Use Molecular Markers Analysis and Sensory Methods in the Revelation of Fragrance in Iraqi Rice

Abstract

Allele-Specific Amplification together with four primers External Sense Primer ESP, External Antisense Primer EAP, Internal Fragrant Antisense Primer IFAP, and Internal Non fragrant Sense Primer INSP) and sensory valuation with grains and leaves were executed to differentiate aromatic rice varieties. Our results such as The PCR results showed that band 355bp in seven varieties of rice represent the nonfragrant allele, and showed bands of 257 bp in nine varieties of rice represent the fragrant allele. While sensory test showed six varieties of nonfragrant and ten varieties fragrant. Sensory evaluation or molecular analysis alone couldn't define aromatic condition fully. The incorporation of sensory methods with molecular marker analysis was observed as fast and reliable for a check of aromatic varieties.

Full text

Use Molecular Markers Analysis and Sensory Methods in the
Revelation of Fragrance in Iraqi Rice
Zeina Thamer Abd ULHussein ALrufaye1, Ban A.H.AL Khafaji2, Warqa'a Muhammed Sheriff Al-sheikh3
1 Department of biology, College of science, University of kerbala, Iraq
2 Department of biology, College of Education for pure sciences, university of kerbala, Iraq.
3 College of Dentistry, University of Al- Qadisiya, Iraq
Abstract Allele-Specific Amplification together with four primers External Sense Primer ESP, External Antisense Primer EAP, Internal Fragrant
Antisense Primer IFAP, and Internal Non fragrant Sense Primer INSP) and sensory valuation with grains and leaves were executed to
differentiate aromatic rice varieties. Our results such as The PCR results showed that band 355bp in seven varieties of rice represent the
nonfragrant allele, and showed bands of 257 bp in nine varieties of rice represent the fragrant allele. While sensory test showed six varieties of
nonfragrant and ten varieties fragrant. Sensory evaluation or molecular analysis alone couldn't define aromatic condition fully. The
incorporation of sensory methods with molecular marker analysis was observed as fast and reliable for a check of aromatic varieties.
Keywords: Oryza sativa L, Allele Specific Amplification (ASA), fgr gene
INTRODUCTION
Aroma is the furthermost important characteristic of rice which
leading a higher price. Consequently aromatic rice is playing a
active role in global rice trading (1,2) .One of the main reasons for
the aromatic emission of cooked rice because it contains various
volatile compounds chemically (3). Aromatic rice varieties release
aroma in vegetative stages more than at mature stages (4). The
investigators used many methods to decide the existence or non-
existence of aroma in rice, for instance, analyzing the aroma using
Sensory test, gas chromatography and dilute KOH (5), and
molecular markers associated with rice fragrance (6). Affording to
many studies that designated the recessive gene (fgr) on
chromosome eighth on chromosome eighth of rice which contains
an 8bp deletion 5 GATTATGG3 and 3 single nucleotide
polymorphisms in the exon 7 of the badh2gene encoding betaine
aldehyde dehydrogenase2 BAD2 made a nonfunctional Betaine
Aldehyde Dehydrogenase 2 (BADH2) enzyme with amplified
levels of 2-acetyl-1-pyrroline 2AP is the chief composite
responsible for the aroma (7). consequential in aroma in rice. On
the other hand, there are other conservative aromatic rice
varierties which display different volatile compounds other than
2AP such as propanol, 2-butanone, acetaldehyde, hexanol,
pentanal etc (8). Several Several molecular markers for instance
iso-enzymes, SSR, STSs, RAPDs, and RFLPs have been
improved for aromatic rice identification and selection (9).
Furthermore, an idealistic marker technique called Allele Specific
Amplification was advanced by(6). This technique was considered
beneficial for distinguish aromatic and non aromatic rice in
breeding databases( 1).
In this study we evaluate the efficiency of molecular markers and
incorporation of sensory approaches for the recognition of
aromatic in diverse varieties of Iraqi rice.
MATERIALS AND METHODS
The sixteen varieties were collected from Mashkhab Research
Center in AL-Najaf Iraq Tab.1
Extraction of DNA.
