Sequence Mutation of Exon 7 of Badh2 Gene and Aroma Identification of Fragrant Rice Varieties

Qingjie TANG Jianxiao CHEN Liqiong TANG Yisheng HAN Xiaowei YAN

Abstract In this study， we performed amplificaion and sequence analysis of exon7 of gene Badh2 of 12 fragrant rice materials， and identified the aroma of fragrant rice materials by the method of seed chewing and KOH soaking， so as to analyze the sequence mutation of exon 7 in the Badh2 gene of rice material and its corresponding relation with the flavor character. The results showed that an 8 bp deletion （aaaa--t---ggc） and a mutation of SNP （g→t） in exon 7 of Badh2 gene were found in 10 materials， including Xiangnuo， Lvjinxiang， Meixiangzhan 2， Huaxiang， Yuexiangxuan 1， Hongyuxiang， Meixiangxuan 1， Baxiangxuan 1， Taixiangxuan 1， Taixiangxuan 2. This mutation was consistent with the mutation of EU155083 sequence in GenBank and was reported for the first time in Chinese rice materials. In these 10 fragrant rice materials with mutation， Huaxiang and Meixiangxuan 1 were identified as the heterozygote genotype， and Hongyuxiang was identified as non-fragrant rice， so the sequence mutation in exon 7 of Badh2 gene in fragrant rice materials did not correspond to aroma traits one by one; and 7 materials were identified as fragrant rice， and the brown rice of Meixiangzhan 2 and Yuexiangyuan 1 had sweet taste. The results could provide a reference for the research on the genetic mechanism of rice aroma character and the promotion of fragrant rice varieties.

Key words Fragrant rice; Badh2; Sequence mutation; Aroma identification

Fragrant rice is a special kind of rice with a special aromatic smell. The rice is fragrant and delicious and rich in nutrition. According to Compendium of Materia Medica， fragrant rice can nourish the heart and lungs and prolong life after a long term of consumption. Fragrant rice is widely loved by consumers and has high economic value. Traditional fragrant rice varieties generally have poor resistance and low yield， and are difficult to promote on a large scale. There is an urgent need for new fragrant rice varieties with stronger adaptability and higher yield in agricultural production.

The formation of rice aroma is not only controlled by nuclear genes[1]， but also affected by factors such as environmental temperature， humidity， soil nutrient element content， and irradiation[2-5]. Up to now， researchers have mapped rice fragrance genes （fgr） on chromosomes 3， 4， 5， 8， 9， 11， and 12 of rice. Among them， the Badh2 gene located on chromosome 8 is one of the hot spots in fgr research[6-7]. Bradbury et al.[8] found that the 8 bp deletion （gattatgg） and the 3 bp mutation （a→t， c→t） in exon 7 of Badh2 gene on chromosome 8 of fragrant rice resulted in early termination of translation， producing non-functional betaine aldehyde dehydrogenase （Betaine aldehyde dehydrogenase 2 （Badh2）， which might cause the increase of 2-acetyl-1-pyrroline （2-AP） precursor substances and accumulation of 2-AP and finally produce fragrance. Shi et al.[9]， Shao et al.[10] and Jiang et al.[11] obtained results consistent with Bradbury et al. by studying a variety of fragrant rice materials. However， Bourgis et al.[12] found that exon 7 of the Philippine fragrant rice variety Azucena Badh2 had an 8 bp deletion （aaaa--t---ggc） and a 1 bp mutation （g→t）， and Amarawathi et al.[13] found that an 8 bp deletion （aagat---ggc） and a 2 bp mutation （a→t， g→a） occurred in exon 7 of the Badh2 gene of the Indian fragrant rice variety Pusa 1121， indicating that there may be many types of sequence mutations in exon 7 of the Badh2 gene in fragrant rice， and its genetic origin and control mechanisms for aroma traits need to be studied in depth.

In this study， the sequences of exon 7 of the Badh2 gene in 12 fragrant rice materials were compared， and the corresponding relationship between the sequence mutation of exon 7 of Badh2 gene of fragrant rice material and aroma traits was investigated through the aroma identification of the fragrant rice materials by seed chewing and KOH soaking， aiming to provide reference for the research on the genetic mechanism of rice aroma traits and the promotion of aromatic rice varieties.

Materials and Methods

Materials

The tested rice materials were 12 fragrant rice materials introduced and selected by the Cereal Crops Institute， Hainan Academy of Agricultural Sciences （see Table 1）. On January 16， 2018， the above-mentioned materials were planted in the base of Hainan Academy of Agricultural Sciences， Yongfa Town， Chengmai County， Hainan Province. 100 plants of each variety were planted with a row spacing of 16.5 cm×19.8 cm， and they were managed according to conventional rice cultivation techniques.

