Genetic Characteristics of Amylose
Content in Medium-Grain Rice (Oryza
sativa)
Bui
Phuoc Tam1,4†, Pham Thi Be Tu2* and Nguyen Thi Pha3†
1PhD student, Can Tho University, Can Tho city 94000,
Vietnam
2College of Agriculture, Can Tho University, Can Tho city
94000, Vietnam
3Institute of Food and Biotechnology, Can Tho University,
Can Tho city 94000, Vietnam
4Loc Troi
Agricultural Research Institute, An Giang Province 90000, Vietnam
*For correspondence:
ptbtu@ctu.edu.vn; phuoctam1987@gmail.com
†Contributed equally
to this work and are co-first authors
Received
09 October 2023; Accepted 11 January 2024; Published 18 March 2024
Abstract
Amylose content (AC) is one of the key traits related to
rice grain quality. It decided the hard or soft grain rice. Consumers generally
prefer cooked rice with a soft and sticky texture. AC is a single-gene trait
and is controlled by the Waxy gene
located on chromosome 6. Genetic studies related to AC in medium-grain rice
varieties underpin the identification of genes and linked molecular markers
that few previous studies mentioned. The study’s purpose is to investigate the
genetic characteristics of AC in medium-grain rice on 12 chromosomes. A rice
diversity panel of 114 genotypes was applied to evaluate AC and its correlation
with the agronomic and quality characteristics. Genome-Wide Association Study
(GWAS) was utilized to identify significant SNPs. The results showed that the
AC of varieties had a large range from 10.83 to 30.12%, in which, the group of
varieties with low AC (less than 20%) accounted for 21.9%. AC was strongly
correlated with gel consistency (GC) but not with gelatinization temperature
(GT) and agronomic traits. The GWAS
analysis of AC identified 9 significant SNPs on chromosomes 1, 2, 4, 8, 9, 11
and 12. The frequency of alleles widely ranged from 0.94 to 56.60%. On
chromosome 6, 74 significant SNPs had allele frequencies ranging from 3.8 to
67.0%. In which, two SNPs fd7 (1764762 bp) and fd8 (176707 bp) are located in
the Waxy gene region. These findings
provided the basic genetic foundation for high-quality medium-grain rice
breeding programs. © 2024 Friends Science Publishers
Keywords: Allele frequency; Amylose content
(AC); Chromosome; Medium-grain rice; Single nucleotide polymorphism (SNP)
Introduction
Rice (Oryza sativa
L.) grain consists of approximately 90% starch and AC is widely recognized as
the most important factor affecting eating and cooking quality (Tian et al. 2009; Cai et al. 2015; Zhou et al.
2018). AC in rice is classified into high (>25%), intermediate (20–25%), low
(12–20%), very low (5–12%), or waxy (0–2%) (Juliano et al. 1981; Yu et al.
2012). Rice with a high AC tends to cook hard and dry, whereas rice with a
intermediate AC tends to be softer and stickier, and rice with a low AC is generally
quite soft and sticky. The major gene for amylose synthesis in rice is the Waxy (Wx) gene located on chromosome 6, which encodes the granule bound
starch synthase (Septiningsih et al.
2003; Fan et al. 2005; Tian et al. 2009). Other minor QTLs were also
detected on the 12 chromosomes of rice in previous studies.
AC has various tend in different rice
species. According to Feng et al.
(2017), evaluating 635 rice varieties (including pure rice and hybrid rice),
the AC of japonica rice (short grain) was significantly lower (P < 0.05)
than indica rice (long grain). This result was also recorded by IRRI (2013).
Sano (1984) showed that the Wx locus
has three different alleles, Wxa,
Wxb and wx, in the rice species of indica (or
long grain), japonica (or short grain), and sticky rice, respectively.
