Cereal Chem - 2023 - Bao.pdf

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Jinsong Bao ORCID iD: 0000-0002-9606-6615
Molecular and genetic bases of rice cooking and eating quality:
an updated review
Jinsong Baoa,b
, Bowen Denga,b
, Lin Zhanga,b
a
Hainan Institute, Zhejiang University, Yazhou Bay Science and Technology City,
Yazhou District, Sanya 572025, China
b
Institute of Nuclear Agricultural Sciences, Key Laboratory of Nuclear Agricultural
Sciences of Ministry of Agriculture and Zhejiang Province, Zhejiang University,
Zijingang Campus, Hangzhou, 310058, China
Abstract
Background and objectives:
Rice grain quality is a primary determinant of its market price and consumer
acceptance. Although milling quality, appearance quality, cooking and eating quality
(CEQ), and nutritional quality represent the main features of grain quality, rice CEQ
is of the most economic importance. Starch physicochemical properties and sensory
evaluation have been widely applied to predict and assess rice CEQ. Understanding
the genetic and molecular bases of CEQ formation will facilitate rice quality
improvement through molecular breeding strategy.
Findings
The major genes responsible for rice CEQ formation have been characterized long
before. Waxy (Wx) encoding granule-bound starch synthase I (GBSSI) controls
apparent amylose content (AAC), starch synthase IIa (SSIIa) controls gelatinization
temperature, and fragrant gene (fgr) controls the aroma of cooked rice. Many natural
variations (allelic variants) have been identified in these genes among rice germplasm.
Protein content in rice grain is not only responsible for the nutritional quality, but also
affects CEQ. Two major genes controlling protein content have been identified and
cloned. Pyramiding of different alleles by marker assisted selection and creation of
new alleles by genome editing technology have facilitated improvement of new rice
varieties with desirable CEQ.
Conclusions
In addition to updating the advances made in the important CEQ genes, we identified
some future challenges. These include: the need to exploit new alleles in these genes,

especially in Wx, to confer low AAC with transparent appearance need to be exploited;
identifying alleles in Wx suitable for improving the texture of cooked rice with high
AAC and high resistant starch; how to manipulate genes and modify agronomic
practices to reduce protein content to improve CEQ; how to breed climate smart rice
with good, stable CEQ in changing environments.
Significance and Novelty
This article identifies some priorities for future research, which should enhance our
understanding of the molecular basis of CEQ for improving this important rice quality
attribute.
Keywords: Amylose, eating quality, fragrance, gelatinization temperature, protein,
rice
1. INTRODUCTION
The importance of the tiny rice grain in human daily life cannot be over-stressed,
since half of the world population takes rice as a staple food and eats rice every day.
With population growth, the demand for more rice production is ever increasing.
However, people’s demand for high quality rice and for a better life is also increasing.
Rice grain quality improvement has become one of the top priorities in rice breeding
programs, so that understanding the genetic control of rice cooking and eating quality
(CEQ) is necessary to improve rice grain quality.
Rice CEQ can be indirectly evaluated by the starch physicochemical properties, such
as AAC, gel consistency, gelatinization temperature (GT) and pasting viscosity.
However, sensory properties of cooked rice, such as texture, aroma and flavor are the
most important factors determining rice acceptability and value in the market (Bao,
2016). Waxy (Wx) gene encoding granule-bound starch synthase I (GBSSI) is
responsible for amylose biosynthesis and is the major gene controlling the AAC, gel
consistency and pasting viscosity, which are the main physicochemical parameters for
eating quality. Starch synthase IIa (SSIIa) functions to elongate amylopectin short
chains with degree of polymerization (DP) ≤12 (A chains) to B1 chains (13 ≤ DP ≤
24), so it is the major genetic factor responsible for GT, which is the main
physicochemical parameter for cooking quality (Gao et al., 2003; Nakamura et al.,
2005; Umemoto et al., 2002; Umemoto et al., 2004). The fragrance gene (fgr)
regulates the biosynthesis of 2-acetyl-1-pyrroline (2-AP), a chemical contributing to
the popcorn aroma, which is a major gene responsible for the sensory quality (Chen et

al., 2008). Protein content in rice grain is negatively correlated with eating quality and
palatability (Bao, 2016). Two major genes, qPC1 and qGPC-10, have been cloned
from rice germplasm showing different protein contents (Peng et al., 2014a; Yang et
al., 2019). Thus, molecular marker assisted selection (MAS) breeding using natural
variation in the above-mentioned genes is expected to be the basis for designing new
rice with good CEQ. Gene editing technology, especially the clustered regularly
interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)
genome editing tool, is another strategy to quickly generate new alleles with desired
traits to improve CEQ.
As many facets of rice grain quality have been comprehensively reviewed (Bao, 2014;
2016, 2019; Lau et al., 2015; Zhou et al., 2020; Zhou et al., 2022), this review will
focus on the above-mentioned genes to update current knowledge of the molecular
and genetic basis of CEQ in rice (Bao, 2012) with references mostly published in the
past decade. Future challenges and priorities of rice research in the field of rice CEQ
have been identified.
2. Important CEQ gene: Wx
Amylose is the most important factor affecting the cooking and eating quality (CEQ)
of milled rice (Bao 2012). Since amylopectin long branch chains can also be
complexed with iodine, the amylose content measured with formation of the
iodine-amylose complex is called apparent amylose content. AAC in waxy rice is
lower than 2%, whereas common rice has an AAC ranging from very low (5-12%)
(so-called soft rice), low (12-20%), intermediate (20-25%) to high (25%-33%) (Bao
2012). The AAC of amylose extender (ae) mutant can be over 35% (Yano et al 1985).
At present, there are no rice genotypes that approach the AAC of amylomaize (i.e.,
70%) (Bao and Bergman, 2018).
Amylose is synthesized by the action of granule-bound starch synthase I (GBSSI) in
the rice endosperm, encoded by the Waxy gene (Wx). Natural allellic variations in Wx
have been discovered, including wx, Wxa
, Wxin
, Wxb
, Wxmw/la
, Wxmp
, Wxmq
, Wx°p
, Wx/hp
and Wxlv
, which correspond to diverse AAC variation among rice germplasm (Fig. 1)
(Cai et al., 1998; Liu et al., 2009; Mikami et al., 1999, 2008; Hoai et al., 2014; Sato et
al., 2002; Zhang et al., 2019, 2021; Zhou et al., 2021). The alleles Wxa
in indica rice

(high AAC rice with a G single nucleotide polymorphism (SNP)) and Wxb
in japonica
rice (low AAC rice, T SNP) differ by a single base pair mutation (G to T) at the 5′
junction of the first intron (Int1-1 SNP, Fig. 1) (Hirano et al., 1998). This G/T
mutation also affects the levels of mature Wx mRNA (Cai et al., 1998). The wx allele
is a null Wx type present in glutinous rice (Cai et al., 1998; Hirano et al., 1998).
Fig. 1. Summaries of major Wx alleles and their relation with AAC. (A) The
functional markers in the Wx gene; (B) Sequence variation in different Wx alleles and
AAC.
It has been known for a long time that rice genotypes with the similar AAC can have
different CEQ, but the underlying reason has not been clarified. To solve this problem,
rice chemists have designed the gel consistency and viscosity tests to differentiate
those rice genotypes with similar AAC. An SNP at Ex10-115 site of Wx locus has
been discovered by association analyses of rice germplasm to be associated with soft
gel consistency and low viscosity phenotypes (Chen et al., 2008, Tran et al., 2011,
Traore et al., 2011, Teng et al., 2012, Teng et al., 2013, Hoai et al., 2014). Recently,
Zhang et al. (2019) also discovered that the Ex10-115 site of Wx allele, called Wxlv
, is
responsible for low viscosity by map-based cloning approach. They found that all the
rice with Wxa
allele have a T nucleotide at the Wxlv
locus, while all other rice
accessions have C at the Ex10-115 site. The nucleotides G in Int1-1 combined with C
in Ex10-115 in Wxlv
allele are the causal SNPs resulting in high AAC and low
viscosity. They also confirm that this C nucleotide in Ex10-115 is ancestral and may
play an important role in Wx domestication in rice, because it presents in the Wx gene

