The Efficacy of Molecular Markers Analysis with Integration of Sensory Methods in Detection of Aroma in Rice

Allele Specific Amplification with four primers (External Antisense Primer, External Sense Primer, Internal Nonfragrant Sense Primer, and Internal Fragrant Antisense Primer) and sensory evaluation with leaves and grains were executed to identify aromatic rice genotypes and their F1 individuals derived from different crosses of 2 Malaysian varieties with 4 popular land races and 3 advance lines. Homozygous aromatic (fgr/fgr) F1 individuals demonstrated better aroma scores compared to both heterozygous nonaromatic (FGR/fgr) and homozygous nonaromatic (FGR/FGR) individuals, while, some F1 individuals expressed aroma in both leaf and grain aromatic tests without possessing the fgr allele. Genotypic analysis of F1 individuals for the fgr gene represented homozygous aromatic, heterozygous nonaromatic and homozygous nonaromatic genotypes in the ratio 20 : 19 : 3. Genotypic and phenotypic analysis revealed that aroma in F1 individuals was successfully inherited from the parents, but either molecular analysis or sensory evaluation alone could not determine aromatic condition completely. The integration of molecular analysis with sensory methods was observed as rapid and reliable for the screening of aromatic genotypes because molecular analysis could distinguish aromatic homozygous, nonaromatic homozygous and nonaromatic heterozygous individuals, whilst the sensory method facilitated the evaluation of aroma emitted from leaf and grain during flowering to maturity stages.


Introduction
Aroma is the most important quality trait of aromatic rice which commands a higher price than nonaromatic rice. Thus, aromatic or scented rice plays a vital role in global rice trading [1][2][3]. Several chemical constituents including different volatile compounds are the major causes of aroma in cooked rice [4][5][6]. Moreover, Bradbury et al. [7] reported that a recessive gene (fgr) on chromosome 8 of rice which contains an 8 bp deletions and 3 Single Nucleotide Polymorphism (SNPs) produced a nonfunctional Betaine Aldehyde Dehydrogenase 2 (BADH2) enzyme resulting in aroma in rice. Many molecular markers such as RFLPs, RAPDs, STSs, and iso-enzymes have been developed for fragrant rice selection and identification [8]. Meanwhile, two types of molecular marker that is, Simple Sequence Repeat (SSR) and Single Nucleotide Polymorphism (SNP) were identified as promising marker, because they are genetically linked to aroma [6,[9][10][11]. In Addition, a perfect marker technique named Allele Specific Amplification (ASA) was developed by Bradbury et al. [12] for aroma genotyping and discriminating aromatic and nonaromatic rice. This technique was considered useful for selection of aromatic and nonaromatic rice genotypes in rice breeding programs [1]. In Malaysia, some constraints including high temperature during grain filling and ripening stage are slowing down the effectiveness of Maker-assisted selection for the improvement of aromatic rice varieties. So, aroma analysis throughout the life cycle using a combined sensory and molecular marker approach, may overcome these constraints by facilitating selection of the most appropriate parental materials for breeding programmes [13,14]. In this study, we evaluated the efficacy of molecular markers and integration of sensory methods with these molecular markers for the detection of aroma in different rice genotypes.      [12] mentioned that it is possible to differentiate nonfragrant from fragrant rice varieties and to identify fragrant homozygous, nonfragrant homozygous and nonfragrant heterozygous genotypes by using this method.
In  The Scientific World Journal During this screening process, homozygous aromatic, heterozygous nonaromatic, and homozygous nonaromatic genotypes appeared in the ratio 20 : 19 : 3, which suggests that there are 20 aromatic and 22 (19 + 3) nonaromatic F 1 individuals (Figure 2). Mohamad et al. [18] also observed a similar amplification pattern while used EAP, ESP, INSP, and IFAP primers (STS markers) in multiplex PCR condition and they identified 28 homozygous aromatic: 2 heterozygous nonaromatic: 45 homozygous nonaromatic, indicated 28 aromatic and 47 nonaromatic rice individuals. Meanwhile, another group of researchers, Bounphanousay et al. [17], detected 36 homozygous aromatic: 3 heterozygous nonaromatic: 17 homozygous nonaromatic whilst Sarhadi et al. [1] found 10 aromatic: 18 nonaromatic, also demonstrating the efficiency of these markers and 100% accuracy to detect this aroma allele. The results also confirmed the previous findings of Bradbury et al. [7] who demonstrated that the fragrance of basmati or jasmine rice were associated with the presence of a gene (fgr) on chromosome 8 of rice encoding nonfunctional BADH2.