GWAS mapping makes it possible to
simultaneously screen a very large number of accessions for genetic variation
underlying diverse complex traits. One great advantage of the GWAS design for
rice is the homozygous nature of most rice varieties, which makes it possible
to employ a “genotype or sequence once and phenotype many times over” strategy,
whereby once the lines are genomically characterized, the genetic data can be
reused many times across different phenotypes and environments (Zhao et al. 2011). In rice, GWAS is gaining
widespread use and there are reports (Huang et
al. 2010; Zhao et al. 2011;
Vanniarajan et al. 2012; Courtois et al. 2013; Ueda et al. 2014; Kumar et al.
2015).
The objectives of this study were to:
(1) evaluate the agronomic traits and analyze the amylose content,
gelatinization temperature and gel consistency, (2) identify the correlation of
amylose content with agronomic traits and other quality indexes, and (3)
investigate single nucleotide polymorphisms (SNPs) in the whole of rice
chromosomes and the target chromosome.
Materials and Methods
Place and time
The experiments were conducted at the experimental
station of CanTho University, CanTho city, Vietnam in the Summer-Fall season of
2020.
Materials
The group of medium-grain rice varieties included 114
varieties selected from the 44k-SNP rice diversity panel of the International
Rice Research Institute (IRRI). The data set of 44,100 SNP were referenced from
Zhao et al. (2011).
Evaluation of the
agronomic characteristics
The rice plants were grown on the alluvial soil in the
2020 Summer-Fall season. The experiment was performed as a randomized complete
block design with three replications, each replication was 3 m2.
Each rice plant individual was separetaly transplanted in a 20 × 20 cm
distance. Rice farming was applied the alternate wetting and drying method with
the fertiziler formula of 80 N: 60 P2O5: 60 K2O
(kg/ha). Agronomic characteristics were
evaluated including duration (days), plant height (cm), panicle length (cm),
number of panicles per square meter (panicles), number of filled grain per
panicle (grains), percentage of unfilled grains, 1000 grain weight (gram), and
yield (tons/ha).
Analysis of
amylose content (AC)
The AC of the milled rice samples was determined
according to the method of Juliano (1971); Graham (2002). Milled rice flour
(100 mg) was soaked in 1 mL of 95% ethanol and 9.0 mL of 1 N NaOH in a 50-mL
glass test tube and allowed to stand undisturbed overnight for 16 h.
Afterwards, distilled water (90 mL) was added to bring the solution up to 100 mL,
and 0.5 mL aliquot was transferred into a 20-mL test tube containing 5 mL
distilled water. Then, 0.1 mL of 1 M CH3COOH was added and the
solution was mixed thoroughly using a vortex mixer followed by addition of 0.2
mL of iodine solution (0.15% I2 in 1.5% KI). The solution was then
diluted to 10 mL using 4.2 mL distilled water. To develop the calibration
curves for the determination of amylose content in a rice sample, 40 mg Avebe
potato amylose (standard amylose) put in a 50 mL test tube and proceeded as
described above. Then, 0.1, 0.2, 0.3, 0.4, 0.5 mL of the standard amylose
sample solution were transferred into 20 mL test tubes and proceeded in the
same way as for the test sample. Construction of the calibration curve was
carried by converting from the spectral reading to the percentage of amylose
content according to the followed formula (y = ax + b, where y is the
absorbance OD, and x is the amount of amylose in the measured sample (mg/L)
(Graham 2002).
Analysis of
gelatinization temperature (GT)
The GT was determined using the alkali digestion test
(IRRI 2014). A duplicate set of six whole-milled kernels without cracks was
selected and placed in a petri dish (8.0 cm in diameter). Ten mL of 1.7% KOH
solution was added. The samples were arranged to provide enough space between
kernels to allow for spreading. The dishes were covered and incubated for 23 h
at 30°C. The starchy endosperm was rated visually based on a seven-point
numerical spreading scale as a standard evaluation system for rice: high (1–2),
high or intermediate (3), intermediate (4–5), and low (6–7) (IRRI 2014).