of most wild rice accessions (Oryza rufipogon) and the orthologs encoding GBSSI
from monocotyledon species like maize (Zea mays), sorghum (Sorghum bicolor),
millets (Setaria virdis, Setaria italica), Panicum Hallii, Panicum virgatum,
Brachypodium stacei and Brachypodium distachyon.
Although low AAC rice has better CEQ and acceptability for consumers in many
regions (Calingacion et al., 2014), rice varieties with a lower AAC, usually <13%, has
a dull or opaque grain appearance (Zhang et al., 2021; Zhou et al., 2021; Fu et al.,
2022), which offsets its commercial value because of unacceptable visual quality.
Recently, a novel Wx allele, Wxla
(Zhou et al., 2021) or Wxmw
(Zhang et al., 2021) has
been discovered. Rice with Wxla/mw
exhibits a low AAC (11-14%) and transparent
endosperm. Rice with this allele has reduced GBSSI activity and thus is responsible
for a transparent appearance and good eating quality (Zhang et al., 2021; Zhou et al.,
2021). Ando et al. (2010) reported that a 37 bp deletion in intron 10 of Wx could
reduce AAC by 7.8%.
The evolutionary origin of the different Wx alleles during the domestication of O.
sativa was proposed by Zhang et al. (2021) (Fig. 2). Assuming wild rice has the
Wxlv-w
haplotype from which the Wxlv
allele evolved, this then could have divided into
four (Zhang et al., 2019) or five haplotype groups (Zhou et al., 2021), i.e., Wxlv
-I to V,
due to artificial selection or de novo domestication. Since there are many wild rice
populations with a high level of genetic differentiation worldwide (Vaughan et al.,
2008), five Wx haplotype groups are shown in Fig. 2. The Wxb
allele originated from
the Wxlv
-V group as a result of one functional SNP at Int1-1, which is always found in
japonica subspecies. The Wxin
allele originated from the Wxlv
-IV group as a result of
one functional SNP at Ex6-62 in indica rice. However, japonica Wxin
allele can be
formed by recombination. The Wxa
and Wxop
alleles were selected after a base-pair
substitution at Ex10-115 or Ex4-77 site in the Wxlv
-II and Wxlv
-III group, respectively.
A 23-bp insertion at Ex2-113 site into the Wxb
allele generated the null wx allele,
resulting in a glutinous mutant and the indica waxy rice was derived from japonica
waxy rice by introgression of the wx allele (Yamanaka et al., 2004, Muto et al., 2016),
because mutation from G to T was required for origin of glutinous rice (Olsen and

Purugganan, 2002). The Wxmp/mq
allele also originated directly from the Wxb
allele
(Yang et al., 2013). Wxla/mw
allele originated from recombination between two alleles,
Wxb
and Wxin
in the japonica subspecies (Zhang et al., 2021; Zhou et al., 2021).
Fig. 2 The evolutionary relationships among Wx alleles and haplotypes in rice.
Redrawn according to Zhang et al. (2019; 2021) and Zhou et al. (2021).
Besides Wx which controls AAC, other genes or modifiers may genetically modify
AAC, although many of these affect AAC through interaction with Wx. Zhang et al.
(2014) mapped the AAC in a set of chromosome segment substitution lines (CSSLs)
derived from the heat resistant indica 9311 in the heat-sensitive japonica Nipponbare
background. Four quantitative trait loci (QTLs), qHAC4, qHAC8a, qHAC8b and
qHAC10, on chromosome 4, 8, 8 and 10, respectively, can reduce the deleterious
effects of amylose content at high temperature. The CSSLs carrying the qHAC8a9311
,
qHAC8b9311
and qHAC49311
have high pre-mRNA splicing efficiency of Wx gene,
which likely leads to stable amylose content at high temperature. Takemoto-Kuno et
al. (2015) revealed a novel QTL, designated qAC2, on the long arm of chromosome 2
that contributed to the low AC of japonica rice Kuiku162. The qAC2 Kuiku
allele has an

epistatic interaction with Wxa
and has epistatic interactions with two loci, dull1 (du1)
and du2, but it has an additive effect to that of du3 on the Wxb
background.
A high-to-low AAC selection trend in the course of rice domestication (Zhang et al.,
2019) and in current global rice breeding programs is manifest, since low amylose
rice or soft rice has become more and more popular (Calingacion et al., 2014; Yang et
al., 2022; Zhou et al., 2022). In current rice breeding programs, the major Wx alleles
in modern cultivated rice are Wxa
, Wxb
, and Wxin
(Zhang et al. 2019). Over the past 50
years in developing hybrid rice in China, it was found that almost all sterile lines are
with Wxa
allele, explaining why the hybrid rices have a low CEQ (Pan et al., 2022).
Introduction of other alleles into new varieties may facilitate breeding rice with low
AAC, soft texture of cooked rice, and transparent grain appearance. Introgression of
Wxmp
or Wxmq
alleles (AAC 8−12 %) into commercial rice cultivars can be used to
breed elite ‘soft rice’ with high palatability and win a high acceptance in the market.
When Wxmw
was introduced into a high‐yielding japonica cultivar via molecular
marker‐assisted selection, the new lines exhibited clear improvements in CEQ and
endosperm transparency (Zhang et al. 2021). This indicates that the Wxla/mw
is a
promising allele to improve CEQ and grain transparency in the japonica rice breeding
programs. The AAC is generally correlated with high resistant starch content. The
Wxlv
allele can be introduced into the high resistant rice mutants which are derived
from the defective starch synthase IIIa (SSIIIa) (Ying et al. 2023) or starch branching
enzyme IIb (SBEIIb) genes (Ying et al. 2022; Hu et al., 2023), to produce new rice
varieties with softer texture since high resistant starch rice generally has firm texture
in the cooked form (Zhang et al., 2019).
Gene editing technology such as CRISPR/Cas9 has been widely applied in rice
breeding to modify the target genes, and Wx is a good candidate in many studies to
modify AAC and then improve rice CEQ. Editing of coding sequences of Wx gene by
CRISPR/Cas9 technology usually results in a waxy mutant (Zhang et al. 2018; Zhang
et al., 2023; Teng et al. 2021; Fu et al. 2022), but waxy rice is already available in
germplasm and current cultivars, so these waxy mutants obtained in this way only
increases the pool of rice germplasm. However, editing conserved cis-acting elements

in the promoter of Wx gene may be expected to reduce Wx expression levels, resulting
in a lower AAC and enhancement of CEQ. Some novel Wx alleles with reduced
amylose levels by promoter editing using CRISPR/Cas9 system have been
successfully obtained in rice either carrying the Wxb
allele (Huang et al. 2020) or Wxa
allele (Zeng et al. 2020; Yang et al., 2022). Targeting these sequences is useful in CEQ
improvement via modification of expression of Wx gene. Base editing is another good
way to modify a single nucleotide by changing only one amino acid of the Wx protein,
which can create new alleles such as Wxop/hp
, Wxmq/mp
and Wxla/mw
. Using a cytidine
base editor, Xu et al (2020) have successfully obtained very low amylose rice with the
AAC ranging from 1.4 to 11.3% (the AAC of wildtype is 14.4%), using sgRNAs that
target the third to fifth exons of Wxb
. Using an adenine base editor, Huang et al.
(2021a) successfully obtained modified rice with sgRNAs targeting the seventh and
tenth exons of Wxb
. Some lines obtained were waxy rice, many lines had similar AAC
as wild type (AC of 19.87%), one line had a typical “soft rice” AAC (11%). However,
there were a few lines showing unexpectedly high AAC (21-30%) (Huang et al.,
2021a). Editing the Wx gene by deleting its 1st intron using CRISPR/Cas9 technology
can generate rice with AAC significantly increased from 13.0% to approximately 24.0%
in rice carried the Wxb
allele, but no significant difference in AAC was observed
between wild-type plants and mutant lines carrying the Wxa
and Wxlv
alleles (Liu et al.,
2022).
Wx gene is also responsible for the genetic basis of starch properties, such as gel
consistency, pasting viscosity and retrogradation properties (Bao et al., 2000; Hsu et
al., 2014; Wang et al., 2007; Wang et al., 2017; Xu et al., 2015, 2016). In a candidate
gene association analysis (Yang et al., 2014; Li et al., 2017), Wx was found to be a
major main-effect QTL for pasting viscosity, gel texture, and retrogradation property.
Wx in a starch synthase IIa (SSIIa) background was significant for conclusion
temperature and enthalpy of gelatinization as measured by differential scanning
calorimetry (Yang et al., 2014).
Genetic interaction between Wx and other genes has attracted great interest to
understand the genetic basis of CEQ. Luo et al. (2015) investigated the allelic (indica

