Int. J. Biosci.

2016

International Journal of Biosciences | IJB |

ISSN: 2220-6655 (Print), 2222-5234 (Online)
http://www.innspub.net
Vol. 8, No. 1, p. 47-54, 2016

RESEARCH PAPER OPEN ACCESS

Identification of fragrance gene in some elite advance lines of

rice cultivated in foothills of the Himalayas

Hamid Ali1*, Fida Muhammad Abbasi 1, Habib Ahmad 1, Aziz-Ud-Din1 , Abzar 1,

Abdullah Khan1 , Muhammad Abid Khan 2 , Irfan Ullah1 , Aqib Zeb1 , Adnan Sarwar 1

1
Department of Genetics, Hazara University Mansehra, Pakistan
2
Department of Botany, Hazara University Mansehra, Pakistan

Key words: Oryza sativa, Aroma, Grain Quality, Polymerase Chain Reaction (PCR).

http://dx.doi.org/10.12692/ijb/8.1.47-54 Article published on January 20, 2016

Abstract
A molecular survey was conducted for the screening of fragrance (fgr) gene in some elite advance lines of rice
developed by Dr. Fida Muhammad Abbasi, Professor at Department of Genetics Hazara University Mansehra.
Sequence Tag Site (STS) marker RG 28L was used in this study that amplified 140 and 120 bp fragment in
fragrant and non-fragrant genotypes, respectively. Among the cultivated varieties Basmati-385 and Swat-1
showed the presence of fgr gene (140 bp amplicon) while IRBB59, JP-5, Fakhre Malakand, and IR24 were
lacking this gene. Among the advance lines 12 genotypes showed the presence of fgr gene (140), two genotypes
(NPT-86 and Line 36) were segregating while the remaining 16 genotypes were lacking this gene. Grain length of
genotypes was also measured that ranges from 4.67 t0 8.10 mm. On the basis of grain length the genotypes were
categorized into short, medium, long and extra-long. In this study 4 genotypes possessed extra-long, 17 long, 13
medium and only 2 have short grains.
* Corresponding Author: Hamid Ali  biotechd.ali@gmail.com

47 Ali et al.
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Introduction properly assessed, therefore the present study was

Rice (Oryza sativa L.) is a staple food for more than being proposed with the aims to identify fragrance
half of the world's population (Marathi et al., 2012). (fgr) gene in these advance lines.
In rice physical grain quality plays an important role
in consumer preference. Juliano and Duff (1991) Materials and methods
concluded that improvement of physical grain quality Plant material
is the second major objective of rice breeding Plant material was comprised of 6 cultivated varieties
programs after yield in many rice producing countries viz. Basmati-385, IRBB59, JP-5, Fakhre Malakand
of the world. The physical grain quality of rice is a and 30 elite advance lines of rice. Seeds of cultivated
complex trait that is composed of many components varieties were obtained from the Gene Bank of Plant
such as appearance quality, cooking quality, eating Genetic Resource Institution (PGRI), NARC (National
quality and nutritional quality. Each one of these Agriculture Research Centre), Islamabad, Pakistan
components also consists of many attributes whose while the advance lines were provided by Dr. Fida
values are determined not only by their physio- Muhammad Abbasi that have been developed at
chemical properties but also by the history and Hazara University, Mansehra, Pakistan. These lines
cultural traditions of the people in the human were planted at National Tea and High value crops
communities who consume the rice (Tan et al., 1999). Research Institute (NTHRI), Shinkiari Mansehra
One of the most valuable traits in high-quality rice is Pakistan (Fig. 1).
aroma or fragrance, which is important for consumer
preference and global trade (Singh et al., 2000). DNA extraction and PCR analysis of advance lines of
Fragrant rice emits specific scent in the fields at the rice for the presence of (fgr) gene
time of flowering, at harvesting, in storage, during Five seeds of each genotype were taken in 1.5 ml
milling, cooking and eating. Economically, it Eppendorf tubes and 500µl of 2x CTAB buffer (50mM
possesses an extraordinary position in the global Tris-HCl, pH 8.0, 25mM EDTA, 300mM NaCl and
business sector because of its pronounced, pleasant 2% CTAB) was added to it, incubated at 65° C for 1
and unique scent and mouth feeling taste after hour and then crushed with squashing needles. 500µl
cooking. Fragrant rice is preferred over non-fragrant Phenol: Chloroform: Isoamyl alcohol (25: 24: 1) was
rice due to special occasions and for export, and thus added and leave at room temperature for 30 minutes.
they command a higher market price. A better Centrifuge the tubes at 8000 rpm for 15 minutes.
understanding of the factors that contribute to the 400µl clear supernatant was transfer to new tubes
overall grain quality of rice will lay the foundation for and equal volumes of 2-propanol was added and
developing new breeding and selection strategies for incubated at – 20° C for 2 to 3 hours. Spin at 8000
combining high quality, with high yield. This is rpm for 10 minutes to form DNA pellet. Supernatant
necessary to meet the growing global demand for high was discarded and pellet was washed with 70%
quality rice while offering producing countries ethanol. The tubes were kept invert for few hours to
additional opportunities for generating higher export become dried. Then 50µl TE buffer was added to each
revenues. tube. 5µl DNA sample of each genotype was checked
on electrophoresis on 1% agarose gel and stained with
Pakistan is famous for exporting high quality basmati ethidium bromide. The concentration of extracted
rice but rice yield is stagnant from the previous few genomic DNA was measured by Spectrophotometer
decades. Dr. Fida Muhammad Abbasi Professor at and was adjusted from 20 to 50 ng/µl by using
Department of Genetics, Hazara University Mansehra sterilized distilled water and stored in Eppendorf
Pakistan has developed advance lines of rice in order tubes at 4°C for further use.
to break yield stagnation. These lines are high
yielding but their physical grain quality has not been Amplification of fgr gene was carried out using allele

