of 1 sample was invalid (Figure 2B). Combining the results of the two markers, the results of 75 tests were consistent with the sequencing results (Figure 2; Table 1), the success rate was 93.75%, indicating that fluorescence molecular markers of badh2-E4-5 and badh-E7 were successfully developed in this study.

1.3 Two molecular markers were successfully used for genotyping of fragrant rice

To further verify the validity of the two developed markers, we identified 50 Guangxi local variety materials. The results showed that no genotype was detected in serial No. 1, and a total of 11 materials with serial No. 2-12 were badh2-E7 genotype. Serial number 13~22 material is badh2-E4-5. Among 28 previously unidentified materials with serial number 23-50, 4 were unidentified genotypes of non-fragrant rice, 11 were badh2-E7 genotypes of fragrant rice, 14 were badh2-E4-5 deleted genotypes of fragrant rice, and 3 were badh2-E4-5 and badh2-E7 genotypes (Table 3). Compared with Table 4, the identification results of the first 22 materials were consistent with those of the previous sequencing method and SSR molecular markers, and the two fluorescence markers could also be used to detect the unknown genotypes of fragrant rice. Therefore, the fluorescence markers developed in this study based on real-time fluorescence PCR technology could be applied to genotyping identification of fragrant rice.

2 Discussion

Rice breeding has entered the genomic era with the deepening of studies on genes and their interrelationships (Bao et al., 2008). More and more phenotypic differences are being shown to be related to markers or single nucleotide changes on the genome. In breeding, it is very important to select individuals with target traits effectively from the population, which will greatly improve the efficiency and economy of breeding. Traditional rice breeding mainly adopts qualitative methods such as chewing method, potassium hydroxide soaking method and hot water method to select fragrant rice (Ye et al., 2013, Modern Agricultural Science and Technology, (6): 9-10), and gas chromatography-mass spectrometry (GC-MS) was used to quantify aroma substances (Nataporn et al., 2017). However, these methods cost a lot of manpower and time. In rice breeding, marker-assisted breeding has been proved to be an effective and labor-saving method (Wang et al., 2011).

In studies of fragrant rice, Badh2 gene mutation has been widely proved to be the main cause of aroma in rice (Bradbury et al., 2008; Okpala et al., 2019). Mutation detection of Badh2 gene through molecular biological methods has been widely used in fragrant rice breeding, and the current main methods are Sanger sequencing and SSR molecular marker, PCR electrophoresis analysis (He et al., 2015). Due to its advantages of high flux, low cost and high sensitivity, fluorescence probe Real-time PCR is widely used in SNP detection (Broccanello et al., 2018). In 2017, Inam et al. (2017) developed a set of molecular markers for the detection of badh2-E7 using dye-based real-time fluorescence PCR technology. In 2007, Lopez (2008) developed a real-time fluorescence PCR assay based on Taqman probe, which designed a set of primers for Badh2 gene and could be used to identify the authenticity of basmati rice. In this study, two common molecular markers of Guangxi fragrant rice were developed by real-time PCR with probe method, which had strong practicability.

In this study, 40 samples of Guangxi fragrant rice Badh2 gene were detected by direct sequencing, and 92.5% of the samples were found to have 806 bp deletion of exons 4 and 5. AAAAGATTATGGC was mutated to TATAT (8 bp deletion and 3 bp mutation) at 4 138-4142 bp in exon 7 of Badh2 gene in 20% of them. Based on previous studies (Zeng et al., 2017), it can be seen that the flavor genotypes of local fragrant rice materials in Guangxi mainly include badh2-E4-5 and badh2-E7. The 806 bp deletion of exon 4 and exon 5 found in this study is the same as the 806 bp deletion type (badh-E4-5.1) of exon 4 and exon 5 reported by Shao et al. (2013). Meanwhile, compared with 803 bp deletion type of exon 4 and exon 5 (badh-E4-5.2) reported by Shao et al. (2011), exon 4 and exon 5 are both 94 bp deletion type and 19 bp deletion type, and only contain 3 bp more, indicating that 806 bp and 803 bp deletion type of exon 4 and exon 5 are identical in sequence.

By developing fluorescent markers based on real-time fluorescent PCR, we verified a total of 90 samples, which proved that the two fluorescent markers based on real-time fluorescence PCR has high practicability and can successfully type fragrant rice materials with these two deletion mutation types. This set of fluorescent markers can be applied to fragrant rice selective breeding and improve breeding efficiency.

Previous studies have shown that more than 200 compounds are involved in aroma of fragrant rice (Mahattanatawee and Rouseff, 2014; He et al., 2015). Out of a total of 90 materials verified by fluorescence markers, 2 and 9 of the badh2-E4-5.1 aromatic rice were particularly fragrant and fragrant, respectively. This suggests that in addition to 2AP, there may be other aromatic substances involved in aroma formation of fragrant rice. Mahattanatawee and Rouseff (2014) detected the aroma of lasmine, Basmati and lasmati rice varieties, and found 5 new aroma active substances, including 3-methyl-2-butene-1-mercaptan, geranyl acetate, β-Damasone and α-ionone, which add nutty flavor and sweetness to the aroma of rice; Hoffmann et al. (2018) tested seven fragrant rice and one non-fragrant rice in Brazil, and found that in addition to 2AP, there were five compounds, including decanal, 2-hexanone, 2-amyl furan, 1-hexanol and hexanal, which could be used to distinguish fragrant rice from non-fragrant rice. It can be seen that different studies have different conclusions, which may be related to the origin of fragrant rice varieties. In addition, 3 of the 40 fragrant rice materials were not clear about their flavor genotypes, suggesting that other genes may regulate flavor formation.