vs japonica) effects of six starch biosynthetic genes on starch functional properties in
a recombinant inbred line population. They found that the Wxa
allele played a major
role in the increase of amylose content, whereas indica SSI allele, japonica SSIIa
allele and indica BEI allele had minor roles. Interactions between Wx, SSIIa and
floury2 (flo2) are significant for many starch or flour physicochemical properties
(Zhang et al., 2022a; Zhao et al., 2022). Wx may also interact with SSI to affect grain
quality and starch physicochemical properties (Zhang et al., 2022b).
Wx is a pleiotropic gene. In addition to affect the AAC and starch physicochemical
properties, the Wx locus is a major gene for crude fat content in rice. Lipids or fat
content has an impact on rice CEQ (Tong and Bao, 2019). Rice with higher crude fat
content is generally accompanied with brighter luster and better eating quality (Xia et
al., 2022). Using genome-wide association analysis and linkage analysis of 533
diverse cultivars and an F2 population, Xia et al. (2022) found a major QTL on
chromosome 6, qFC6, which was allelic with Wx, affected crude fat content of rice.
qFC6 positively affects bound lipid content and negatively regulates free lipid content.
The Int1-1 SNP of Wx was identified to be strongly associated with albumin content
of rice protein (Chen et al., 2018). Significant negative correlations were observed
between albumin content and AAC or quantity of 2.3 kb mature Wx RNA (Table 2),
suggesting that Wx may negatively regulate albumin content (Chen et al., 2018).
Kashiwagi and Munakata (2018) also detected the association between Wx locus and
protein content in a rice chromosome segment substitution line population.
Furthermore, it was found that Wx is an important genetic factor for grain fissure
resistance and head rice yield as revealed by a genome-wide association study, so Wx
also has an effect on the milling quality (Deng et al., 2022).
3. Important CEQ gene: SSIIa
The starch gelatinization temperature (GT) is mainly controlled by the SSIIa, or
alkaline degenerate (ALK) gene, also known as SSII-3, which is located on
chromosome 6 (Gao et al., 2003; Bao et al., 2006; Umemoto et al., 2002; Umemoto et
al., 2004; Waters et al., 2006). There are four non-synonymous SNPs in the SSIIa
gene (Fig. 3A). The first one is at 264 bp in Exon 1 of the sequence AY423717, where

a change from G to C results in change of glutamate to aspartate at amino acid (aa) 88.
The second site is at 3799 bp, where glycine (at 604 aa) encoded by GGC is replaced
by serine encoded by AGC. The third site is at 4198 bp, where valine (at 737 aa)
encoded by GTG is replaced by methionine encoded by ATG. The fourth site is at
4330 bp, glycine-leucine at 780-781 aa encoded by GGGCTC is replaced by
glycine-phenylalanine encoded by GGTTTC (Bao et al., 2006; Umemoto et al., 2004;
Waters et al., 2006). Nakamura et al (2005) indicated from gene fragments shuffling
experiments that Val-737 and Leu-781 are essential, while Gly-604 plays some role in
SSIIa catalytic activity when Leu-781 is replaced by Phe. Glu-88 apparently does not
play an equally important role in determining SSIIa activity. Gao et al. (2011) also
showed that when replacing the valine-737 with methionine-737, the enzyme activity
decreased with the lowering of GT, which indicated that the A SNP is essential in
decreasing GT. The third and fourth SNP indicate there are three haplotypes available
in rice germplasm (Fig. 3B). The G/GC haplotype rice has a high or intermediate GT,
while the A/GC or G/TT haplotype rice has a low GT (Bao et al., 2006; Bao et al.,
2009; Nakamura et al., 2005; Waters et al., 2006). Chen et al. (2020) further showed
that G/TT allele starch had a slightly lower GT but had a wider distribution among
rice subpopulations than A/GC. Zhang et al. (2020) indicated that an SNP in Exon 1
combined with G/GC in a high-GT indica rice had a higher GT and improved
retrogradation properties. The high or low GT in each haplotype generally concurs
with the SSIIa activity, as seen from the shuffling constructs (Nakamura et al., 2005).
However, SSIIa activity of A/GC (0.13 mol ADPglucose/min/mg protein) allele was a
little lower than that of the G/TT (0.15 mol ADPglucose/min/mg protein) allele.
Zheng et al. (2020) found a sequence variation, i.e. a 9 bp insertion/deletion (InDel),
in the promoter region of SSIIa. The 9 bp insertion at position -741 bp of the promoter
existed in most of indica and japonica rices that have three haplotypes (A/GC, G/GC
and G/TT) in the third and fourth SNPs. The absence of this 9 bp sequence exists in
most of Aus/boro and Basmati ecotypes with only the G/GC haplotype. Since G/GC
rices have high or intermediate GT (Bao et al., 2006; Bao, 2011, 2019), whether this
InDel can further differentiate high or intermediate GT rice is an interesting topic that
deserves to be studied.

Fig. 3. Summaries of major SSIIa haplotypes and their relation with gelatinization
temperature (GT). (A) The functional markers in the SSIIa gene; (B) Three haplotypes
and their GT.
However, it should be mentioned that the frequency of the A SNP at 4198bp is
especially low (Bao et al., 2006; Zhou et al., 2016) and A/TT haplotype has not been
found in the rice germplasm. The origin of the A SNP is an interesting issue. Song et
al. (2022) indicated that the A SNP or A/GC haplotype only presented in the temperate
japonica rice; they speculated that the temperate japonica rice with A SNP in SSIIa
might undergo positive selection for low GT starch during its domestication under a
small selection pressure. At the very beginning of domestication, all the wild rice
located in southern China had the G SNP at 4,198 bp and both GC/TT at 4,329/4,330
bp. After the diversification of indica and japonica, a mutation with the A SNP at site
4,198 substituted for the G SNP (Fig. 4). Another intriguing question is raised, that is,
why does it need two ways to make its GT low? On one hand, low GT rice requires
less energy to be cooked, and maybe there was not enough energy to cook rice in
temperate region due to cold climate, so that low GT rice is positively selected. On
the other hand, it is possible that our ancestors preferred eating low GT rice, which
has a better eating quality, and then applied a pressure to select the low GT rice. It
should be mentioned that the A SNP may have originated recently, because its allele
frequency is very low in rice germplasm (Bao et al., 2006; Umemoto et al., 2004;
Waters et al., 2006; Zhou et al., 2016).

Fig. 4. The flow of SSIIa-G/A SNP between rice and its wild relative constructed
based on the single-origin hypothesis of rice. Adapted from Song et al. (2022).
SSIIa functions to elongate amylopectin short chains with degree of polymerization
(DP) ≤12 (A chains) to 13 ≤ DP ≤ 24 (B1 chains), which is responsible for low or high
GT (Nakamura et al., 2005; Miura et al., 2018; Fujita et al., 2022). High percentage of
B1 chains favors formation of longer double helices as found in indica rice with the
G/GC haplotype, whereas A chains can only form a short double helix as found in
japonica rice with the A/GC or G/TT haplotypes. Although the A/GC and G/TT rices
have a low SSIIa activity, whether the complete deficiency of SSIIa can further
increase amylopectin short chains and reduce GT is unknown. Miura et al. (2018)
screened an ss2a mutant with no SSIIa activity or SSIIa protein following
N-methyl-N-nitrosourea mutagenesis of a japonica rice Kinmaze, and found that this
mutant showed more A chains, a 5.6 o
C lower GT and 3.4 % higher AAC than the
wildtype. An SSIIa knock out mutant in another japonica rice, Nipponbare, with
CRISPR/Cas9 technology also results in more A chains and a lower GT, but the AAC
was not affected (Huang et al., 2021b).
In addition to controlling the GT, SSIIa also affects other phycochemical properties of
starch. Among non-waxy rice, SSIIa is a major main-effect QTL for retrogradation
properties, but a minor main-effect QTL for some pasting viscosity parameters, such
as breakdown, gel consistency, among others (Gao et al., 2011; Yang et al., 2014).
Among the wx rice accessions, SSIIa is a major genetic factor controlling pasting
viscosities, swelling volume and retrogradation properties besides GT (Xu et al.,
2013), suggesting that the functional SNPs of SSIIa are useful in molecular breeding
of high quality waxy and non-waxy rice. Rice cakes developed from ss2a waxy rice