The KOH used was of analytical grade and was produced by Shanghai Aibi Chemical Reagent Company. Sangon Biotech （Shanghai） Co.， Ltd. was entrusted to synthesize PCR primers. Rice genomic DNA extraction kit was purchased from Sangon Biotech （Shanghai） Co.， Ltd. 2×EasyTaq PCR SuperMix was purchased from TransGen Biotech.

Genomic DNA extraction， PCR amplification and product electrophoresis detection

Genomic DNA was extracted from 50-100 mg of fresh tender leaves of rice materials at the seedling stage using a rapid extraction kit and used as templates. The functional marker GRFM04 （sequence： 5′-GTTAGGTTGCATTTACTGGGAG-3′， 5′-GAATGATGCTCAAAGTGTCT-3′） located on both sides of exon 7 of the Badh2 gene designed and developed by Wang et al.[14] was used as the primer pair for the PCR implication of exon 7 of Badh2 gene in the rice materials. The PCR reaction system （50 μl） included template （with a concentration of 50-100 ng/μl） 2 μl， forward and reverse primers （with a concentration of about 10 μmol/L） 2 μl each， 2×EasyTaq PCR SuperMix 25 μl， and double distilled water 19 μl . The reaction procedure was ① 94 ℃ for 4 min， ② 30 cycles of 94 ℃ for 45 s， 57 ℃ for 45 s， and 72 ℃ for 1 min， and ③ 72 ℃ 8 min. Then， 10 μl of the amplified product was taken for electrophoresis detection on a 2% agarose gel at 140 V for 40 min. The remaining 40 μl of amplified product was used for sequence determination.

Sequencing and sequence analysis of PCR amplification product

Using the functional marker GRFM04 as the sequencing primers， 40 μl of the above PCR product was sent to BGI for sequencing. The sequencing results were compared with the reference sequence NC_029263.1 （from Oryza sativa Japonica Group） on GenBank using the blastn function of the Nucleotide BLAST module on the NCBI website. The website address is https：//blast.ncbi.nlm.nih.gov/Blast.cgi. According to the comparison results， the sequence mutation of exon 7 of Badh2 gene was analyzed.

Seed chewing

According to the method of Zeng et al.[15]， individual ears of mature rice were collected， and 16 grains of one ear were randomly selected and shelled after sun-drying， giving the brown rice which was chewed one by one. If the 16 grains were not fragrant， the rice would be identified as a non-fragrant rice variety. If there were five consecutive grains with aroma， the rice would be identified as a fragrant rice variety. If there were both fragrant and non-fragrant grains in the 16 grains， it would be considered that there was a segregation of aroma traits， and the rice was a heterozygous genotype. Three persons identified the grains separately， and disputed materials would be identified again.

KOH soaking

According to the method of Zeng et al.[15]， 2 g of the leaves from single plant of each rice material at the tillering stage was taken and cut into small pieces， which were put in a 50 ml centrifuge tube， and then added with 10 ml of 1.7% KOH solution and covered. The sample was stood at room temperature for 10 min， followed by removing the cover and smelling the sample immediately. Three persons identified the sample， separately， and disputed materials would be identified again.

Results and Analysis

PCR product detection

Using the functional marker GRFM04 as primers and the leaf genomic DNA of 12 fragrant rice materials as templates for PCR amplification， amplified products containing the sequence of exon 7 of the Badh2 gene of each experimental material were obtained. The electrophoresis results are shown in Fig. 1. Ten materials， namely Xiangnuo， Lvjinxiang， Meixiangzhan 2， Huaxiang， Yuexiangxuan 1， Hongyuxiang， Meixiangxuan 1， Baxiangxuan 1， Taixiangxuan 1， and taixiangxuan 2， all showed a shorter band， approximately 201 bp in length. Two materials， Yuexiangzhan and Haixiangzhan， showed a longer band with a length of about 209 bp. Compared with the material with the longer band， the materials with the shorter band had an 8 bp deletion.

Sequence analysis of exon 7 of Badh2

The above PCR products were sent to BGI for sequencing. The sequencing results had been submitted to GenBank， and the sequence accession numbers were： MK064228， MK064229， MK064230， MK064231， MK064232， MK064233， MK064234， MK064235， MK064236， MK064237， MK064238， and MK064239. Using the blastn function of the Nucleotide BLAST module on the NCBI website， the above amplified product sequences were compared with the reference sequence NC_029263.1 （from Oryza sativa Japonica Group） on GenBank respectively. The results are shown in Fig. 2. The sequences of exon 7 of the Badh2 gene of such two materials as Yuexiangzhan and Haixiangzhan were completely consistent with that of the non-fragrant rice Oryza sativa Japonica Group. The exon 7 of the Badh2 gene of such 10 materials as Xiangnuo， Lvjinxiang， Meixiangzhan 2， Huaxiang， Yuexiangxuan 1， Hongyuxiang， Meixiangxuan 1， Baxiangxuan 1， Taixiangxuan 1， and taixiangxuan 2 all had an 8 bp deletion （aaaa--t---ggc） （blue base in Fig. 2） and a 1 bp mutation （g→t） （red base in Fig. 2）， consistent with the electrophoresis detection results of the PCR products， but different from the 8 bp deletion （gattatgg） and the 3 bp mutation （a→t， c→t） in exon 7 of the Badh2 gene of fragrant rice discovered by Bradbury et al.[8].