In this study, we directly sequenced the Badh2 gene of 40 fragrant rice samples from Guangxi, and found that there were two major mutations in Badh2 gene, that was, 806 bp deletion in exon 4 and exon 5 (badh2-E4-5.1) and 8 bp deletion in exon 7 (badh2-E7). The two fluorescent markers based on real-time PCR were designed for these two mutation types with high accuracy, which was suitable for the identification and screening of fragrant rice genotypes and played an important auxiliary role in fragrant rice breeding.

3 Materials and Methods

3.1 Test materials

Forty local fragrant rice materials from Guangxi without fragrance genotypes were selected (12 from Hechi City, 6 from Chongzuo City, 9 from Baise City, 3 from Nanning City, 2 from Qinzhou City, 3 from Liuzhou City, 1 from Yulin City, 1 from Guilin City, 1 from Fangchenggang City, and 1 from Laibin City, 1 from Beihai city) and used to detect SNP loci of Badh2 gene by direct sequencing method, and Nipponbare was used as non-fragrant rice control material (Table 5). All the materials were soaked and accelerated to bud. After the grains became white, the seeds were sown in plastic boxes. When the seedlings grew to 1.5 to 2.5 leaves, 10 plants with neat growth were kept and the leaves of 3 plants were taken from each material for subsequent experiments.

Fifty Guangxi native variety materials were used for molecular marker verification (Table 4). Among them, 1 was fragrant rice with 7 bp deletion of the second exon of Badh2 gene identified in previous studies, and 11 were fragrant rice with 8 bp deletion and 3 bp mutation (badh2-E7) of the seventh exon of Badh2 gene identified in previous studies. Ten fragrant rice materials were identified as 803 bp deletion type of exon 4 and exon 5 of Badh2 gene in previous studies, and 28 fragrant rice materials were to be identified (4 non-fragrant rice and 24 fragrant rice were newly collected from the third National Crop Germplasm Resources Survey and collection). Leaves of 3 plants were also taken from each material at the 4-leaf stage for subsequent experiments. 

3.2 Total DNA extraction

Refer to the CTAB method of Murray and Thompson (1980) to extract the total DNA of fragrant rice leaves. 2~3 g of leaves were crushed into a sterilized 2.0 mL centrifuge tube, frozen with liquid nitrogen and ground into powder, then preheated 700 μL 2% CTAB extract was added and mixed, then the same volume of chloroform/isopentyl alcohol (24:1) was added and mixed and centrifuged (repeat twice), then 600 μL of supernatant was absorbed and placed in sterilized 1.5 mL centrifuge tube. The same volume of pre-cooled isopropyl alcohol was added, mixed, and placed in the refrigerator at -20℃. The precipitated DNA was placed for more than 2 h, and the precipitated DNA was retained by centrifugation. 400 μL 70 % ethanol was added to rinse twice to remove impurities, and sterilized ultrapure water was added to dissolve DNA after drying.

3.3 Primer design

When SNP locus of Badh2 gene was detected by direct sequencing method, primer information for amplification of the entire Badh2 gene sequence was slightly supplemented on the basis of Shi et al. (2008) (Table 6).

3.4 PCR, sequencing and mutation type classification

All PCR reactions were performed using a 20 µL system, consisting of 10 µL 2×Ex Taq (Takara Bio, Dalian), 1 µL of upstream and downstream primers (10 µmol/L), 1 µL of genomic DNA, and 7 µL of ddH₂O. PCR cycle conditions: pre-denaturation at 95℃ for 5 min, denaturation at 95℃ for 10 s, annealing at 58℃ for 30 min, and extension at 72℃ for 1 min. There were 35 cycles of denaturation, annealing and extension. The PCR products were detected by AGAR gel electrophoresis and Sanger sequencing.

ContigExpress software (Invitrogen, USA) was used to splicing all the sequencing results, and the sequences were compared with AlignX software. Based on the comparison results, the mutations carried by Badh2 gene of the tested material were analyzed. Mutation types were classified according to the classification method reported by He et al. (2015).

3.5 Design and validation of two fluorescence markers based on real-time fluorescence PCR

According to the mutation results detected by direct sequencing method of Badh2 gene, real-time PCR primers and probes were designed for the 806 bp deletion of exon 4 and exon 5 and 8 bp deletion and 3 bp mutation of exon 7 of Badh2 gene, and the two molecular markers were commissioned to synthesize by Shanghai Sangon Biotech Co., Ltd. The sequence information of molecular marker primers is shown in Table 2. Using the designed molecular markers, 40 sequencing materials and 50 fragrant rice and non-fragrant rice materials to be verified were detected by real-time fluorescence PCR using qTOWER2.2 (AnalytikJena, Germany). PCR system: genomic DNA 1 µL, upstream and downstream primers (10 µmol/L) 0.4 µL, Taqman probe (10 µmol/L) 0.2 µL, ddH₂O supplement 20 µL. PCR cycle conditions: pre-denaturation for 5 min at 95℃, denaturation for 10 s at 95℃, annealing and extension for 1 min at 60℃. A total of 35 cycles of denaturation and extension were conducted, and fluorescence signal was collected after each extension.

Authors’ Contributions

ZY and LDT are the experimental design and executor of this study. ZY completed data analysis and wrote the first draft of the paper; YXH, XXZ, NBX, ZZQ, XZJ, CC, DGF participated in experimental design and analysis of experimental results; LDT is the architect and principal of the project, directing experimental design, data statistics, paper writing and revision. All authors read and approved the final manuscript.

Acknowledgments

This study was supported by the Basic Scientific Research Project of Guangxi Academy of Agricultural Sciences (Guinongke2019M12), the Guangxi Natural Science Foundation of China (2019JJA130104), and the Guangxi Specific Crops Experimental Station (Experimental Station of Shangsi Fragrant Rice).

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