Evaluation of the Fragrance Gene (fgr) in Self-Supplied Seed
Lots of Black Rice (Oryza sativa L.) from Thailand and Laos
Fragrance is the most important trait among the domesticated characteristics
of rice (O. sativa L.). The recessive fragrance gene on chromosome
8 is associated with rice fragrance. The gene for fragrance in a fragrant
rice variety shows the presence of a mutation portion (i.e., an eight
base pair deletion in exon 7). This allele is responsible for rice fragrance.
In the present study, 65 self-supplied seed lots of black rice (O.
sativa L.) from Thailand and Laos were assessed for purity of the
fragrance grain of each sample for this locus using a PCR assay. The results
indicate that black rice germplasm were genotyped for homozygous fragrant,
heterozygote and non-fragrant homozygous. The data from the 65 samples
show that 7.7 and 23% of the samples were heterozygous and non-fragrant
homozygous, respectively. Heterozygous individuals, black rice plants
that carry both the fragrant allele and non-fragrant allele of the fragrance
gene and non-fragrant seeds need to avoid because they are non-fragrant
and give rise to a mixture of fragrant and non-fragrant seed lots. Therefore,
domestication of black rice in order to maintain grain aroma would require
the use of quality black rice seed germplasm.
Colored rice, most of which is red or black, is not the most commonly
consumed; that distinction belongs to white rice. Colored rice has been
considered a health food; black rice pigment fraction has strong preventive
effects against atherosclerotic disease or coronary heart disease (Ling
et al., 2001, 2002; Xia et al., 2003).
Black rice is popular in Asian countries where it is mixed with white
rice prior to cooking to enhance the flavor, color and nutritional value
(Yang et al., 2008). Historically, black rice has been reserved
for use in festival foods and desserts in Asian countries. Typically,
black rice grains are aromatic and because grain fragrance is an important
feature of premium-value rice, it commands higher prices in domestic and
international markets. In Southeast Asia, especially in Lao PDR and the
north and northeastern regions of Thailand, black rice serves as the staple
food; Khao Kam is the traditional name of black rice in these regions.
Black rice is classified into two categories: grain with purple pigmentation
on glumes and various color shades on the pericarp and grain with straw
glumes and purple pericarp. Its color can be attributed to anthocyanins
(cyaniding 3-glucoside and peonidin 3-glucoside) found in surface cells
of the grain (Xia et al., 2006).
Fragrance in rice is a highly valued trait and known to be primarily
associated with grain 2-acetyl-1-pyrroline. It has been previously determined
that the fragrance gene is located on chromosome 8 that controls the level
of aromatic compound 2-acetyl-1-pyrroline (Bradbury et al., 2005a).
The structure of the fragrance gene (fgr) comprises 15 exons interrupted
by 14 introns. Fragrance is a recessive trait, the alleles from fragrant
varieties all showed the presence of mutations (i.e., the 8 bp deletion
in exon 7), resulting in a loss of function of the fragrance gene product.
Interestingly, the concentration of 2-acetyl-1-pyrroline (2-AP) was high
in cooked black rice (Yang et al., 2008). Seed of local rice varieties
maintained by farmers are genetically diverse (Saito et al., 2007).
Analysis of molecular diversity using molecular techniques allows variation
to be evaluated between individual plants, particularly the aromatic character
of the black rice of saved farmer seeds, by using a polymerase chain reaction
The goal of this study is to genetically characterize the farmers` seed
germplasm. The results from this study can be used to enhance the efficiency
and effectiveness of black rice germplasm maintenance for the aromatic
character, promote farmer awareness of the value of collecting seeds and
encourage seed conservation of black rice varieties.
MATERIALS AND METHODS
Sample collection: Seeds of black rice samples from different
farmers` self-supplied seed lots in several regions of Thailand and Laos
were collected. A total of 65 samples were collected and evaluated. Four
monthly collections were made over a period of two years: during March
2006 and 2007 and December 2006 and 2007. The summary information of black
rice samples are listed in Table 1.
DNA extraction and polymerase chain reaction: Mature seeds of
each seed lot were germinated and grown in pots at a green house at Mahasarakham
University. Five seedlings generated from each seed lot were randomly
selected and bulked for use in the analysis. Genomic DNA was extracted
from the bulked samples of the young leaf according to the protocol of
Doyle and Doyle (1987). PCR amplification of the fgr gene was performed
using the DNA sequences of oligonucleotide primers (i.e., Os2AP-exon7.1F:
5`-TGCTCCTTTGTCAT CACACC-3` and Os2AP-exon7.1R: 5`-TTTCCACCAAGTTCC AGTGA-3`),
which were used previously to amplify the fragrance gene located on chromosome
8. In addition, this DNA marker can be used in breeding for fragrant rice
varieties (Shi et al., 2008). The oligonucleotide primers were
synthesized by BSU (BioService Unit, National Center for Genetic Engineering
and Biotechnology, National Science and Technology Development Agency,
The PCR reaction was performed in a 20 μL reaction mixture containing
2 μL of DNA solution, 50 pmol each of the primer pairs, 2.0 mM MgCl2,
2 units Taq polymerase (Promega), 0.1 mM dNTPs. Cycling conditions
were 94°C (5 min); then 40 cycles of 94°C (1 min), 60°C (1
min), 72°C (1.5 min) and a final extension of 72°C (5 min). Using
these primer pairs, the DNA template from fragrant rice, Khao Dawk Mali
105 and a non-fragrant rice, Chai Nart 1 (CN1), were used as positive
and negative controls, respectively, in the experiment for comparison
of bands resulting from PCR between fragrant and non-fragrant rice. The
PCR products were separated in 4.5% polyacrylamide denaturing gels of
200x125x1 mm (lengthxwidthx thickness). After electrophoresis, the bands
were stained with silver-stain. The PCR product of approximately 396 bp
obtained from Thai jasmine rice (Khao Dawk Mali 105) was present in every
sample with the recessive allele (the 8 bp deletion), whereas the dominant
allele gave a product of approximately 404 bp from the Thai non-fragrant
rice (CN 1). From the PCR assay, heterozygotes can be discriminated by
the presence of both PCR products. The genotypic and allelic frequencies
were computed based on Hardy-Weinberg formulations. Goodness-of-fit statistics
were calculated for the figure observed compared to values expected using
the Hardy-Weinberg equilibrium.