Analysis of gel
consistency (GC)
Analysis of gel consistency according to the method of
Tang et al. (1991). Milled rice flour (100 mg) was put into a glass test
tube (13 x 100 mm). Then, 0.2 mL of 95% ethanol containing 0.03% green thymol
was placed into the test tube. 2 mL of 0.2 N KOH was further added to the test
tube and shaken well on a vortex machine. The test tube was covered and placed
in a pot of boiling water (100oC) for 8 min. Test tubes were cooled
to room temperature for 5 min and placed in an ice bath for 20 min. Test tubes
were removed and placed horizontally for 1 h. The gel consistency is the length
of the moved gel measured from the bottom of the test tube to the end of the
gel. The classification of gel consistency is applied according to the standard
evaluation system for rice of IRRI (2014): soft (61–80 mm), medium (41–60 mm),
and hard (< 40 mm).
Investigation of
single nucleotide polymorphisms (SNPs)
A 44,100 SNP genotyping data (Zhao et al. 2011) were used for association of SNP variants with the
phenotypes. GWAS by GAPIT (Genomic Association and Prediction Integrated Tool)
was done using R-studio 3.2.4 software (Zhiwu Zhang Laboratory 2020). The GAPIT
tool optimized enriched compressed mixed linear model approach (eCMLM). To
combine association results, the significance threshold was set to P <
0.0001. Information on SNPs was collected using the Rice SNP-Seek database
(https://snp-seek.irri.org/). The QTL mapping of related SNPs was made using
the Ritchie Lab Visualization database
(http://visualization.ritchielab.org/phenograms/plot). The steps of GWAS
analysis were based on the GAPIT tool, which is depicted in Fig. 1.
Statistical
methods
Data were collected and
stored, and graphs were designed using the Microsoft Office Excel 2013 program.
Descriptive statistics of traits were carried using STAR 2.0.1 software (IRRI)
(http://bbi.irri.org/products). GWAS by GAPIT was analyzed by R-studio 3.4.1
software (https://rstudio.com/products/rstudio/download). A chart of a
correlation matrix was displayed by R-studio 3.4.1 software with the
'PerformanceAnalytics' package.
Results
Evaluation of the
agronomic characteristics
The agronomic characteristics of the medium-grain rice
varieties are showed in Table 1. Rice varieties had short to average growth
duration and were not affected by photoperiod. The agronomic characteristics
had a wide variance and a small standard error.
Determination of
AC, GT and GC
Amylose content has a large difference among rice
varieties, ranging from 10.83 to 30.12% (Table 2). Cooked rice kernels with
high AC (>25%) occupying 36.0% of the total of rice varieties were dry,
separate, less tender, and become hard upon cooling, whereas those with low
(≤ 20%) with 21.9% were glossy, soft, and sticky. Intermediate AC (20–25%)
rice made up 42.1% was soft and flaky (Fig. 2). The experiment recorded that GT
in the medium-grain rice varied from score 3 to score 7 (Table 2). GT in the
high group (≤ score 3) accounted for about 4.4%, in the intermediate
group (score 4–5) accounted for 23.7% and the rest was the low group (score 6–7)
made up the majority, about 71.9%. For GC, most varieties were in the medium
classification (41–60 mm), accounting for 51.8%. The GC in the soft group was
34.2% while this in the hard group was about 14.0%.
Correlation
between AC and agronomic traits and quality characteristics in medium grain
rice
Fig. 1:
Investigation of single nucleotide polymorphism (SNP) for rice genotypes
with different amylose content on chromosomes of rice
Fig. 2: Groups of AC in medium
grain rice varieties
Notes: AC:
Amylose content
Fig. 3: Correlation between AC and
agronomic and quality characteristics of medium-grain rice varieties
Notes: Statistics at 95% significance level; TGST:
Duration; Caocay: Plant height; Daibong: Panicle length; Sobong: Number of panicles
per m2; Hatchac: Number of filled grains per panicle; Tllep:
Percentage of unfilled grain; W1000: 1000 grain weight; AC: Amylose content;
GT: Gelatinization temperature; GC: Gel consistency
The
correlation between agronomic and quality characteristics was showed at Fig. 3.
