What is the Molecular Basis of Aroma in Biriyanicheera - A Tropical Aromatic Rice Genotype

The aromatic rice cultivars possess excellent aroma generally when grown in their favourable and specic environments. An early maturing selection from a Kerala aromatic local landrace with short grains, named ‘Biriyanicheera’, when grown in normal tropical conditions was suciently fragrant. The present study focused on the analysis of aroma in ‘Biriyanicheera’ rice genotype through molecular methods. The seeds of two aromatic rice varieties viz., Biriyanicheera and Gandhakasala (from Palakkad, Thrissur and Ernakulam districts) along with one non-aromatic rice variety Triveni (control) were used for the study. The BADH2 gene was amplied in all the three rice varieties. Upon sequencing the amplied PCR products of genomic DNA, the mutation in BADH2 gene was detected. The sequencing results of aromatic varieties revealed the presence of 8 base pair mutation in exon 7 in Biriyanicheera and Gandhakasala, whereas this mutation was absent in the non-aromatic variety Triveni. This indicated that aroma production in Biriyanicheera variety is due to similar mutation in BADH2 gene as that of the popular scented rice Basmati.


Introduction
Aromatic rice, is considered as a special group of rice due to the presence of excellent aroma and superior grain quality. India holds the pride for being the homeland for the highly popular famous nature's gift, Basmati rice. With its superior, scented, long slender grain rice, Basmati is acclaimed as the major aromatic rice genotype around the globe and fetches premium price in national and international markets.
At the same time, India is the homeland for many traditional aromatic rice cultivars also, which are comparable with basmati genotypes in their aroma, making many of these unrecognized landraces as an attractive low-cost alternative to the highly priced Basmati. The short grains, low yield, and lack of prominent improvement methods have kept the non-basmati cultivars, behind in the line. However, there is an immense potential for many non-basmati genotypes to hold a major share in aromatic rice market, if these genotypes are improved with high yield and other preferred qualities (Mondal et al., 2021) Buttery et al. (1983) was the rst person to analyse the aroma of cooked rice, in which 2-acetyl-1-pyrroline (2AP) was identi ed as a major contributor of aroma. But some studies had revealed the presence of many other volatile compounds, other than 2-AP to be responsible for the aroma in rice grains (Jezussek et al., 2002;Sansenya et al., 2018). The aroma present in different aromatic genotypes originated from diverse places and its level of expression tends to vary with genetic as well as environmental parameters.
The genomic history of the aromatic population was unraveled from the diversity found in the unique genome-wide sativa-ru pogon data set (Civan et al., 2020). Several molecular markers had been developed to utilise the aroma trait in rice breeding programmes (Ahn et al., 1992;Garland et al., 2000;Jin et al., 2003). Among the 25 polymorphic SSR markers across 24 aromatic rice genotypes, RM527 might be the best marker for identi cation and diversity estimation of aromatic genotypes followed by RM1, RM22565 and RM207 markers (Kumar et al., 2018).
The fragrance nature in rice is controlled by a recessive trait in homozygous condition, on chromosome 8 (Bradburry et al., 2005). The mutation in BADH2 gene leads to non-functional enzyme synthesis, which is responsible for aroma production. Bradbury et al. (2005a) showed the presence of a gene having homology with a gene coding for betaine aldehyde dehydrogenase (BAD), consisting signi cant polymorphisms in the coding region of fragrant plants. Later the accumulation of 2-acetyl-1-pyrroline in fragrant rice was proposed to be due to the presence of mutations resulting in loss of function of the 'fgr' gene by introducing a stop codon in conserved amino acid sequence of BADs.
In Kerala, Wayanad Jeerakasala' and Wayanad Gandhakasala', have already been GI tagged and gained considerable attention in the international market for their suitability in multinational cuisines. However, these aromatic rice cultivars are found to possess higher fragrance, when grown in their own speci c geographical niches, for their growth and proper expression of aroma and other unique characteristics.
There has been decrease in aroma when the same cultivars were grown in regions other than their normal traditional growing regions. This posed di culty in popularizing them in other major rice growing tracts of the country. If a genotype with good aroma that can be grown in normal tropical conditions is available, it can be introduced in other parts also. The early maturing selection from a Kerala aromatic local landrace with short grains, named 'Biriyanicheera', when grown in normal tropical conditions was observed to have su cient aroma. It has the capacity to produce su cient aroma wherever it is grown, irrespective of the geographical or environmental conditions. In this context, the present study focuses on the molecular analysis of aroma gene in 'Biriyanicheera' rice genotype through gene sequencing.