||List of self-supplied seed of black rice (O. sativa
L.) of different origins and their PCR-based genotypes of the fragrance
A PCR assay was used to evaluate 65 self-supplied seed lot plants which
derived from seed germination. The assay predicted the genotype of each
of the individuals within a seed lot. The different genotypes and the
distribution of the allele of the fgr gene in the black rice samples
used in the present study are shown in Table 1. Overall,
a total number of 45, 5 and 15 seed lots were genotyped for DD (allele
D, 8 bp deletion), ND (heterozygote) and NN (allele N, non-deletion),
respectively. The data from the 65 combined seed lots show that 31% of
the samples were heterozygous and homozygous non-fragrant (Fig
1). The overall allelic frequencies were 0.731 and 0.269 for D and
N alleles, respectively. The distribution did differ significantly from
that expected under the Hardy-Weinberg equilibrium (Goodness-of-fit χ2
= 42.3, p<0.01) (Table 2). One of the most important
causes that may underline the inconsistent results from the Hardy-Weinberg
equilibrium is artificial selection of black rice genotype by traditional
farmers. The artificial selection causes changes in allele frequencies
of the fragrance gene in black rice populations.
||Statistical analysis of fragrance gene (fgr)
in the 65 self-supplied seed samples of black rice from Thailand and
|Goodness-of-fit testing showed significant difference
suggesting that locus of the fgr gene is not conformed to Hardy-Weinberg
A number of sensory methods have been utilized to assist breeders in
selecting fragrant rice, but limitations occur when processing large numbers
of samples. In addition, these methods are labor intensive, difficult
and unreliable (Bradbury et al., 2005b). DNA markers are pieces
of DNA that associate the presence or absence of particular traits. Selection
for the trait can be undertaken on the basis of molecular techniques.
For the fragrance trait in rice, a DNA-based marker situated within the
fragrance gene was developed (Bradbury et al., 2005b; Shi et
al., 2008). The marker-assisted selection can separate fragrant and
non-fragrant rice varieties. An example of the DNA marker approach for
screening black rice grain aroma was reported by Bounphanousay et al.
(2008). They reported that the fragrant diagnostic band is associated
with aromatic character which is indicated by the level of 2-AP in the
examined grains. All accessions of their sample black rice showed either
homozygous fragrant or homozygous non-fragrant. This means that the seed
of black rice used in their report contained a mixture of non-fragrant
seed and fragrant seed.
The major finding of the present study was that farmer saved seed lots
of black rice had dramatically contaminated (5 out of 65 seed lots) seed
of black rice with genotypes non-fragrant and heterozygous.
PCR products in a 4.5% polyacrylamide gel showing three
genotypes (NN = No-deletion of the 8 base pair; ND = Heterozygote
and DD = Deletion of the 8 base pair) of the fgr gene in black
rice (O. sativa L.). Lane M shows DNA molecular weight
(base pair, bp). Lane, putative samples and their genotypes (respectively)
are as follows: NN: 1, 2, 13, 14, 20, 27; ND: 5, 7, 8, 10, 11, 15,
18, 22, 23, 25, 29, 31, 34, 35; DD: 3, 4, 6, 9, 12, 16, 17, 19, 21,
24, 26, 28, 30, 32, 33, 36
Heterozygous individuals, black rice plants that carry both the fragrant
allele and non-fragrant allele of the fragrance gene, need to be avoided
because they are non-fragrant and give rise to a mixture of fragrant and
non-fragrant seed lots. Domestication of black rice in order to maintain
grain aroma would require the use of quality black rice seed germplasm.
The assay can also identify mixtures of fragrant and non-fragrant plants
which is useful for pure seed maintenance. Farmer and industry awareness
of seed quality can be enhanced by facilitating the development of marketplace
demand-driven germplasm improvement. An emphasis on the development of
the production of black rice seed quality should become a priority. The
small scale farming sector stands to benefit from the development through
an increase in productivity, profitability and sustainability.
This study was supported by Mahasarakham University. The author is grateful
to Thai and Laos farmers for providing valuable black rice samples and
to V. Pilap for his technical assistance.
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