For agronomic characteristics, AC was positively correlated on average (+0.44)
with number of panicles/m2, and negatively correlated with plant
height, panicle length and yield. However, AC was not significantly or not
correlated with duration, number of filled grains per panicle, percentage of
unfilled grain, and 1000 grain weight. AC was not strongly correlated with the
degree of GT and this correlation tended to be negative, the correlation
coefficient was (-0.19). AC was negatively associated with GC. The correlation
coefficient recorded between these two indicators was (-0.85).
Investigation of
single nucleotide polymorphism (SNP) for varieties with different AC on
chromosomes of rice
Through analysis of AC in the medium-grain rice
accessions by GWAS, nine significant SNPs (LOD score ~ 3) were identified on
chromosomes 1, 2, 4, 8, 9, 11 and 12 (Fig. 4, 5). These SNPs significantly
linked the target gene at P ≤ 0.001 and their allele frequencies ranged
from 10.13 to 56.60% (Table 3). This shows that there was a difference in
amylose content among medium grain rice varieties. Based on the Rice SNP-Seek
database (IRRI), among nine identified SNPs, two (id2000259 and id2000249) on
chromosome 2 and one SNP (id12001229) on chromosome 12 were selected. SNP
id2000259 at position 349130 bp had the substitution of the T allele by the A
allele with the frequency of 10.13% and explained 15.2% of the variations in AC
of the medium-grain rice varieties. Similarly, SNP id2000249 has a substitution
of the T allele by the C allele at position 346742 bp on chromosome 2. This
allele had the substitution frequency of 11.99% and explained 13.9% of the
variations. On chromosome 12, SNP id12001229 had a substitution of A by C at
position 2966521 bp, the frequency of substitution allele was 34.00% and the
SNP explained 13.4% of the variations in phenotype. Among 3265 SNPs located on
chromosome 6, 74 SNP positions were identified with LOD score ≥ 3 at the
significance level p ≤ 0.001 (Fig. 6). The frequency of occurrence of
alternative alleles of SNPs varied widely, from 3.8 to 67.0% (Table 4).
When investigation of SNPs focusing on
chromosome 6, among 3265 discovered SNPs, 74 SNP positions were identified with
LOD score ≥ 3 at the significance level p ≤ 0.001 (Fig. 6). The
frequency of occurrence of alternative alleles of SNPs varied widely, from 3.8
to 67.0% (Table 4). For the level of significance of the SNPs, a total of 74
SNPs were selected with LOD values ranging from 3.0 to 11.0. In which, 27 SNPs
were with a LOD value of 3.0 ≤ LOD < 4.0 (accounting for 36.5%), 25
SNPs with 4.0 ≤ LOD < 5.0 (accounting for 33.8%), and 22 SNPs with LOD
value ≥ 5.0 (accounting for 29.7%). Especially, the study showed that two
SNPs fd7 (1764762 bp) and fd8 (1770007 bp) were located on the Waxy gene region (Fig. 7). The SNP fd7
had the highest LOD value among the SNPs (LOD = 10.96) that had the replacement
of the G allele with the T allele at the frequency of 17.9% and explained
11.63% of the variations in AC. The SNP fd8 with a LOD value of 5.65 had a
replacement of allele A with allele C at the frequency of 14.2% and explained
9.26% of phenotypic variations.
Discussion
The agronomic characteristics of these rice varieties
had a big range and a large phenotypic variance. This is a rich source
of variation that allowed the exploitation of desirable traits. Based on the
morphological and agronomic characteristics, the breeder can predict and orient
the hybridization between rice varieties to achieve the highest breeding
efficiency. Anwar et al. (2009) and
Oladosu et al. (2018) suggested that
path analysis for rice yield can be calculated directly through directly or
indirectly measurement by yield components. For rice varieties, short-duration
help to increase the number of crops per year, moderate plant height to prevent
falling, and yield components are required such as long panicle, good tillering,
high number of filled seeds, and big seed weight. The most respected
characteristic is the actual yield, high yield helps to increase production
efficiency and increase profits for farmers.