could maintain softness and elasticity for up to 6 days when stored at low temperature
(Nakano et al, 2023). Under BEIIb mutant background, no matter what the SSIIa
allele, the starch has a high GT and low paste viscosities, suggesting an interaction
between SSIIa and BEIIb (Hu et al., 2023).
4. Important CEQ gene: fragrance (fgr)
Cooked rice aroma and flavor are sensory features of CEQ that are popular with
consumers. Aroma is becoming an increasingly important target in modern rice
breeding programs. Basmati and Jasmine aromatic rices dominate in the international
trade market. Basmati type is mainly grown in India and Pakistan, whereas Jasmine
type is grown primarily in Thailand (Calingacion et al., 2014; Daygon et al., 2017).
More than 200 volatile compounds have been identified in cooked rice, of which only
a few may relate to the aroma and flavor of cooked rice (Bao, 2016). It is difficult to
determine which volatile compounds are responsible for the perceived aroma/flavor of
rice. Only one compound, 2-acetyl-1-pyrroline (2-AP; popcorn aroma) has been
confirmed to contribute a characteristic aroma. Furthermore, 2-AP is the only volatile
compound in which the relationship between its concentration in rice and sensory
intensity has been established. The fragrance gene (fgr) regulating the biosynthesis of
2-AP on chromosome 8, which encodes the betaine aldehyde dehydrogenase 2
(BADH2), has been cloned. A deletion of 8 bp in exon 7 makes it non-functional,
leading to accumulation of 2-AP in the rice grain. This 8 bp deletion in exon 7
(badh2-E7) is the predominant allele in most aromatic varieties including the Jasmine
and Basmati fragrant rice (Bradbury et al., 2005a; Shi et al., 2008; Kovach et al.,
2009). An additional 17 alleles in fgr have been explored by sequencing the fragrant
rice germplasm (Shao et al., 2013; He and Park 2015) (Table 1). Among these, five
alleles have been genetically validated for co-segregation with aroma in rice grain, but
others have not been validated (He and Park, 2015). As expected, more
polymorphisms can be identified by sequencing more rice accessions. Phitaktansakul
et al. (2022) identified 26 alleles in the BADH2 coding region where eight alleles
were previously reported. Those alleles showing co-segregation with aroma can be
directly used in selection and breeding of aromatic rice cultivars (Jin et al., 2010; Lau

et al., 2017). By targeting BADH2 using CRISPR/Cas9 technology, modern rice
cultivars including both of three lines and two lines hybrid fragrant rice varieties have
been successfully bred (Ashokkumar, et al., 2020; Tang, et al., 2021; Hui, et al., 2022;
Imran, et al., 2023; Tian et al., 2023; Zhang et al., 2023). Some studies targeted Wx
and BADH2 gene together to quickly generate waxy or low AAC fragrant rice (Tian et
al., 2022; Zhang et al., 2023)
Table 1 Summary of the exon polymorphisms in Badh2
Alleles Locatio
n
Variatio
n
Marker
develope
d
Co-segregatio
n tested
Reference
s
badh2-p-5′ UTR 5′ UTR 3-bp
deletion
N – Shi et al.
(2014)
badh2-E1.1 Exon 1 2-bp
deletion
N – Kovach et
al. (2009)
badh2-E1.2 (256) Exon 1–
intron 1
junction
G/A snp Y Y Ootsuka et
al. (2014)
badh2-E2.1(454-460) Exon 2 7-bp
deletion
Y Y Shi et al.
(2008)
deletion
N – Shao et al.
(2013)
badh2-E4-5.1 Exon 4
to exon
5
806-bp
deletion
N – Shao et al.
(2013)
badh2-E4-5.2 Exon 4
to exon
5
803-bp
deletion
Y N Shao et al.
(2011)
badh2-E7 (3039-3046) Exon 7 8-bp
deletion
Y Y Bradbury
et al.
(2005a,b),
Shi et al.
(2008);
Kovach et
al. (2009)
badh2-E8 Exon 8 7-bp N – Amarawat

insertion hi et al.
(2008)
insertion
N – Kovach et
al. (2009)
deletion
N – Kovach et
al. (2009)
badh2-E10.3 Exon 10 G/T snp N – Kovach et
al. (2009)
badh2-E10.4 Exon 10 G/A snp N – Shao et al.
(2013)
badh2-E12
(5241-5243)
Exon 12 3-bp
deletion
Y Y He and
Park
(2015)
badh2-E13.1(5380-538
2)
Exon 13 3-bp
insertion
Y Y Kovach et
al. (2009)
badh2-E13.2 Exon 13 C/T snp N – Kovach et
al. (2009)
insertion
Y N Kovach et
al. (2009)
badh2-E14.2 Exon 14 G/T snp N – Kovach et
al. (2009)
Adapted from He and Park (2015).
As for the origin of fragrant rice, Kovach et al. (2009) have revealed the badh2-E7
allele originated within the japonica varietal group and that of indica rice was
introgressed from japonica rice. Basmati-like accessions were nearly identical to the
ancestral japonica haplotype, demonstrating a close evolutionary relationship between
Basmati varieties and the japonica gene pool. However, it should be mentioned that
the badh2 gene is not the only gene responsible for all the aroma differences observed
in aromatic rice accessions. The amount of 2-AP in most uniform fgr genotypes (i.e.,
badh2-E7 allele with 8 bp deletion) was not significantly different from that in
aromatic genotypes with non-badh2-E7 allele, but several badh2-E7 genotypes
accumulated exceptionally large amounts of 2-AP (Fitzgerald et al., 2008). This raised

the question “Is there a second gene for fragrance in rice” by Fitzgerald et al., (2008),
who speculated that the exceptionally large amounts of 2-AP in fragrant rice may be
driven by alleles of at least two different genes, not by different alleles of the same
fragrance gene. This question is still waiting for answers since no second gene has so
far been reported. One good answer has been the detection of a minor-effect QTL on
chromosome 1 by association mapping of 2-AP content (Daygon et al., 2017). The
candidate gene is likely TPK1 which encodes thiamine pyrophosphate kinase,
catalysing the conversion of thiamine to thiamine pyrophosphate. Another good
answer is from multi-omics technology including whole-genome resequencing, and
transcriptomic and metabolomic analyses, of 475 accessions in the Korean World
Rice Collection. Phitaktansakul et al. (2022) identified an array of expression QTLs
(eQTLs) and trans-protein QTLs (pQTLs) associated with badh2 expression and
protein accumulation which are likely regulators mediating 2-AP variation in fragrant
rice. However, the function of these QTLs in determining the 2-AP needs further
investigation.
5. Genes for protein content
Protein is the second-most abundant component of rice endosperm and
constitutes 6-10 % of the dry matter in the milled rice. It is well known that protein
content of milled rice has negative effects on CEQ. High-protein rice has much firmer
texture, less stickiness and weaker flavor of cooked rice than low-protein rice, and the
GBSSI protein also has a negative correlation with cooked rice stickiness (Bao, 2016).
As mentioned above, Wx might be a minor genetic factor controlling the protein
content. Many QTLs have been detected for protein content (Chen et al., 2018, 2023;
Xu et al., 2015, 2016; Wang et al., 2017; Kashiwagi and Munakata, 2018)
Two major QTLs controlling protein content have been cloned. qPC1 encodes a
putative amino acid transporter OsAAP6, which functions as a positive regulator of
GPC in rice, such that higher expression of OsAAP6 is correlated with higher GPC
and its fractions, glutelins, prolamins, globulins, albumins (Peng et al., 2014).
qGPC-10 encoding glutelin precursors regulates the glutelin synthesis and
accumulation through differential transcription expression intensity, leading to higher

GPC in indica rice than in japonica rice (Yang et al., 2019). Both genes could serve as
targets for manipulation through gene editing or molecular marker-aided selection in
rice quality improvement. For example, knockout mutants of OsAAP6 in japonica
varieties using the CRISPR/Cas9 system showed decreased GPC and AAC, indicating
the CEQ is improved (Wang et al., 2020). However, high protein content means better
nutritional quality. How to balance CEQ and nutritional quality is a question. New
strategies for balancing nutritional quality and eating and cooking quality in rice grain
need to be explored.
6. Future challenges
We understand that many other factors may affect CEQ through genetic or other
means. Many regulatory elements that modify starch biosynthesis will also modify
CEQ, such as floury and dull genes (Bao et al., 2022; Takemoto-Kuno et al., 2015).
However, there are still some issues that need to be clarified. (1) Low amylose rice
(soft rice) with transparent appearance is favored by consumers, so finding additional
Wx alleles conferring low amylose is necessary to breed rice matching the demand of
consumers; however, are such new alleles still available in rice germplasm. (2) Which
Wx alleles are suitable to improve the hard texture of the cooked high-AAC and high
resistant starch rice? (3) Which genetic factors determine the high or intermediate GT
carrying the G/GC allele? (4) How do the interactions between starch biosynthesis
genes and enzymes regulate the starch physicochemical properties and, in turn, CEQ?
(5) Is there a second fragrant gene for cooked rice aroma? (6) How many genes
control the protein content? Commercial varieties, such as Koshihikari, usually have a
low protein content, so how low a protein content gives rice its best eating quality,
and how can protein content be reduced to improve the CEQ?
Many famous commercial varieties, such as Koshihikari, Basmati, Khao Dawk
Mali 105, among others (Calingacion et al., 2014; Bin Rahman and Zhang, 2022),
could not produce expected premium quality when planted in areas other than their
original ecological niches, suggesting a great effect of environmental conditions.
Genetic dissections of the internal or external factors that make up the premium
quality of these famous rice cultivars are necessary (Calingacion et al., 2014; Bin