48 Ali et al.
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specific primers RG 28L (Table 1). Amplification absence of fgr linked DNA fragments.
reactions was carried out in 16 ul reaction volumes
containing 1µl genomic DNA (20 – 50 ng/µl), 0.5µl Determination of grain size
each of forward and reverse primers (10 μM / µl), Physical grain quality including grain size was
1.2µl of dNTPs (25 mM each) , 0.4 µl of Taq DNA determined by measuring the grain length of ten
Polymerase (2 units, Enzynomix), 1X Taq Buffer and unbroken milled kernels. On the basis of average
1.6µl MgCl2 (2.5 mM). length, the grains were classified by using the scale as
reported by Khush et al., 1979 (Table 2).
PCR amplification was carried out in DNA Thermal
Cycler (Applied Bio System) set at: an initial Results and discussions
denaturation of 5 min at 94 C; 32 cycles of 94 C for DNA Extraction
Genomic DNA was extracted from fresh seeds of
45 sec, 55 C for 45 sec, and 72 C for 1:30 sec. One
cultivated varieties and advance lines of rice using
additional cycle of 7 min at 72 C was used for final CTAB method as reported by Delaporta et al., 1983,
extension. Amplification products were resolved by with few modifications. High quality genomic DNA
electrophoresis on 3% agarose gel run in 1 X TAE was obtained as shown in Fig. 2. The concentration of
buffer. The amplified products were observed under DNA samples were adjusted from 20 to 50 ng/µl with
UV light after staining with ethidium bromide (10 the help of Spectrophotometer.
ug/ml). The data was scored for the presence or

Table 1. Primer sequence of RG28L used in this study.

Forward 5´-GATCTCACTCCAAGTAAACTCTGAC-3’
Reverse 5´-ACTGCCATTGCTTCTGTTCTC-3´

Table 2. Scale for measurement of grain size of rice.

Scale Size category Length in mm
1 Extra-long More than 7.5
3 Long 6.61 to 7.5
5 Medium 5.51 to 6.6
7 Short less than 5.5