Aroma identification and its correspondence with mutation in the sequence of exon 7 of Badh2 gene

Table 2 shows the aroma identification results of the grain chewing method and KOH soaking method. According to the analysis of grain chewing method， Xiangnuo， Lvjinxiang， Meixiangzhan 2， Yuexiangxuan 1， Baxiangxuan 1， Taixiangxuan 1， and Taixiangxuan 2 were identified as fragrant rice due to aroma in continuous 5 grains of brown rice. The brown rice of Huaxiang and Meixiangxuan 1 were identified as the heterozygous genotype due to some grains with aroma and some grains without aroma， and they had segregation of aroma traits. The brown rice of Meixiangzhan 2 and Yuexiangxuan 1 were sweet when chewing. Through the KOH soaking method analysis， Huaxiang， Meixiangxuan 1 and 7 materials identified as fragrant rice by the grain chewing method were identified as fragrant rice （9 materials in total） because of their aroma in leaves. Among the 10 materials with a mutation in exon 7 of the Badh2 gene， Huaxiang and Meixiangxuan 1 were identified as the heterozygous genotype， and Hongyuxiang was identified as non-fragrant rice， so the sequence mutation in exon 7 of Badh2 gene in fragrant rice materials did not correspond to aroma traits one by one （Table 2）.

Conclusions and Discussion

Since Bradbury et al. found the specific 8 bp deletion and 3 bp mutation of exon 7 of the fragrant rice Badh2 gene， the specific 7 bp deletion of exon 2 of the Badh2 gene， the 803 bp deletion of exons 4 and 5， the 7 bp insertion of exon 8， the SNP and 3 bp insertion of exon 13， the 8 bp insertion and MITE deletion in the promoter region， and the 3 bp deletion in the 5 non-coding region were found successively in different fragrant rice materials from different regions. These mutations sometimes appear alone， sometimes in different combinations， and the correlation between each other is not clear. In some rice populations， the specificity of Badh2 gene mutations is not strong， because some materials that have mutations are non-fragrant rice， while some of the materials without mutations are fragrant rice[7-10，12-13，16-19]. In this study， the PCR product detection results and sequence analysis of Huaxiang and Meixiangxuan 1， which were identified as the heterozygous genotype by the grain chewing method， showed that the exon 7 of the Badh2 alleles of these two materials had mutations， and the exon 7 of the Badh2 gene of Hongyuxiang， which was identified as non-fragrant rice by the grain chewing method and KOH soaking method， also had a mutation. These results are consistent with Fitzgerald et al.[20]. It shows that the mutation of gene Badh2 or the exon 7 of gene Badh2 is not the only factor that controls the aroma traits of rice. It may control the expression of aroma traits through interaction with other genes or other mutations of Badh2 gene and environmental factors.

In this study， the 8 bp deletion （aaaa--t---ggc） and the 1 bp mutation （g→t） in exon 7 of the Badh2 gene of the fragrant rice materials are different from the 8 bp deletion （gattatgg） and 3 bp mutation （a→t， c→t） in exon 7 of the Badh2 gene in fragrant rice discovered by Bradbury et al.[8]， but consistent with the mutation of EU155083 sequence submitted to Genbank by Boulgis et al. in 2016[12]. It was first discovered in China. Kovach et al.[21] found that different types of mutations in the Badh2 gene have unique geographic and genetic origins. The experimental materials used in this study may have a similar genetic origin to the Philippine local fragrant rice variety Azucena studied by Bourgis et al.[12]. Further research on the diversity of Badh2 gene sequence and the genetic origin of experimental materials is of great significance for exploring the genetic control mechanism of rice aroma traits.

In this study， the grain chewing method and the KOH soaking method jointly identified 7 fragrant rice varieties from the 12 experimental materials， and the brown rice of Meixiangzhan 2 and Yuexiangxuan 1 tasted sweet when chewing， indicating the quality of fragrant rice materials is excellent， and some varieties have higher promotion value. The two materials， Huaxiang and Meixiangxuan 1 were identified as the heterozygous genotype， and there was segregation of aroma traits， which might be caused by natural hybridization during the planting process. The next step requires multi-generation single plant selection and identification of these two varieties， so as to obtain homozygous genotype fragrant rice germplasm materials as soon as possible.

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