Besides the agronomic
characteristics, cooking and eating quality is one of the important factors
determining the value of rice grains. AC, GT and GC are three important quality
parameters closely related to the softness of rice grains when cooked as well
as cooled. AC among rice varieties was diversified and this result is similar
to many previous studies (Manners 1979; Juliano 1992; Patindol et al. 2015). Through analysis of AC, it
was found that in medium grain rice varieties, the group of varieties with low
AC accounted for a lower percentage than other groups of amylose. This as a
potential amylose group for breeding new rice varieties with low AC. Similarly,
the value of GT and GC in the medium-grain rice varieties was a large range.
These results inferred that there are diversified quality characteristics in
the medium rice to permit exploitation for rice breeding programs. A rice
variety is considered a good cooking quality when converging factors such as
low AC (≤ 20%), low GT (score 6 to 7), soft GC (> 60 mm), aroma, and
other valuable nutritious properties. This was also mentioned by Custodio et al. (2019).
Fig. 4: Analysis results of
single nucleotide polymorphism (SNP) based on GWAS for AC in medium-grain rice varieties on 12
chromosomes in rice
Fig. 5: QTL mapping of candidate SNPs related to amylose content, gel consistency, and gelatinization
temperature in medium-grain rice varieties over 12 chromosomes
Notes: chr.: chromosome; AC: Amylose Content; GT: Gelatinization Temperature;
GC: Gel Consistency; The region coverred
by pink color was the expected region of Waxy gene
Fig. 6: Analysis results of single
nucleotide polymorphism (SNP) based on GWAS for amylose content in medium-grain
rice varieties on the chromosome 6 in rice
Fig. 7: QTL map of candidate SNPs related to AC in medium-grain rice varieties on chromosome 6
In the correlation between AC
and other traits, AC did not have a strong correlation with agronomic
characteristics. This correlation was similarly noted in previous studies
(Graham-Acquaah et al. 2018; Sharifi
2018).
AC was lowly correlated with GT
and this result was
similar to the conclusions of Jennings et
al. (1979), Hossain et al.
(2009); and Pang et al. (2016). AC
was strongly associated with GC. This implied that the lower the AC, the
greater the GC, the more flexible the rice, and vice versa. This result was
similarly recorded in previous studies (Lapitan et al. 2009; Ritika et al.
2010; Zhang et al. 2020).
Table 4: Significant SNPs related to amylose content located
on the chromosomes 6 in medium-grain rice varieties
No. |
SNP ID |
Position(bp) |
P-value |
LOD-value |
O- allele |
R- allele |
F (%) |
R2 |
1 |
fd7 |
1764762 |
1.09 × 10-11 |
10.96 |
G |
T |
17.9 |
11.63 |
2 |
id6002622 |
3251812 |