Materials And Methods
Rice varieties were used in this study are as detailed below : a) Biriyanicheera -test variety (newly identi ed and recommended aromatic rice variety for tropical areas)

Plant materials
The aromatic rice genotypes Biriyanicheera and Gandhakasala (check genotype) grown in of Kerala along with a Non-aromatic genotype Triveni grown in Mulangunnathukavu from Thrissur, Kerala during the period, 2017-2018 were used for the study. The average days for maturity of rice genotypes Biriyanicheera, Gandhakasala and Triveni variety were 105, 120 and 105 days respectively. Pusa Basmati 1, the highly popular and well studied aromatic rice variety was used for the validation of speci c primers designed for BADH2 gene.

Raising of rice plants
Seeds of aromatic rice genotypes, Biriyanicheera and Gandhakasala and the non-aromatic rice genotype Triveni were germinated in petri plates lined with moist tissue paper and allowed to germinate. After a week, the germinated seedlings were transferred to a pot. Later, 21 days old seedling were transplanted into new pots at three seedlings per pot.

Molecular characterization 2.3.1 DNA isolation
Genomic DNA was isolated from young leaves of rice plants grown in pots. Fresh and green leaves yielded good quality of DNA in required quantity. The leaves were collected early in the morning from all the three genotypes. The collected leaves were immediately covered in aluminium foil and carried to the laboratory.
The extraction of genomic DNA from the leaves was performed through CTAB method (Dellaporta et al., 1983).

Quantity and quality determination of genomic DNA
The purity of DNA was analysed using NanoDrop ND-100 spectrophotometer. Nucleic acids and proteins show absorption maxima at 260 nm and 280 nm respectively. NanoDrop recorded the absorbance at wavelengths 260 and 280 nm and purity was determined by the ratio OD (A 260/280 ). The OD values obtained between 1.8 and 2.0 shows the purity of the DNA, which is free from proteins. The quantity of DNA in the sample was mentioned as ng/µl. The quality was checked by agarose electrophoresis on 0.8 per cent agarose gel. The gel pro le was examined for intactness, clarity of DNA band, the presence of contaminants such as RNA and proteins.

Designing of primers for PCR
The BADH2 gene sequence (size = 6.1 Kb) retrieved from the Rice Genome Annotation Project was suitably divided into seven distinct regions based on the position of exons for convenient PCR ampli cation of exons. The primer details are mentioned in Table 1.

Polymerase Chain Reaction (PCR)
The total volume of the PCR reaction was 10 µl constituted of Taq assay buffer, dNTPs, forward and reverse primers, Taq DNA polymerase, template DNA and sterile distilled water. The ampli cation pro le was as follows: The initial denaturation was carried out at 95˚C for 3 minutes (min). The denaturation (94˚C for 50 sec), primer annealing (around 57˚C for 30 sec) and extension (72˚C for 1 min), were carried out for 35 times.
Final extension was carried out at 72 ˚C for 10 min.

PCR protocol using Dimethyl Sulphoxide (DMSO) :
The total PCR reaction volume was 50 µl. Three per cent of DMSO (1.5 µl for 50 µl reaction) was used for the ampli cation in addition to Taq assay buffer, dNTPs, forward and reverse primers, Taq DNA polymerase, template DNA and sterile distilled water. The initial denaturation was carried out at 95˚C temperature for 8 min. The denaturation (94˚C for 3 min), primer annealing (around 55˚C for 30 sec) and extension (72˚C for 1 min) steps were repeated for 35 times. The quality of the ampli ed PCR product was checked on 1.2 per cent agarose gel by agarose gel electrophoresis. The ampli ed PCR products were sequenced at AgriGenome Labs Pvt. Ltd., Kochi, Kerala.