DNA was extracted from Young leaves after 25 days from
agriculture, according to (10 ) Quality of DNA was verified by
Gel electrophoresis and quantification was achieved by using the
bio drop technique .The sequences of primers were as follows
TTGTTTGGAGC TTGCTG ATG (ESP),
ATAGGAGCAGCTGAAATA TATACC (IFAP),
CTGGTAAAAAGAT TATGGC TTCA (INSP), and
AGTGCTTTACA AAGTCC CGC (EAP) According to this
reference (6)
Table 1: List of Iraq Oryza Sativa L. cultivars analyzed in this
research
No.
Name of
Varieties
Source Pedigree
1 Aba 1
Mashkhab Research
Center in AL-Najaf
Aba Research
Center
2
Eanber
albaraka
Mashkhab Research
Center in AL-Najaf
Indian
3 Forat 1 Mashkhab Research
Center in AL-Najaf
Research
Intemational Rice
Institute
4 Ghadeer Mashkhab Research
Center in AL-Najaf
Research
Intemational Rice
Institute
5 Barnamaj4 Mashkhab Research
Center in AL-Najaf
Research
International Rice
Institute
6 Yasmine
Mashkhab Research
Center in AL-Najaf
Vietnam
7 Buhooth1
Mashkhab Research
Center in AL-Najaf
Turkey
8 Eanber 33
Mashkhab Research
Center in AL-Najaf
Local
9 Dijlah
Mashkhab Research
Center in AL-Najaf
Chinese
10 Meshkhab 2 Mashkhab Research
Center in AL-Najaf
Research
Intemational Rice
Institute
11 Meshkhab1 Mashkhab Research
Center in AL-Najaf
Research
Intemational Rice
Institute
12 First genotype
(T85) Mashkhab Research
Center in AL-Najaf
Research
Intemational Rice
Institute
13
Second
genotype( HT-
1)
Mashkhab Research
Center in AL-Najaf
Research
Intemational Rice
Institute
14
Third
genotype (LT-
2)
Mashkhab Research
Center in AL-Najaf
Research
Intemational Rice
Institute
15
Fourth
genotype (BT-
7)
Mashkhab Research
Center in AL-Najaf
Research
Intemational Rice
Institute
16 Fifth genotype
(aromatic 64) Mashkhab Research
Center in AL-Najaf
Research
International Rice
Institute
Zeina Thamer Abd ULHussein ALrufaye et al /J. Pharm. Sci. & Res. Vol. 10(10), 2018, 2531-2533
2531

PCR amplification
The volume of the PCR reaction mixture is 20μL that included
2.0 L of 10X reaction buffer (with 20mM Mg+), 0.2 L of
10mM dNTPs mix, 0.25 L of YEA taq DNA Polymerase
(Yeastem Biotech Co. Ltd., Taiwan), DNA template 5.0 L , 0.4
L of each primer EAP, and ESP, 0.5 L of primer INSP and 0.5
L of IFAP, Amplification was carried out using a thermal cycler.
The amplification procedure is as follows 5min at 94℃ followed
by 35 cycles of 30 s denaturation at 94 , of 30 s annealing at 53℃
and 1min extension at 72℃, concluding with the final extension of
7min at 72℃. Amplification products were electrophoresed on 2%
agarose gels and stained with ethidium bromide (EB). was done to
analyse PCR products. PCR fragment size was estimated through
100 bp ladder( Bioneer – USA). The bands representing aromatic,
non aromatic, for fgr gene were analyzed by Allele Specific
Amplification technique
RESULTS AND DISCUSSION
Aroma Estimation by Sensual Methods In this research grain and
leaf of selected16 varieties were used for aroma evaluation The
results showed that ten varieties is aromatic and six varieties non
aromatic character in (Table 2).
The four primers in single tube Allele Specific Amplification
(ASA)
aroma analysis for sixteen varieties by Allele Specific
Amplification resulted THIS Aba 1, Barnamaj4 ,Buhooth1
,Dijlah, Meshkhab 2,First genotype (T85), Fifth genotype
(aromatic 64) varieties 355 bp bands showed a PCR product
amplified through two Primers (External Antisense (EAP),
Internal Nonfragrant Sence (INSP) )as of the nonfragrant allele.