3.27 × 10-11 |
10.49 |
C |
T |
18.9 |
9.57 |
3 |
id6002910 |
3638340 |
1.40 × 10-10 |
9.85 |
G |
A |
20.8 |
9.08 |
4 |
id6002257 |
2905046 |
1.33 × 10-9 |
8.88 |
T |
C |
17.9 |
8.23 |
5 |
id6002745 |
3330294 |
1.89 × 10-8 |
7.72 |
G |
A |
22.6 |
7.47 |
6 |
id6001346 |
1793697 |
4.12 × 10-8 |
7.39 |
C |
T |
8.5 |
7.10 |
7 |
id6002767 |
3364148 |
7.54 × 10-8 |
7.12 |
G |
T |
24.5 |
6.79 |
8 |
id6002778 |
3377594 |
7.54 × 10-8 |
7.12 |
A |
G |
24.5 |
6.79 |
9 |
id6002798 |
3408987 |
1.36 × 10-7 |
6.87 |
A |
G |
25.5 |
6.86 |
10 |
id6002753 |
3332532 |
1.55 × 10-7 |
6.81 |
G |
A |
22.6 |
6.51 |
11 |
id6002804 |
3414296 |
1.94 × 10-7 |
6.71 |
A |
G |
24.5 |
6.40 |
12 |
id6002613 |
3244706 |
3.32 × 10-7 |
6.48 |
T |
A |
23.6 |
6.21 |
13 |
id6003812 |
5976978 |
3.37 × 10-7 |
6.47 |
T |
A |
12.3 |
6.20 |
14 |
id6002690 |
3289852 |
5.42 × 10-7 |
6.27 |
C |
A |
23.6 |
6.28 |
15 |
id6000293 |
418306 |
7.15 × 10-7 |
6.15 |
G |
A |
16.0 |
5.97 |
16 |
id6002888 |
3599544 |
2.08 × 10-6 |
5.68 |
G |
A |
12.3 |
5.47 |
17 |
id6001434 |
1892145 |
2.09 × 10-6 |
5.68 |
G |
A |
8.5 |
10.19 |
18 |
fd8 |
1767007 |
2.22 × 10-6 |
5.65 |
A |
C |
14.2 |
9.26 |
19 |
wd6000096 |
3564084 |
2.59 × 10-6 |
5.59 |
A |
G |
25.5 |
5.48 |
20 |
id6001256 |
1658758 |
4.10 × 10-6 |
5.39 |
C |
G |
27.4 |
5.31 |
21 |
id6001209 |
1612742 |
4.76 × 10-6 |
5.32 |
G |
T |
27.4 |
5.12 |
22 |
id6012152 |
23441321 |
5.59 × 10-6 |
5.25 |
C |
A |
16.0 |
5.45 |
23 |
id6012159 |
23442920 |
1.11 × 10-5 |
4.95 |
C |
G |
65.1 |
4.77 |
24 |
id6002810 |
3427968 |
1.13 × 10-5 |
4.95 |
G |
A |
27.4 |
4.76 |
25 |
id6001645 |
2194965 |
1.23 × 10-5 |
4.91 |
G |
A |
14.2 |
5.10 |
26 |
id6002750 |
3330720 |
1.43 × 10-5 |
4.84 |
C |
T |
23.6 |
7.73 |
27 |
id6010574 |
20037449 |
1.58 × 10-5 |
4.80 |
C |
T |
40.6 |
4.62 |
28 |
id6011692 |
22493522 |
1.93 × 10-5 |
4.71 |
T |
A |
28.3 |
4.58 |
29 |
id6002701 |
3312612 |
2.03 × 10-5 |
4.69 |
G |
A |
24.5 |
4.51 |
30 |
id6002807 |
3427237 |
2.55 × 10-5 |
4.59 |
T |
A |
25.5 |
5.02 |
31 |
id6002874 |
3589970 |
3.24 × 10-5 |
4.49 |
T |
C |
21.7 |
4.47 |
32 |
id6011886 |
23086388 |
3.28 × 10-5 |
4.48 |
T |
C |
26.4 |
4.91 |
33 |
id6001672 |
2234192 |
4.27 × 10-5 |
4.37 |
G |
A |
22.6 |
4.37 |
34 |
id6012170 |
23444308 |
4.89 × 10-5 |
4.31 |
C |
T |
3.8 |
4.13 |
35 |
id6012182 |
23464841 |
4.89 × 10-5 |
4.31 |
T |
C |
3.8 |
4.13 |
36 |
wd6003011 |
23402917 |
4.92 × 10-5 |
4.31 |
T |
C |
3.8 |
4.17 |
37 |
id6012207 |
23610475 |
5.17 × 10-5 |
4.29 |
G |
A |
3.8 |
4.30 |
38 |
id6002447 |
3078075 |
5.64 × 10-5 |
4.25 |
C |
G |
11.3 |
4.07 |
39 |
id6001376 |
1832609 |
6.39 × 10-5 |
4.19 |
A |
G |
13.2 |
4.02 |
40 |
ud6000315 |
6602235 |
8.16 × 10-5 |
4.09 |
T |
C |
31.1 |
3.93 |
41 |
id6004221 |
6601428 |
8.33 × 10-5 |
4.08 |
A |
T |
31.1 |
4.09 |
42 |
id6001567 |