Analysis of sequence data
The forward and reverse DNA sequences obtained from sequencing results were used to construct contigs using CAP3 software. The contigs were then aligned with the reference BADH2 sequence retrieved from the Rice Genome Annotation Project using CLUSTAL OMEGA software to analyze the possible mutations in the exonic regions of genomic DNA.

Validation of speci c primers of BADH2 gene :
The speci c primers designed for BADH2 gene were evaluated for region 1 covering the exons 1 and 2 in the three aromatic rice genotypes Biriyanicheera, Gandhakasala and Pusa Basmati 1 along with the nonaromatic rice genotype Triveni. The genomic DNA was used for the ampli cation of PCR products.

DNA isolation and PCR
Fresh and young leaves of Biriyanicheera, Gandhakasala, and Triveni collected during early morning hours were used for DNA isolation. The quality of DNA was assessed using agarose gel electrophoresis. Single intact band of good intensity was obtained indicated that the isolated DNA was of good quality. The DNA was free from RNA and protein contamination. The concentration of the DNA was between 300 and 800 ng/µl. The A260/A280 ranged between 1.8 and 2.0 indicating good quality of genomic DNA.

Analysis of sequence data
The genomic DNA sequence of BADH2 gene present in all genotypes was aligned by Multiple Sequence Alignment (MSA) by using CLUSTAL OMEGA software. The reference sequence, Locus Id: LOC_Os08g32870, obtained from Rice Genome Annotation Project, was used for the comparison of sequences. MSA identi ed the presence of 8 base pair mutation and 3 SNPs in the exon 7 of both the genotypes Biriyanicheera and Gandhakasala. The mutation was absent in non-aromatic genotype Triveni. The sequences were further compared with the reported exon 7 sequence of Basmati genotype available in NCBI. The MSA showed that mutation in both Biriyanicheera and Gandhakasala were similar to the reported mutation in Basmati cultivars as mentioned Table 2. Table 2 Sequences of exon 7 mutation in aromatic and non aromatic rice varieties Page 8/15

Validation of speci c primers of BADH2 gene
The speci c primers designed for region 1 covering the exons 1 and 2 of BADH2 gene was used for the ampli cation of genomic DNA. The ampli cation was successful only in non-aromatic genotype Triveni, whereas aromatic genotypes Biriyanicheera, Gandhakasala and Pusa basmati 1 showed no ampli cation (Plate 5). The primer 1 produced PCR band with desired band size of 846 bp in Triveni genotype.

Discussion
The aromatic rice varieties, 'Basmati' (popular around the globe) and 'Gandhakasala' (popular in Kerala, India) are having GI tags, indicating that for their growth and proper expression of aroma and other unique characteristics, they need their own speci c geographical niches. Whereas, the test variety is having the capacity produce su cient aroma in wherever it is grown, irrespective of the geographical or environmental conditions. Aroma detection of rice grains was performed by Sensory evaluation test by DUS (Distinctness, Uniformity, Stability) protocol prescribed by IIRR, Hyderabad. During the analysis of grains, Biriyanicheera had good to strong aroma, Gandhakasala had Medium to good aroma while Triveni had no aroma.
The genetic basis of rice aroma trait (fgr) explored by many researchers in early 1992, revealed the recessive nature of the gene and it was located at 4.5 cM distance from the marker RG28 (Ahn et al., 1992). Later, the aroma gene fgr was located to be between markers RM223 and RM342 (Wanchana et al., 2005). The map-based cloning and sequencing of the fgr region identi ed signi cant difference in the Badh2 gene between scented and non-scented cultivars. The mutation in 7th exon of Badh2 gene, which led to loss of Badh2 protein, was thought to be controlling the aroma in rice. Similarly, the ampli cation of BADH2 (or fgr) gene was attempted to understand the actual molecular reasons for the special nature of Biriyanicheera genotype. The sequencing of the genomic DNA consisting of exons of BADH2 gene helped to unravel the molecular nature in Biriyanicheera. The occurrence of previously reported 8bp mutation (TATATATTT) in the exon 7 of BADH2 gene in both the Biriyanicheera and Gandhakasala genotypes suggests that the aroma gene present in them must have followed similar path of evolution and