Rahman and Zhang, 2022). In doing so, it would be possible to transfer the quality
traits into high yielding cultivars using MAS technology
Climate change has an adverse effect on rice production and rice grain quality.
The world is experiencing rising global temperatures and CO2 concentration, and
change of precipitation patterns resulting in more droughts or flooding (Bin Rahman
and Zhang, 2022). All these factors may modify starch, protein and lipid biosynthesis,
and hence starch structure and CEQ (Zhang et al., 2022c). Development of
climate-smart rice varieties that display stable rice production and grain quality under
stressed conditions become a main target in current breeding programs (Sreenivasulu
et al., 2015). Understanding of the genetic basis of the resistance to climate change is
a first step to breeding climate-smart rice varieties.
Acknowledgements
The authors sincerely thank Prof. Les Copeland for his constructive comments and
careful corrections of the grammars. This work was financially supported by the
Hainan Provincial Natural Science Foundation (323MS066), AgroST Project
(NK2022050102) and Zhejiang Provincial Natural Science Foundation
(LZ21C130003).
Reference
Amarawathi, Y., Singh, R., Singh, A.K., Singh, V.P., Mohapatra, T., Sharma, T.R., Singh, N.K.
(2008) Mapping of quantitative trait loci for basmati quality traits in rice (Oryza sativa L.).
Molecular Breeding, 21, 49-65.
Ando, I., Sato, H., Aoki, N., Suzuki, Y., Hirabayashi, H., Kuroki, M., Shimizu, H., Ando, T., &
Takeuchi, Y. (2010) Genetic analysis of the low-amylose characteristics of rice cultivars
Oborozuki and Hokkai-PL9. Breeding Science, 60(3), 187–194.
Ashokkumar, S., Jaganathan, D., Ramanathan, V., Rahman, H., Palaniswamy, R., Kambale, R.,
Muthurajan, R. (2020) Creation of novel alleles of fragrance gene OsBADH2 in rice through
CRISPR/Cas9 mediated gene editing. PLoS One, 15, e0237018.
Bao, J.S. (2012) Toward Understanding the Genetic and Molecular Bases of the Eating and
Cooking Qualities of Rice. Cereal Foods World, 57, 148-156.
Bao, J.S. (2014) Genes and QTLs for rice grain quality improvement. In: Yan, W.G., Bao, J.S.
(Eds.), Rice - Germplasm, Genetics and Improvement (pp. 239-278). InTech.
Bao, J.S. (2016) Rice: Eating quality. In: Wrigley, C., Corke, H., Seetharaman, K., and Faubion, J.
(Eds.), Encyclopedia of Food Grains, 2nd Edition (pp. 166-175). Oxford: Academic Press.
Bao, J.S. (2019) Rice starch. In: Bao JS (ed) Rice Chemistry and Technology, 4th edition (pp.
55-108). Elsevier.
Bao, J.S., Bergman, C.J. (2018) Rice flour and starch functionality. In: Sjoo M, Nilsson L. (Eds.),
Starch in Food: Structure, function and application, 2nd Edition (pp. 373-419). Elsevier
Press.
Bao, J.S., Corke, H., Sun, M. (2006) Nucleotide diversity in starch synthase IIa and validation of
single nucleotide polymorphisms in relation to starch gelatinization temperature and other
physicochemical properties in rice (Oryza sativa L.). Theoretical and Applied Genetics, 113,
1171-1183.

Bao, J.S., Xiao P, Hiratsuka M, Sun, M., Umemoto T., 2009. Granule-bound SSIIa protein content
and its relationship with amylopectin structure and gelatinization temperature of rice starch.
Starch/Starke, 61, 431-437.
Bao, J.S., Zheng, X.W., Xia, Y.W., He, P., Shu, Q.Y., Lu, X., Chen, Y., Zhu, L.H. (2000) QTL
mapping for the paste viscosity characteristics in rice (Oryza sativa L.). Theoretical and
Applied Genetics, 100, 280-284.
Bao, J.S., Zhang, Y., Zhao, J., Chen, Y., Wu, W., Cao, L., Xu, F. (2022) Identification of new allele
of FLOURY ENDOSPERM2 in white-core endosperm mutant of rice. Rice Science, 29,
407-411
Bin Rahman, A.N.M.R., Zhang, J. (2023) Trends in rice research: 2030 and beyond. Food and
Energy Security, 12, e390.
Bradbury, L.M.T., Fitzgerald, T.L., Henry, R.J., Jin, Q., Waters, D.L.E. (2005a) The gene for
fragrance in rice. Plant Biotechnology Journal, 3, 363-370.
Bradbury, L.M.T., Henry, R.J., Jin, Q., Reinke, R.F., Waters, D.L.E. (2005b) A perfect marker for
fragrance genotyping in rice. Molecular Breeding, 16, 279-283.
Cai, X. L., Wang, Z. Y., Xing, Y., Zhang, J. L., Hong, M. M. (1998) Aberrant splicing of intron 1
leads to the heterogeneous 5’ UTR and decreased expression of waxy gene in rice cultivars of
intermediate amylose content. Plant Journal, 14, 459-465.
Calingacion M, Laborte A, Nelson A, Resurreccion A, Concepcion JC, Daygon VD, Mumm R,
Reinke R, Dipti S, Bassinello PZ et al (2014) Diversity of global rice markets and the science
required for consumer-targeted rice breeding. PLoS One, 9, e85106.
Chen P., Lou G., Wang Y., Chen J., Chen W., Fan Z., Liu Q., Sun B., Mao X., Yu H., Jiang L.,
Zhang J., Lv S., Xing J., Pan D., Li C., He Y. (2023) The genetic basis of grain protein
content in rice by genome-wide association analysis. Molecular Breeding, 43, 1.
Chen Z, Lu Y, Feng L, Hao W, Li C, Yang Y, Fan X, Li Q, Zhang C, Liu Q (2020) Genetic
dissection and functional differentiation of ALKa and ALKb, two natural alleles of the
ALK/SSIIa gene, responding to low gelatinization temperature in rice. Rice, 13, 39.
Chen, M.-H., Bergman, C., Pinson, S., Fjellstrom, R. (2008) Waxy gene haplotypes: Associations
with apparent amylose content and the effect by the environment in an international rice
germplasm collection. Journal of Cereal Science, 47, 536-545.
Chen, P., Shen, Z., Ming, L., Li, Y., Dan, W., Lou, G., Peng, B., Wu, B., Li, Y., Zhao, D., Gao, G.,
Zhang, Q., Xiao, J., Li, X., Wang, G., He, Y. (2018) Genetic basis of variation in rice seed
storage protein (albumin, globulin, prolamin, and glutelin) content revealed by genome-wide
association analysis. Frontiers in Plant Science, 9, 612.
Chen, S., Yang, Y., Shi, W., Ji, Q., He, F., Zhang, Z., Cheng, Z., Liu, X., Xu, M. (2008) Badh2,
encoding betaine aldehyde dehydrogenase, inhibits the biosynthesis of 2-acetyl-1-pyrroline, a
major component in rice fragrance. Plant Cell, 20, 1850-61.
Chen, Z., Lu, Y., Feng, L., Hao, W., Li, C., Yang, Y., Fan, X., Li, Q., Zhang, C., Liu, Q. (2020)
Genetic Dissection and Functional Differentiation of ALKa and ALKb, Two Natural Alleles
of the ALK/SSIIa Gene, Responding to Low Gelatinization Temperature in Rice. Rice, 13,
39.
Daygon, V.D., Calingacion, M., Forster, L.C., Voss, J.J.D., Schwartz, B.D., Ovenden, B., Alonso,
D.E., McCouch, S.R., Garson, M.J., Fitzgerald, M.A. (2017) Metabolomics and genomics
combine to unravel the pathway for the presence of fragrance in rice. Scientific Reports, 7,
8767.
Deng Z., Liu Y., Gong C., Chen B., Wang T. (2022) Waxy is an important factor for grain fissure
resistance and head rice yield as revealed by a genome-wide association study. Journal of
Experimental Botany, 73, 6942-6954.
Fitzgerald, M.A., Sackville Hamilton, N.R., Calingacion, M.N., Verhoeven, H.A., Butardo, V.M.
(2008) Is there a second fragrance gene in rice? Plant Biotechnol. J, 6, 416-423.
Fu, Y., Luo, T., Hua, Y., Yan, X., Liu, X., Liu, Y., Liu, Y., Zhang, B., Liu, R., Zhu, Z., Zhu, J. (2022)
Assessment of the Characteristics of Waxy Rice Mutants Generated by CRISPR/Cas9.
Frontiers in Plant Science, 13, 881964.
Fujita, N., Miura, S., Crofts, N. (2022) Effects of various allelic combinations of starch
biosynthetic genes on the properties of endosperm starch in rice. Rice, 15, 24.
Gao, Z., Zeng, D., Cheng, F., Tian, Z., Guo, L., Su, Y., Yan, M., Jiang, H., Dong, G., Huang, Y.,
Han, B., Li, J., Qian, Q. (2011) ALK, the key gene for gelatinization temperature, is a