Nonaromatic rice cultivars have what seems to be a completely
practical copy from the gene encoding BADH2 for the synthesis
of γ-Aminobutyric acid (GABA) , while Eanber albaraka, Forat 1
,Ghadeer ,Yasmine , Eanber 33, Meshkhab1 ,Second genotype(
HT-1) ,Third genotype (LT-2) ,Fourth genotype (BT-7) varieties
showed bands of 257 bp indicating fragrant a PCR product
amplified through two Primers ( External Sence Primer (ESP),
Internal Fragrant Antisense (IFAP)) from the fragrant allele
(fgr),Consistent with the resuts (11 ). Fragrant varieties are
possessing a copy from the gene encoding BADH2 which owns
the SNPs and deletion, leading to a frameshift that generates the
premature stop codon that apparently disables the BADH2
enzyme that results in the synthesis of 2-acetyl-1-pyrroline
(2AP).This polymorphism affords an opportunity for the
production of a complete marker for aroma in rice previously, (6)
revealed that it is potential to distinguish non aroma from aroma
rice varieties by using this method.
Sensory Methods enable the detection of fragrant and
nonaromatic varieties while molecular marker promotion to
recognize specific allele in single tube for detection of aroma in
rice .In this investigation, we used molecular marker methods and
sensory analyses for the revealing of the absence or presence of
aroma in sixteen varieties, 8 varieties which were categorized as
possessing the aroma alleles by the analysis of molecular marker,
also discovered the presence of aroma in sensory tests.
while the Meshkhab1 varieties did not show aroma in sensory
methods while transferring fgr gene in molecular marker
analysis.First genotype (T85) and Fifth genotype (aromatic 64)
varieties did not display aroma in fgr gene in molecular marker
assay while presented presence of aroma in sensory tests may be
because of a presence of another aromatic compound.
Table 2: Aromatic and non aromatic characters of Iraqi
rice
No.
Name of Varieties
Sensory
1
Aba 1
non aromatic
2
Eanber albaraka
Aromatic
3
Forat 1
Aromatic
4
Ghadeer
Aromatic
5
Barnamaj4
Non aromatic
6
Yasmine
Aromatic
7
Buhooth1
Non aromatic
8
Eanber 33
Aromatic
9
Dijlah
Non aromatic
10
Meshkhab 2
Non aromatic
11
Meshkhab1
Non aromatic
12
First genotype (T85)
Aromatic
13
Second genotype( HT-1)
Aromatic
14
Third genotype (LT-2)
Aromatic
15
Fourth genotype (BT-7)
Aromatic
16
Fifth genotype (aromatic 64)
Aromatic
Fig. 1: Aroma analysis for sixteen cultivars rice with PCR marker (Allele Specific Amplification).
Lane 1 DNA marker( 100–2,000 bp) , Lanes 2 control , Lanes3 Aba 1. Lanes4 Eanber albaraka, Lanes5 Forat 1, Lanes6 Ghadeer, Lanes7 Barnamaj4,
Lanes8 Yasmine, Lanes 9 Buhooth1, Lanes10 Eanber 33, Lanes11 Dijlah, Lanes12 Meshkhab 2, Lanes13 Meshkhab1, Lanes14 First genotype (T85),
Lanes15 Second genotype( HT-1) , Lanes16 Third genotype (LT-2), Lanes17 Fourth genotype (BT-7) , Lanes18 Fifth genotype (aromatic 64)
respectively.
355bp
257bp
Zeina Thamer Abd ULHussein ALrufaye et al /J. Pharm. Sci. & Res. Vol. 10(10), 2018, 2531-2533
2532

The difference in the sensory analyses may result from
environmental factors or minor genes and that some rice varieties
may transfer minor QTLs which own an effect on rice aroma. The
difference in the sensory analyses may because of environmental
factors or minor genes and that some rice cultivars may transfer
minor QTLs which own an redounds on rice aroma Consistent
with the results(12 ) .These results indicated that only sensory
methods or molecular marker analysis could not clarify the
complete aromatic conditions. Consistent with the results (13)
stated that the molecular marker outcomes accepted well with the
chemical analysis in the majority of the rice cultivars, except
some covariance results( 1) reported chance between results from
1.7% KOH sensory testing and molecular marker analysis for the
taxonomy of non-aromatic and aromatic rice .In this study the
fragrant variety ware identified can be used improving breeding
programs.