2091584 |
9.19 × 10-5 |
4.04 |
G |
A |
10.4 |
3.86 |
43 |
id6003135 |
4211403 |
9.63 × 10-5 |
4.02 |
A |
G |
17.0 |
3.84 |
44 |
wd6000289 |
4683858 |
9.63 × 10-5 |
4.02 |
T |
C |
17.0 |
3.84 |
45 |
id6003349 |
4732170 |
9.63 × 10-5 |
4.02 |
C |
T |
17.0 |
3.84 |
46 |
id6003422 |
4929359 |
9.63 × 10-5 |
4.02 |
G |
A |
17.0 |
3.84 |
47 |
id6012242 |
23696829 |
9.76 × 10-5 |
4.01 |
T |
G |
42.5 |
3.85 |
48 |
id6010440 |
19662774 |
1.09 × 10-4 |
3.96 |
A |
G |
29.2 |
3.82 |
49 |
id6006125 |
9652883 |
1.57 × 10-4 |
3.80 |
G |
A |
13.2 |
3.62 |
50 |
id6006227 |
9931739 |
1.57 × 10-4 |
3.80 |
C |
A |
36.8 |
3.94 |
51 |
id6002818 |
3430848 |
1.70 × 10-4 |
3.77 |
A |
G |
26.4 |
3.75 |
52 |
id6003402 |
4869557 |
1.78 × 10-4 |
3.75 |
A |
T |
16.0 |
3.85 |
53 |
id6002912 |
3638528 |
1.94 × 10-4 |
3.71 |
G |
T |
59.4 |
3.50 |
54 |
ud6000539 |
12067053 |
2.01 × 10-4 |
3.70 |
A |
G |
65.1 |
3.60 |
55 |
id6006423 |
10381968 |
2.12 × 10-4 |
3.67 |
C |
G |
44.3 |
3.59 |
56 |
id6008098 |
13486344 |
2.29 × 10-4 |
3.64 |
T |
G |
67.0 |
3.43 |
57 |
id6004211 |
6595944 |
2.32 × 10-4 |
3.63 |
G |
C |
30.2 |
3.43 |
58 |
id6003901 |
6176014 |
2.33 × 10-4 |
3.63 |
T |
C |
29.2 |
3.87 |
59 |
id6011935 |
23107424 |
2.87 × 10-4 |
3.54 |
T |
A |
64.2 |
3.36 |
60 |
id6011933 |
23106969 |
3.73 × 10-4 |
3.43 |
T |
C |
60.4 |
3.51 |
61 |
id6002930 |
3648304 |
3.75 × 10-4 |
3.43 |
C |
T |
53.8 |
3.80 |
62 |
id6002885 |
3598148 |
3.92 × 10-4 |
3.41 |
A |
G |
24.5 |
3.41 |
63 |
id6010438 |
19661511 |
4.30 × 10-4 |
3.37 |
C |
T |
30.2 |
3.17 |
64 |
id6011277 |
21501803 |
4.46 × 10-4 |
3.35 |
A |
T |
29.2 |
3.90 |
65 |
id6003463 |
5035086 |
4.90 × 10-4 |
3.31 |
G |
C |
15.1 |
3.35 |
66 |
id6003170 |
4317102 |
5.16 × 10-4 |
3.29 |
C |
T |
17.0 |
3.01 |
67 |
id6013459 |
25054910 |
5.56 × 10-4 |
3.25 |
A |
T |
43.5 |
3.44 |
68 |
ud6000850 |
20159426 |
6.27 × 10-4 |
3.20 |
C |
T |
22.6 |
3.07 |
69 |
id6004306 |
6724842 |
8.29 × 10-4 |
3.08 |
G |
C |
59.4 |
2.95 |
70 |
id6004317 |
6732352 |
8.44 × 10-4 |
3.07 |
T |
C |
60.4 |
2.91 |
71 |
id6003109 |
4162872 |
9.39 × 10-4 |
3.03 |
C |
A |
21.7 |
3.00 |
72 |
id6012253 |
23700701 |
9.72 × 10-4 |
3.01 |
C |
T |
54.7 |
3.18 |
73 |
id6001216 |
1632684 |
9.80 × 10-4 |
3.01 |
T |
C |
16.0 |
2.88 |
74 |
id6011826 |
22864363 |
10.2 × 10-4 |
2.99 |
G |
A |
53.8 |
3.66 |
Notes: Chr.: Chromosome; LOD: Logarithm of the odds
ratio; O-allele: Original allele; R-allele: Replaced allele; F: Allele
frequency; R2: The ratio of
expression of each QTL to the total expression of the corresponding
characteristic
Therefore, when determining the AC or
GC, the breeders can predict the range of the remaining parameter. Moreover,
within the same AC group, rice varieties with softer GC were preferred
(Morgante and Olivieri 1993; Hirano et
al. 1998).