modifier gene for gel consistency in rice. J Integrat Plant Biol, 53, 756-765.
Gao, Z., Zeng, D., Cui, X., Zhou, Y., Yan, M., Huang, D., Li, J., Qian, Q. (2003) Map-based
cloning of the ALK gene, which controls the gelatinization temperature of rice. Science
China C Life Science, 46, 661-668.
He, Q., Park, YJ. (2015) Discovery of a novel fragrant allele and development of functional
markers for fragrance in rice. Molecular Breeding, 35, 217.
Hirano HY, Eiguchi M, Sano Y (1998) A single base change altered the regulation of the Waxy
gene at the posttranscriptional level during the domestication of rice. Molecular Biology and
Evolution, 15, 978-987.
Hoai, T.T.T., Matsusaka, H., Toyosawa, Y., Suu, T.D., Satoh, H., and Kumamaru, T. (2014).
Influence of single-nucleotide polymorphisms in the gene encoding granule-bound starch
synthase I on amylose content in Vietnamese rice cultivars. Breeding Science, 64, 142-148.
Hsu YC, Tseng MC, Wu YP, Lin MY, Wei FJ, Hwu KK, Hsing YI, Lin YR (2014) Genetic factors
responsible for eating and cooking qualities of rice grains in a recombinant inbred population
of an inter-subspecific cross. Molecular Breeding, 34, 655-673.
Hu, Y.Q., Zhang, Y.N., Yu, S.W., Deng, G.F., Dai, G.X., Bao, J.S. (2023) Combined effects of
BEIIb and SSIIa alleles on amylose contents, starch fine structure and physicochemical
properties of indica rice. Foods, 12, 119.
Huang, L., Gu, Z., Chen, Z., Yu, J., Chu, R., Tan, H., Zhao, D., Fan, X., Zhang, C., Li, Q., Liu, Q.
(2021b) Improving rice eating and cooking quality by coordinated expression of the major
starch synthesis-related genes, SSII and Wx, in endosperm. Plant Molecular Biology, 106,
419-432.
Huang, L.C., Li, Q.F., Zhang, C.Q., Chu, R., Gu, Z.W., Tan, H.Y., Zhao, D.S., Fan, X.L., and Liu,
Q.Q. (2020). Creating novel Wx alleles with fine-tuned amylose levels and improved grain
quality in rice by promoter editing using CRISPR/Cas9 system. Plant Biotechnology Journal,
18, 2164-2166.
Huang, X., Su, F., Huang, S., Mei, F., Niu, X., Ma, C., Zhang, H., Zhu, X., Zhu, J.K., Zhang, J.
(2021a) Novel Wx alleles generated by base editing for improvement of rice grain quality. J
Integrative Plant Biology, 63, 1632-1638.
Hui, S., Li, H., Mawia, A.M., Zhou, L., Cai, J., Ahmad, S., Lai, C., Wang, J., Jiao, G., Xie, L.,
Shao, G., Sheng, Z., Tang, S., Wang, J., Wei, X., Hu, S., Hu, P. (2022) Production of aromatic
three-line hybrid rice using novel alleles of BADH2. Plant Biotechnology Journal, 20, 59-74.
Imran, M., Shafiq, S., Tang, X. (2023) CRISPR-Cas9-mediated editing of BADH2 gene triggered
fragrance revolution in rice. Physiol Plant, 175, e13871.
Jin, L., Lu, Y., Shao, Y., Zhang, G., Xiao, P., Shen, S., Corke, H., Bao, J.S. (2010) Molecular
marker assisted selection for improvement of the eating, cooking and sensory quality of rice
(Oryza sativa L.). Journal of Cereal Science, 51, 159-164.
Kashiwagi, T., Munakata, J. (2018) Identification and characteristics of quantitative trait locus for
grain protein content, TGP12, in rice (Oryza sativa L.). Euphytica, 214, 165.
Kovach, MJ, Calingacion MN, Fitzgerald MA, McCouch SR (2009) The origin and evolution of
fragrance in rice (Oryza sativa L.). Proc Natl Acad Sci, 106,14444-14449.
Lau, W.C.P., Rafii, M.Y., Ismail, M.R., Puteh, A., Latif, M.A., Asfaliza, R., Miah, G. (2017)
Development of advanced fragrant rice lines from MR269 × Basmati 370 through
marker-assisted backcrossing. Euphytica, 213, 11.
Lau, W.C.P., Rafii, M.Y., Ismail, M.R., Puteh, A., Latif, M.A., Ramli, A. (2015) Review of
functional markers for improving cooking, eating, and the nutritional qualities of rice.
Frontiers in Plant Science, 6, 832.
Li KH, Bao JS, Corke H, Sun M (2017) Association analysis of markers derived from starch
biosynthesis related genes with starch physicochemical properties in the USDA rice
mini-core collection. Frontiers in Plant Science, 8, 424.
Liu, X., Ding, Q., Wang, W., Pan, Y., Tan, C., Qiu, Y., Chen, Y., Li, H., Li, Y., Ye, N., Xu, N., Wu,
X., Ye, R., Liu, J., Ma, C. (2022) Targeted Deletion of the First Intron of the Wxb
Allele via
CRISPR/Cas9 Significantly Increases Grain Amylose Content in Rice. Rice, 15, 1.
Liu, L., Ma, X., Liu, S., Zhu, C., Jiang, L., Wang, Y., Shen, Y., Ren, Y., Dong, H., Chen, L., Liu,
X., Zhao, Z., Zhai, H., Wan, J. (2009) Identification and characterization of a novel Waxy
allele from a Yunnan rice landrace. Plant Molecular Biology, 71, 609-626.
Luo, J., Jobling, S.A., Millar, A., Morell, M.K., Li, Z. (2015) Allelic effects on starch structure and