Aroma Evaluation through Sensory Methods.
In this investigation, leaf and grain of selected parents (9 genotypes) and their F 1 hybrid individuals were used for aroma evaluation (Table 2). From the leaf aromatic test, the highest mean aroma score was 3 while the lowest was 1. Individuals from five different crosses scored 3, from eight crosses scored 2 and from one cross scored 1. From grain aromatic test, the highest scoring was 2 and the lowest was 1. Individuals from 9 different crosses scored 2 and from 6 different crosses scored 1. Comparing the mean aroma scores from both methods, most produced a better leaf aroma score (Score 3) than the grain (score 2), except for the hybrids from MR 219/Rato basmati, where leaf aroma score was 1 and grain aroma score 2. F 1 s from MRQ 50/Gharib, MR 219/Entry-7, MR 219/Rambir Basmati, MR 219/Sadri had the same aroma score (score 2) in both leaf and grain. Genotypic analysis within F 1 hybrids revealed that homozygous fragrant individuals produced better mean aroma score in leaf and grain than both heterozygous nonfragrant individuals and homozygous nonfragrant individuals, except for F 1 from MRQ 50/Entry-11, MRQ 50/Sadri. Through comparison of both genotypic and phenotypic characteristics of aroma in F 1 rice individuals, it was observed that aroma character from parents was successfully inherited to F 1 individuals.

Integration of Sensory Methods and Molecular Markers for Detection of Aroma in Rice.
Aromatic rice varieties emit aroma from their leaves, grains, and flowering organs at various stages of maturity [19]. There are many approaches used by researchers to determine the presence or absence of aroma in rice, such as evaluating aroma from leaves and grains with dilute KOH [16], analyzing the aroma using gas chromatography [20], and molecular markers related to rice aroma [12]. Sensory method facilitate the identification of aromatic and nonaromatic genotypes while molecular markers assist to identify specific allele but single tube allele specific amplification guides to identify zygosity (Homozygous or heterozygous for fgr gene) of individuals. In this study, we combined sensory tests and molecular marker methods for the detection of the presence or absence of aroma in parents and F 1 hybrids. The F 1 individuals, which were classified as having the aroma alleles (fgr gene) through molecular marker analysis, also showed presence of aroma in sensory tests. However, in less than 40% of the individuals which possessed aroma alleles and showed presence of aroma in sensory tests, the variation was from light aroma to strong aroma in leaf and grain aromatic tests. Less than 10% of the F 1 individuals did not exhibit aroma in grain aromatic test while carrying fgr gene and producing leaf aroma. In another cases, around 30-40% of F 1 individuals that were classified as homozygous nonfragrant (without fgr allele) but produced aroma in both the leaf and grain aromatic tests and <5% produced only grain aroma. Therefore, less than 50% of the F 1 individuals that were classified as aromatic or nonaromatic rice by ASA were scored the same way in both leaf and grain using sensory detection. While more than 50% of the individual's demonstrated aroma by leaf or grain or ASA or in combination of the tests. These results indicated that only molecular marker analysis or sensory methods could not represent the complete aromatic conditions. Bounphanousay et al. [17] reported that the molecular marker results agreed well with chemical analysis in most of the rice varieties, except some contrasting results such as in a local aromatic rice variety, Kai Noi Leuang, which produced aroma but was identified as homozygous nonaromatic by molecular marker analysis. They suggested that different gene location might be responsible for the observed aroma or the presence of another major aromatic compound. Sarhadi et al. [1] reported coincidence between results from 1.7% KOH sensory testing and molecular marker analysis for the classification of aromatic and nonaromatic rice, but occasionally molecular markers could not classify heterozygous and homozygous genotypes. Yi et al. [2] also reported that variation in the sensory score may arise from minor genes or environmental factors and that some rice varieties may carry minor QTLs which have an influence on rice aroma.

Conclusion
Aroma evaluation of rice genotypes is complicated in the tropical environment (countries like Malaysia) because of the large effects of environment and low sense of heritability. The integration of molecular markers and sensory tests can make the evaluation more effective. In allele specific amplification method, Entry-11, Gharib and Sadri was identified as homozygous for the fragrance allele (fgr gene), while the aroma scores were 4 in the sensory test. Genotypes Entry-13, Rato Basmati, Entry-7, Rambir Basmati, and two local checks MRQ 50 and MRQ 72 which scored 3, were also identified as homozygous for fragrance gene, but Kasturi with an aroma score of 3 in the sensory test was found as homozygous nonfragrant. However, homozygous aromatic F 1 individuals possessed higher mean aroma score in leaf and grain compared to heterozygous and homozygous The Scientific World Journal 5