CONCLUSION
Aroma evaluation of rice varieties is complex So,
incorporation of sensory methods with allele specific PCR
amplification of EAP, ESP, IFAP, and INSP primers were
observed as credible, fast, and cost-efficient techniques to Assess
rice aroma in this search, this would play a significant role in the
development of cultivars high-quality rice used in improving
breeding programs
REFERENCES
1- Sarhadi, W. A. ; Hien, N. L. ; Zanjani, M.; Yosofzai, W.; Yoshihashi
, T. and Hirata, Y. “Comparative analyses for aroma and agronomic
traits of native rice cultivars from Central Asia,” Journal of Crop
Science and Biotechnology. 2011, 11, 17–22.
2- Sakthivel, K.; Sundaram, R. M. ; Rani, N. S. ; Balachandran, S. M.
and Neeraja, C. N. “Genetic and molecular basis of fragrance in
rice,”J. Biotechnology Advances. 2009, 27(4), 468–473.
3- Cordeiro,G.M.; Christopher, M. J.; Henry,R. J. and Reinke,R. F.
“Identification of microsatellite markers for fragrance in rice by
analysis of the rice genome sequence. J. Molecular Breeding, 2002,
9(4), 245–250.
4- Vidya H.; Hemant P. and Altafhusain N. Comparative
Characterization of Aroma Volatiles and Related Gene Expression
Analysis at Vegetative and Mature Stages in Basmati and Non-
Basmati Rice (Oryza sativa L.) Cultivars. j. Appl Biochem
Biotechnol. 2015, 178(4)
5- Nguyen L. H. , Tadashi Y. , Wakil A. S. and Yutaka H. Sensory Test
for Aroma and Quantitative Analysis of 2-Acetyl-1-Pyrroline in
Asian Aromatic Rice Varieties. J. Plant Prod. Sci. 2006, 9(3), 294-
297.
6- Bradbury, L. M. ; Henry, R. J. ; Jin, Q.; Reinke, R. F. and Waters,
D. L. “A perfect marker for fragrance genotyping in rice,” Molecular
Breeding. 2005, 16(4), 279–283
7- Subhas, C. R. DNA Barcoding for Wild Rice [Oryza rufipogon
Griff.] of NBU Campus Based on matK gene and Assessment of
Genetic Variation Using DREB and BAD2 Gene Sequences. J. Plant
Gene and Trait, 2015, 6(5), 1-10.
8- Yang, D.S.; Lee, K.S.; Jeong, O.Y.; Kim, K.J.; Kays, S.J. (2008)
Characterization of volatile aroma compounds in cooked black rice.
J. Agricultural and Food Chemistry .56,(2)35–40.
9- Kibria, K.; Islam, M.M. and Begum, S.N. 2008. “Screening of
aromatic rice lines by phenotypic and molecular markers,”
Bangladesh .J. Botany. 37( 2), : 141–147.
10-Dellapporta SL, Hicks JB. 1983 .A new plant DNA minipreparation:
Version II. Plant Mol Biol Rep, 1:19-21.
11-Acga, C. ; Festo, M.; Ismanizan, I.; Mohamad, O.; Habibuddin, H.
2015. High resolution agarose-based system for single-tube
genotyping of fgr and Waxy genes in rice: MAGE to displace
PAGE? Plant omics J.P.O 8(4):348-352.
12- Yi, M.; Nwea, K.T.; Vanavichit, A.; Chai-arree, W. and Toojinda,
T. 2009. “Marker assisted backcross breeding to improve cooking
quality traits in Myanmar rice cultivar Manawthukha,” J. Field Crops
Research. 113,( 2),: 178–186.
13-Bounphanousay, C.; Jaisil, P.; Sanitchon, J.; Fitzgerald, M. and
Hamilton, N. S. 2008. “Chemical and molecular characterization of
fragrance in black glutinous rice from Lao PDR,” Asian J. Plant
Sciences,. 7(1): 1–7.
Zeina Thamer Abd ULHussein ALrufaye et al /J. Pharm. Sci. & Res. Vol. 10(10), 2018, 2531-2533
2533