properties of six starch biosynthetic genes in a rice recombinant inbred line population. Rice,
8, 15.
Mikami I, Aikawa M, Hirano HY, Sano Y. (1999) Altered tissue-specific expression at the Wx
gene of the opaque mutants in rice. Euphytica, 105, 91-99.
Mikami, I., Uwatoko, N., Ikeda, Y., Yamaguchi, J., Hirano, H.Y., Suzuki, Y., Sano, Y. (2008)
Allelic diversification at the wx locus in landraces of Asian rice. Theoretical and Applied
Genetics, 116, 979-989.
Miura, S., Crofts, N., Saito, Y., Hosaka, Y., Oitome, N.F., Watanabe, T., Kumamaru, T., and Fujita,
N. (2018) Starch synthase IIa-deficient mutant rice line produces endosperm starch with
lower gelatinization temperature than japonica rice cultivars. Front. Plant Sci, 9, 645.
Muto, C., Ishikawa R., Olsen K.M., Kawano K., Bounphanousay C., Matoh T., Sato Y.I. (2016)
Genetic diversity of the wx flanking region in rice landraces in northern Laos. Breeding
Science, 66, 580-590.
Nakamura, Y., Francisco, P.B., Hosaka, Y., Sato, A., Sawada, T., Kubo, A., Fujita, N. (2005)
Essential amino acids of starch synthase IIa differentiate amylopectin structure and starch
quality between japonica and indica rice varieties. Plant Mol Biol, 58, 213-227.
Nakano T., Crofts N., Miura S., Oitome N.F., Hosaka Y., Ishikawa K., Fujita N. (2023)Three
starch synthase IIa (SSIIa) alleles reveal the effect of SSIIa on the thermal and rheological
properties, viscoelasticity, and eating quality of glutinous rice. International Journal of
Molecular Sciences, 24, 3726.
Olsen, K.M., M.D. Purugganan (2002) Molecular evidence on the origin and evolution of
glutinous rice. Genetics, 162, 941-950.
Ootsuka K, Takahashi I, Tanaka K, Itani T, Tabuchi H, Yoshihashi T, Tonouchi A, Ishikawa R
(2014) Genetic polymorphisms in Japanese fragrant landraces and novel fragrant allele
domesticated in northern Japan. Breed Sci, 64, 115-124.
Pan, L.-X.; Sun, Z.-Z.; Zhang, C.-Q.; Li, B.; Yang, Q.-Q.; Chen, F.; Fan, X.-L.; Zhao, D.-S.; Lv,
Q.-M.; Yuan, D.-Y.; et al. Allelic Diversification of the Wx and ALK Loci in Indica Restorer
Lines and Their Utilisation in Hybrid Rice Breeding in China over the Last 50 Years.
International Journal of Molecular Sciences, 2022, 23, 5941
Peng B, Kong H, Li Y, Wang L, Zhong M, Sun L, Gao G, Zhang Q, Luo L, Wang G, Xie W, Chen
J, Yao W, Peng Y, Lei L, Lian X, Xiao J, Xu C, Li X, He Y (2014) OsAAP6 functions as an
important regulator of grain protein content and nutritional quality in rice. Nature
Communications, 5, 4847.
Phitaktansakul, R., Kim, K.-W., Aung, K.M., Maung, T.Z., Min, M.-H., Somsri, A., Lee, W., Lee,
S.-B., Nam, J., Kim, S.-H., Lee, J., Kwon, S.-W., Nawade, B., Chu, S.-H., Park, S.-W., Kang,
K.K., Cho, Y.-H., Lee, Y.-S., Chung, I.-M., Park, Y.-J. (2022) Multi-omics analysis reveals
the genetic basis of rice fragrance mediated by betaine aldehyde dehydrogenase 2. Journal of
Advanced Research, 42, 303-314.
Sato, H., Suzuki, Y., Sakai, M., & Imbe, T. (2002). Molecular Characterization of Wx-mq, a Novel
Mutant Gene for Low-amylose Content in Endosperm of Rice (Oryza sativa L.). Breeding
Science, 52(2), 131-135.
Shao G, Tang A, Tang S, Luo J, Jiao G, Wu J, Hu P (2011) A new deletion mutation of fragrant
gene and the development of three molecular markers for fragrance in rice. Plant Breed,
130(2), 172-176.
Shao G, Tang S, Chen M, Wei X, He J, Luo J, Jiao G, Hu Y, Xie L, Hu P (2013) Haplotype
variation at Badh2, the gene determining fragrance in rice. Genomics, 101(2), 157-162.
Shi W, Yang Y, Chen S, Xu M (2008) Discovery of a new fragrance allele and the development of
functional markers for the breeding of fragrant rice varieties. Molecular Breeding, 22(2),
185-192.
Shi Y, Zhao G, Xu X, Li J (2014) Discovery of a new fragrance allele and development of
functional markers for identifying diverse fragrant genotypes in rice. Molecular Breeding,
33(3), 701-708.
Song, J., Hu, Y., Deng, G., Dai, G., Bao, J.S. (2022) The origin of the A/G single nucleotide
polymorphism of starch synthase IIa in rice and its relation to gelatinization temperature.
Cereal Chemistry, 99(2), 275-285.
Sreenivasulu N., Butardo V.M., Misra G., Cuevas R.P., Anacleto R., Kishor P.B.K.(2015)
Designing climate-resilient rice with ideal grain quality suited for high-temperature stress.

Journal of Experimental Botany, 66, 1737 - 1748
Takemoto-Kuno Y, Mitsueda H, Suzuki K, Hirabayashi H, Ideta O, Aoki N, Umemoto T, Ishii T,
Ando I, Kato H et al (2015) qAC2, a novel QTL that interacts with Wx and controls the low
amylose content in rice (Oryza sativa L.). Theoretical and Applied Genetics, 128, 563-573.
Tang, Y.; Abdelrahman, M.; Li, J.; Wang, F.; Ji, Z.; Qi, H.; Wang, C.; Zhao, K. (2021)
CRISPR/Cas9 induces exon skipping that facilitates development of fragrant rice. Plant
Biotechnol. J, 19, 642-644.
Teng, B., Zeng, R., Wang, Y., Liu, Z., Zhang, Z., Zhu, H., Ding, X., Li, W., and Zhang, G. (2012)
Detection of allelic variation at the Wx locus with single-segment substitution lines in rice
(Oryza sativa L.). Mol. Breed, 30, 583.
Teng, K., Wang, X., Guo, X. (2021) Generation of a new glutinous photothermosensitive
genic-male-sterile (PTGMS) line by CRISPR/Cas9-directed mutagenesis of Wx in rice
(Oryza sativa L.). Agriculture, 11, 1044.
Tian, Y., Zhou, Y., Gao, G., Zhang, Q., Li, Y., Lou, G., He, Y. (2023) Creation of Two-Line
Fragrant Glutinous Hybrid Rice by Editing the Wx and OsBADH2 Genes via the
CRISPR/Cas9 System. Int. J. Mol. Sci, 24, 849.
Tong, C., Bao, J.S. (2019) Rice lipids and rice bran oil. In: Bao JS (ed) Rice Chemistry and
Technology, 4th Edition (pp. 131-168). Elsevier.
Tran, N.A., Daygon, V.D., Resurreccion, A.P., Cuevas, R.P., Corpuz, H.M., Fitzgerald, M.A. (2011)
A single nucleotide polymorphism in the Waxy gene explains a significant component of gel
consistency. Theoretical and Applied Genetics, 123, 519-25.
Traore, K., McClung, A.M., Chen, M.H., Fjellstrom, R.G. (2011) Inheritance of flour paste
viscosity is associated with a rice Waxy gene exon 10 SNP marker. Journal of Cereal Science,
53, 37-44.
Umemoto T, Yano M, Satoh H, Shomura A, Nakamura Y., 2002. Mapping of a gene responsible
for the difference in amylopectin structure between japonica-type and indica-type rice
varieties. Theoretical and Applied Genetics, 104, 1-8.
Umemoto, T., Aoki, N., Lin, H.X., Nakamura, Y., Inouchi, N., Sato, Y., Yano, M., Hirabayashi, H.,
Maruyama, S. (2004) Natural variation in rice starch synthase IIa affects enzyme and starch
properties. Functional Plant Biology, 31, 671-684.
Vaughan, D., Lu, B., Tomooka, N. (2008) The evolving story of rice evolution. Plant Science, 174,
394-408.
Wang LQ, Liu WJ, Xu Y, He YQ, Luo LJ, Xing YZ, Xu CG, Zhang QF. (2007) Genetic basis of
17 traits and viscosity parameters characterizing the eating and cooking quality of rice grain.
Theoretical and Applied Genetics, 115, 463-476
Wang S, Yang Y, Guo M, Zhong C, Yan C, Sun S (2020) Targeted mutagenesis of amino acid
transporter genes for rice quality improvement using the CRISPR/Cas9 system. The Crop
Journal, 8, 457-464.
Wang XQ, Pang Y, Zhang J, Wu Z, Chen K, Ali J, Ye G, Xu JL, Li ZK (2017) Genome-wide and
gene-based association mapping for rice eating and cooking characteristics and protein
content. Scientific Reports, 7, 17203.
Waters, D.L.E., Henry, R.J., Reinke, R.F., Fitzgerald, M.A., 2006. Gelatinization temperature of
rice explained by polymorphisms in starch synthase. Plant Biotechnology Journal, 4,
115-122.
Xia, D., Zhou, H., Wang, Y., Ao, Y., Li, Y., Huang, J., Wu, B., Li, X., Wang, G., Xiao, J., Liu, Q.,
He, Y. (2022) qFC6, a major gene for crude fat content and quality in rice. Theoretical and
Applied Genetics, 135, 2675-2685.
Xu, F.F., Zhang, G., Tong, C., Sun, X., Corke, H., Sun, M., Baom J.S. (2013) Association mapping
of starch physicochemical properties with starch biosynthesizing genes in waxy rice (Oryza
sativa L.). Journal of Agricultural and Food Chemistry. 61(42): 10110-10117
Xu, F., Bao, J., He, Q., Park, Y.J. (2016) Genome-wide association study of eating and cooking
qualities in different subpopulations of rice (Oryza sativa L.). BMC Genomics, 17, 663.
Xu, F., Sun, C., Huang, Y., Chen, Y., Tong, C., Bao, J. (2015) QTL mapping for rice grain quality:
a strategy to detect more QTLs within sub-populations. Molecular Breeding, 35, 105.
Xu, Y., Lin, Q.P., Li, X.F., Wang, F.Q., Chen, Z.H., Wang, J., Li, W.Q., Fan, F.J., Tao, Y.J., Jiang,
Y.J., Wei, X.D., Zhang, R., Zhu, Q.H., Bu, Q.Y., Yang, J., and Gao, C.X. (2021) Fine-tuning
the amylose content of rice by precise base editing of the Wx gene. Plant Biotechnol. J, 19,