PCR analysis of cultivated varieties and advance Genotypes that possessed fgr gene in homozygous
lines of rice for the presence of fgr gene condition include line-5, line-34, line-18, line-39,
A molecular survey was conducted for the line-59, line-71, line 87, line-88, line-109, line-111,
identification of fgr gene in advance lines of rice. STS line-130 and line-136.
marker RG 28L was used in this study that amplified
140 and 120 bp fragment in aromatic and non- The rest of advance lines such as line-35, line-38,
aromatic genotypes, respectively. Among the line-42, line-67, line-77, line-82, line-86, line-87, line-
cultivated varieties Basmati-385 and Swat-1 showed 93, line-101, line-103, line-107, line-108, line-154, and
the presence of fgr gene (140 bp amplicon) while line-158 were observed to be non-aromatic as lacking
IRBB59, JP-5, Fakhre Malakand, and IR24 were fgr gene.
lacking this gene (120 bp). Among the advance lines
12 genotypes showed the presence of fgr gene (140), The data was scored using “+” sign for presence of
two genotypes (NPT-86 and Line 36) were gene (fgr) and “–” sign for absence of gene (Table 3
segregating and the rest were non aromatic. and Fig. 3).

49 Ali et al.
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Table 3. Screening of cultivated varieties and advance lines of rice for the presence of fragrance (fgr) gene.
S/No Varieties/ Lines Target gene (fgr) S/No Varieties/ Lines Target gene (fgr)
1 Bas-385 + 19 Line-77 -
2 IRBB-59 - 20 Line-79 -
3 JP-5 - 21 Line-81 -
4 F. Malakand - 22 Line-82 -
5 Swat-1 + 23 Line-86 -
6 IR-24 - 24 Line-87 +
7 Line-5 + 25 Line-88 +
8 Line-34 + 26 Line-93 -
9 Line-18 + 27 Line-101 -
10 NPT-86 ± 28 Line-103 -
11 Line-35 - 29 Line-107 -
12 Line-36 ± 30 Line-108 -
13 Line-38 - 31 Line109 +
14 Line-39 + 32 Line-111 +
15 Line-42 - 33 Line-130 +
16 Line-59 + 34 Line-136 +
17 Line-67 - 35 Line-154 -
18 Line-71 + 36 Line-158 -

Table 4. Grain length of cultivated varieties and advance lines of rice (Oryza sativa L.).
S/No Genotypes Grain Length (mm) S/No Genotypes Grain Length (mm)
1 Bas-385 6.87 19 Line-77 7.16
2 IRBB-59 6.66 20 Line-79 7.40
3 JP-5 4.67 21 Line-81 6.57
4 F. M 6.06 22 Line-82 6.13
5 Swat-1 6.40 23 Line-86 7.20
6 IR-24 6.80 24 Line-87 7.57
7 Line-5 6.60 25 Line-88 7.57
8 Line-34 5.73 26 Line-93 7.10
9 Line-18 8.10 27 Line-101 6.70
10 NPT-86 7.00 28 Line-103 7.07
11 Line-35 6.07 29 Line-107 6.47
12 Line-36 6.60 30 Line-108 7.10
13 Line-38 6.30 31 Line109 7.13
14 Line-39 6.60 32 Line-111 7.17
15 Line-42 6.57 33 Line-130 5.80
16 Line-59 6.77 34 Line-136 7.00
17 Line-67 8.10 35 Line-154 5.00
18 Line-71 7.43 36 Line-158 7.47

Fragrant rice is preferred over non-fragrant rice and aldehydes, ketones, acids, esters, phenols, pyridines,
in this way they summon a higher business sector pyrazines and other compounds have been identified
cost. Fragrance or fine flavor of cooked rice has been in cooked rice. Aroma development in rice grain is
shown to be composed mainly of formaldehydes, influenced by both genetic and environmental factors.
ammonia and hydrogen sulfide. Some researchers The biochemical basis of aroma was identified as 2-
reported that an increase of propanol, pentanal, and acetyl-1-pyrroline (Tanchotikul & Hsieh 1991). The
hexanal during storage seemed to be responsible for conventional methods of plant selection for aroma are
fragrance in rice. As many as 100 volatile aromatic not easy because of the high effects of the
components such as hydrocarbons, alcohols, environment and the very low narrow sense

50 Ali et al.
 Int. J. Biosci. 2016

heritability of fragrance. Molecular approaches such fragrant rice (Cordeiro et al., 2002). In addition, an
as PCR and RFLP analysis using single nucleotide allele specific amplification assay (ASA) allows us to
polymorphism (SNPs), simple sequence repeats distinguish fragrant and non-fragrant genotypes and
(SSRs) and sequence tag site (STS), which are tightly identify homozygous and heterozygous individuals in
linked to aroma and have the advantage of being a population segregating for aroma (Louis et al.,
simple, rapid and only requiring small amounts of 2005).
tissue, have been developed for the selection of

Fig. 1. Advance lines of rice grown at National Tea and High value crop Research Institute (NTHRI), Mansehra,
Pakistan.