In studying the associations between
phenotypic and genotypic variances, LOD score (-log10P) was applied
to select the significant SNPs. LOD scores of 3 or more were often indicative
of a QTL (quantitative trait loci), or a genetic region involved in the
expression of the phenotype (Nurnberger et
al. 2008). On 12 chromosomes in rice, GWAS detected nine candidate SNPs, of
which, two (id2000259 and id2000249) on chromosome 2 and one SNP (id12001229)
on chromosome 12 had the presence of information about a specific base
substitution in the Rice SNP-Seek database. These SNPs related to AC on
chromosomes 2 and 12 were identified in previous studies. QTL SNPs associated
with AC on chromosome 2 had been recognized by the studies of Tan et al. (1999); Lee et al. (2003); Yun et al.
(2016). Additionally, on chromosome 12, many studies also recorded significant
SNPs related to AC including Lee et al.
(2003); Wan et al. (2004); Wada et al. (2006). Therefore, there SNPs
(id2000259, id2000249, and id12001229) have a high possibility of controlling
AC in medium-grain rice.
Focusing on chromosome 6, significant
SNPs were widely distributed on the target chromosome and concentrated near the
Waxy target gene region from position
1,764,623 to 1,769,575 bp (http://shigen.nig.ac.jp).
Among them, two SNP fd7 and fd8 were located right in the Waxy gene region. This indicated that the Waxy gene also contribute to control AC in the medium-grain rice.
The number of SNPs and their significance identified on 12
chromosomes were usually lower than those on only one target chromosome
(chromosome 6). This can be explained by the number of chromosomes, the more
chromosomes are considered, the fewer candidate SNPs are identified, and the
lower the LOD score value of the SNPs.
Conclusion
The AC in the medium-grain rice varieties varied widely
from 10.83 to 30.12%. In which, the group of varieties with low AC of less than
20% accounted for a low percentage. AC in this study was strongly correlated
with GC but not with GT. The genome-wide association study on 12 chromosomes
for AC in medium-grain rice varieties identified three SNPs, id2000259, id2000249
on chromosome 2, and id12001229 on chromosome 12. On chromosome 6, 74 SNPs were
associated with differences in AC of rice varieties. In which, two SNPs, fd7
(1764762 bp) and fd8 (176707 bp), were located in the Waxy gene region. The candidate SNPs are the base to find the
candidate genes and molecular markers related to AC in the medium-grain rice
varieties.
Acknowledgements
The author is highly thankful to the Institute of Food
and Biotechnology and College of Agriculture, Can Tho University for technical
support in laboratory work and their critical comments to improve the
manuscript.
Author Contributions
Bui Phuoc Tam carried out the experimental work,
participated in the sequence alignment, and drafted the manuscript. Pham Thi Be
Tu and Nguyen Thi Pha conceived of the study and participated in its design and
coordination. The authors have read and agreed to the published version of the
manuscript.
Conflicts of Interest
All authors declare no conflict of interest.
Data Availability
Data presented in this study will be available on a fair
request to the corresponding author.
Ethics Approval
Not applicable to this paper.
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