11-13.
Yamanaka S., Nakamura I.,Watanabe K.N.,Sato Y.I. (2004) Identification of SNPs in the waxy
gene among glutinous rice cultivars and their evolutionary significance during the
domestication process of rice. Theoretical and Applied Genetics, 108, 1200-1204.
Yang F, Chen YL, Tong C, Huang Y, Xu FF, Li KH, Corke H, Sun M, Bao JS. (2014) Association
mapping of starch physicochemical properties with starch synthesis-related gene markers in
nonwaxy rice (Oryza sativa L.). Molecular Breeding, 34, 1747-1763.
Yang, J., Wang, J., Fan, F.-J., Zhu, J.-Y., Chen, T., Wang, C.-L., Zheng, T.-Q., Zhang, J., Zhong,
W.-G., Xu, J.-L. (2013) Development of AS-PCR marker based on a key mutation confirmed
by resequencing of Wx-mp in Milky Princess and its application in japonica soft rice (Oryza
sativa L.) breeding. Plant Breeding, 132, 595-603
Yang Y, Guo M, Sun S, Zou Y, Yin S, Liu Y, Tang S, Gu M, Yang Z, Yan C (2019) Natural
variation of OsGluA2 is involved in grain protein content regulation in rice. Nature
Communications, 10, 1949.
Yang, J., Guo, X., Wang, X., Fang, Y., Liu, F., Qin, B., Li, R. Development of soft rice lines by
regulating amylose content via editing the 5′UTR of the Wx gene. International Journal of
Molecular Sciences, 2022, 23, 10517
Yano, M., Okuno, K., Kawakami, J., Satoh, H., Omura, T. (1985) High amylose mutants of rice,
Oryza sativa L. Theoretical and Applied Genetics, 69, 253-257.
Ying, Y., Zhang, Z., Tappiban, P., Xu, F., Deng, G., Dai, G., Bao, J.S. (2022) Starch fine structure
and functional properties during seed development in BEIIb active and deficient rice.
Carbohydrate Polymers, 292, 119640.
Ying, Y.，Hu, Y., Zhang, Y., Tappiban, P., Zhang, Z. W., Dai, G.X., Deng, G.F., Bao, J.S., Xu, F.
(2023) Identification of a new allele of soluble starch synthase IIIa involved in the elongation
of amylopectin long chains in a chalky rice mutant. Plant Science, 328, 111567.
Zeng, D.C., Liu, T.L., Ma, X.L., Wang, B., Zheng, Z.Y., Zhang, Y.L., Xie, X.R., Yang, B.W., Zhao,
Z., Zhu, Q.L., and Liu, Y.G. (2020) Quantitative regulation of Waxy expression by
CRISPR/Cas9-based promoter and 5’UTR-intron editing improves grain quality in rice. Plant
Biotechnol. J, 18, 2385- 2387.
Zhang C, Zhu J, Chen S et al (2019) Wxlv
, the ancestral allele of rice waxy gene. Mol Plant, 12,
1157-1166.
Zhang C., Yun P., Xia J., Zhou K., Wang L., Zhang J., Zhao B., Yin D., Fu Z., Wang Y., Ma T., Li
Z., Wu D. (2023) CRISPR/Cas9-mediated editing of Wx and BADH2 genes created glutinous
and aromatic two-line hybrid rice. Molecular Breeding, 43(4), 24.
Zhang H, Duan L, Dai JS, Zhang CQ, Li J, Gu MH, Liu QQ, Zhu Y (2014) Major QTLs reduce
the deleterious effects of high temperature on rice amylose content by increasing splicing
efficiency of Wx pre‑mRNA. Theoretical and Applied Genetics, 127, 273-282.
Zhang, C., Hao, W., Lu, Y., Yang, Y., Chen, Z., Li, Q., Fan, X., Luo, J., Liu, Q. (2022b) A
comparative evaluation of the effect of SSI and Wx allelic variation on rice grain quality and
starch physicochemical properties. Food Chemistry, 371, 131205.
Zhang, C., Yang, Y., Chen, S., Liu, X., Zhu, J., Zhou, L., Lu, Y., Li, Q., Fan, X., Tang, S., Gu, M.,
and Liu, Q. (2021) A rare Waxy allele coordinately improves rice eating and cooking quality
and grain transparency. Journal of Integrative Plant Biology, 63, 889-901.
Zhang, C.; Yang, Y.; Chen, Z.; Chen, F.; Pan, L.; Lu, Y.; Li, Q.; Fan, X.; Sun, Z.; Liu, Q. (2020)
Characteristics of grain physicochemical properties and the starch structure in rice carrying a
mutated ALK/SSIIa Gene. Journal of Agricultural and Food Chemistry, 68, 13950-13959.
Zhang, J.S., Zhang, H., Botella, J.R. and Zhu, J.K. (2018) Generation of new glutinous rice by
CRISPR/Cas9-targeted mutagenesis of the Waxy gene in elite rice varieties. Journal of
Integrative Plant Biology, 60, 369-375.
Zhang, Y., Zhao, J., Hu, Y., Zhang, Y., Ying, Y., Xu, F., Bao, J.S. (2022a) Combined effects of
different alleles of FLO2, Wx and SSIIa on the cooking and eating quality of rice. Plants,
11(17), 2249.
Zhang, Z., Hu, Y., Yu, S., Zhao, X., Dai, G., Deng, G., Bao, J.S. (2022c) Effects of drought stress
and elevated CO2 on starch fine structures and functional properties in indica rice.
Carbohydrate Polymers, 297, 120044.
Zhao, J., Zhang, Y., Zhang, Y.N., Hu, Y., Ying, Y., Xu, F., Bao, J.S. (2022) Variation in starch
physicochemical properties of rice with different genic allele combinations in two

environments. Journal of Cereal Science, 108, 103575.
Zheng L., Liu P., Zhang S., Li J., Muhammad Y., Yun Y., Hu L., Xue F., Wang Y., Yuan H., Chen
W., Qin P., Ma B., Li S., Tu B., Wang Y. (2020) Favorable allele mining and breeding
utilization of ALK in rice. Molecular Breeding, 40(11), 107.
Zhou H., Xia D., He Y. (2020) Rice grain quality—traditional traits for high quality rice and
health-plus substances. Molecular Breeding, 40(1), 1.
Zhou, H., Xia, D., Zhao, D., Li, Y., Li, P., Wu, B., Gao, G., Zhang, Q., Wang, G., Xiao, J., Li, X.,
Yu, S., Lian, X., He, Y. (2021) The origin of Wxla
provides new insights into the improvement
of grain quality in rice. Journal of Integrative Plant Biology, 63(5), 878-888.
Zhou, L., Zhang, C., Zhang, Y., Wang, C., Liu, Q. (2022) Genetic manipulation of endosperm
amylose for designing superior quality rice to meet the demands in the 21st century. Journal
of Cereal Science, 105, 103481
Zhou, Y., Zheng, H.Y., Wei, G.C., Zhou, H., Han, Y.N., Bai, X.F., Xing, Y.Z., Han, Y.P. (2016)
Nucleotide diversity and molecular evolution of the ALK gene in cultivated rice and its wild
relatives. Plant Molecular Biology Reporter, 34, 923-930.

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