Fig. 2. Genomic DNA extracted from fresh seeds of cultivated varieties of rice using CTAB method and
resolved on 1% agarose gel.

Genetic studies carried out on the inheritance of has facilitated early selection for the presence or
aroma in rice revealed that a recessive nuclear gene absence of scent, and to identify the nature of locus
controls fragrance in rice (Dong et al., 2001). (homozygous or heterozygous condition). It has
Molecular markers that are closely linked to aroma proved very useful for the rapid incorporation of scent

51 Ali et al.
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character into rice breeding lines. Ahn et al., (1992) (nearly isogenic lines). Linkage association of the
reported a DNA marker that is closely linked to clone with the gene was verified using F3 segregating
fragrance in rice and located on chromosome 8. The data. RFLP analysis showed that the gene is linked to
chromosome segments introgressed from the donor a single copy DNA clone, RG28, on chromosome 8 at
genome were distinguished by RFLPs, among NILs a distance of 4.5 cM (Fig. 4).

Fig. 3. PCR analysis of cultivated varieties and advance lines of rice for the presence of fgr gene. (Arrow
showing 140 bp bands linked to fgr gene).

Grain Size
Grain length of advance lines and cultivated varieties
of rice used in this study was also measured that
ranges from 4.6 to 8.1 mm. Maximum grain length
was possessed by line-18 and line-67 (8.10 mm),
followed by line-87 and line-88 (7.57 mm). Minimum
grain length was recorded for cultivated variety JP-5
(4.67 mm) followed by line-154 which was 5 mm.
Among the cultivated varieties three genotypes viz.,
Basmati-385, IRBB59 and IR24 possessed long
grains; two genotypes Fakhre Malakand and Swat-1
medium size grains while only JP-5 showed short
grains. Among the 30 Advance lines 2 possessed
extra- long grains, 16 long, 11 medium and only one

Fig. 4. RFLP map of chromosome 8 of rice showing (line 154) short grains (Table 4 and Fig. 5).

the location of fgr and linked markers. A map of

chromosome 8 was developed by segregation of Grain length is an important agronomic trait for

RFLPs of rice genomic (RG), cDNA (RZ), and oat artificial selection in rice breeding. Breeders tend to

cDNA (CDO) clones based on F3 population derived select plants with large seed size for high yield and

from a cross of aromatic and non-aromatic genotypes. appropriate grain size for milling yield and market

CDO109, RZ562 and RG1034 were not polymorphic preferences. However, it is difficult for breeders to

in the NIL survey but were polymorphic between the improve grain size efficiently by phenotypes, since the

mapping parents (Ahn et al., 1992). traits are quantitatively inherited (McKenzie and

52 Ali et al.
 Int. J. Biosci. 2016

Rutger, 1983). Many quantitative trait loci (QTLs) for characterized recently (Song et al., 2007; Shomura et
grain size have been detected, of which four genes, al., 2008; Wang et al., 2008; Weng et al., 2008).
grain size on chromosome 3 (GS3), grain weight on Among them GS3, is a major QTL for grain length and
chromosome 2 (GW2), grain incomplete filling on weight and minor QTL for grain width and thickness
chromosome 1 (GIF1), and seed width on in rice. A causal C–A mutation in GS3 is highly
chromosome 5 (qSW5/GW5), have been isolated and associated with rice grain length (Fan et al., 2009).

Fig. 5. Grain size of advance lines of rice used in this study. The length of each bar shows number of genotypes.

Conclusions Cordeiro GM, Christopher MJ, Henry RJ,

Most of the advance lines used in this study showed Reinke RF. 2002. Identification of microsatellite
fragrance gene (fgr) and possessed long grains. These markers for fragrance in rice by analysis of the rice
lines can be released as new varieties and could be genome sequence. Molecular Breeding 9(4), 245-
used for further improvement in breeding programs. 250.
However, we recommend these lines for further http://dx.doi.org/10.1023/A:1020350725667
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