Plant Breed. Biotech.

2023 (December) 11(4):271~277 Online ISSN: 2287-9366

https://doi.org/10.9787/PBB.2023.11.4.271 Print ISSN: 2287-9358
RESEARCH ARTICLE

Discovery of Genomic Regions and Candidate Genes for Awn

Length Using QTL-seq in Rice (Oryza sativa L.)

Dongryung Lee1,2†, Hongjia Zhang1†, Yuting Zeng1, Backki Kim1,2, Soon-Wook Kwon1,2*
1
Department of Plant Bioscience, Pusan National University, Miryang 50463, Korea
2
Life and Industry Convergence Research Institute, Pusan National University, Miryang 50463, Korea

ABSTRACT Rice domestication has led to cultivated rice with no or short awns. Discovery of novel genes associated to awn length
is of paramount importance for understanding the molecular mechanisms for the transformation of wild rice long awns to awnless
cultivated rice. In this study, we employed Next-Generation Sequencing based QTL-seq approach to identify genomic regions
associated with awn length using mapping population derived from a cross between awnless Tun Sart and awned Sobaekmangsudo.
QTL-seq analysis identified two awn length QTLs viz. qAwn-4 (12.8-20.3 Mb) and qAwn-8 (22.3-27.2 Mb) on chromosome 4 and 8,
respectively. Based on the sequence comparison between the two parents, Os04g0350700 (bHLH transcription factor) was postulated
to be the candidate of Awn-4 gene. Further discovery of the novel genes in qAwn-8 interval will provide insights into the genetic
architecture of awn length.
Keywords Rice, QTL-seq, Bulk segregant analysis (BSA), Awn length

INTRODUCTION In barley and wheat, awns also photosynthesize which

Rice is originated from wild rice (O. rufipogon Girff.)
through domestication. This considerably changed the
morpho-physiological characteristics of wild rice to in-
crease cultivation efficiency, grain quality, and rice yield
(Fuller et al. 2010; Huang et al. 2012). Compared to wild
rice, cultivated rice typically exhibits favorable charac-
teristics including erect growth, no or short awns, increased
spikelet number per panicle, closed panicle, and reduced
seed shattering and dormancy, all of which changed dramati-
cally during domestication (Kovach et al. 2007; Sweeney
and McCouch 2007).
Awn, a long extension of the lemma tip, helps seed dis-
persal by wind and by sticking to human cloths or animal
fur while providing seed protection from predators under
natural conditions (Elbaum et al. 2007; Svizzero et al. 2019).
contributes to grain filling (Abebe et al. 2009; Maydup et
al. 2010; Guo and Schnurbusch 2016). In contrast, rice
awns are not photosynthetically active because of the
absence of chlorenchyma tissues (Toriba and Hirano 2014;
Ntakirutimana and Xie 2019). Moreover, in agriculture,
awns are inconvenient for harvesting, threshing, packing
and storage (Takahashi et al. 1986). Hence, rice domesti-
cation has led to cultivated rice with no or short awns (Luo
et al. 2013).
Regent studies of the genetic mechanisms underlying the
development of rice awns have suggested that awn devel-
opment is a complex trait controlled by multiple genes. A
total of 21 QTLs with major and minor effect on rice awn
length in rice have been reported in the Gramene database
(https://archive.gramene.org/). However, only a few major
QTLs have been identified and characterized at the molecular

Received November 20, 2023; Revised November 27, 2023; Accepted November 28, 2023; Published December 1, 2023
*Corresponding author Soon-Wook Kwon, swkwon@pusan.ac.kr, Tel: +82-55-350-5506, Fax: +82-55-350-5509
†
These authors contributed equally.

Copyright ⓒ 2023 by the Korean Society of Breeding Science

This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0)
which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
272 ∙ Plant Breed. Biotech. 2023 (December) 11(4):271~277
level. An-1/RAE1 encodes a basic helix-loop-helix (bHLH)
transcription factor that regulates the formation of awn
primordia, cell division and grain length and reduces the
grain number in wild rice (Luo et al. 2013). An-2/LABA1
encodes a cytokinin synthesis enzyme that promotes awn
elongation by increasing cytokinin concentration in the
awn primordia. It reduces the number of grains per panicle
and tiller number per plant (Gu et al. 2015; Hua et al. 2015).
RAE2/GAD1/GLA encodes a secreted signal peptide that
regulates awn development as well as the number of grains
per panicle and grain length (Bessho-Uehara et al. 2016;
Jin et al. 2016; Zhang et al. 2019). The YABBY trans-
cription factor DL, auxin responsive factor OsETTIN2, and
RNA-dependent RNA polymerase SHL2 are also involved
in awn formation (Toriba and Hirano 2014).
To identify novel genomic regions associated with awn
length, we have performed QTL-seq (Takagi et al. 2013), a
combination of bulk segregant analysis (BSA) and whole
genome re-sequencing of DNA pools. An F2 population
derived from a cross between Tun Sart (an awnless cul-
tivar) and Sobaekmangsudo(an awned cultivar) were used.
This study will provide a valuable genetic resource for
future molecular breeding in rice.
MATERIALS AND METHODS
Plant materials
Two japonica rice cultivars [awnless Tun Sart (IT
004483) and awned Sobaekmangsudo (IT 006737)] were
used as parental lines to develop the 197 F2 population. [To
simplify the following description, we represent Tun Sart
as RWG-45 and Sobaekmangsudo as RWG-111]. RWG-45
and RWG-111 are part of the 137 KRICE_CORE pop-
ulation (Kim et al. 2016) and both seeds were received
from the Rural Development Administration (RDA) gene
bank, Korea. F2 population was obtained from Pusan
national University, Miryung, Korea.
Awn length evaluation
Phenotyping was carried out at Pusan University, Miryang,
Korea using 197 individual F2 plants of RWG-45 × RWG-111.
Three main panicles of each plant were used for pheno-
typing and the awn length of the whole panicle was re-
presented by the average of apical spikelets on each primary
branch. Measuring the awn lengths was carried out two
weeks after heading to avoid the awn breakage.
Construction of segregating pools
All young leaves of 197 individual F2 plants were col-
lected separately for total genomic DNA extraction using
CTAB method (Porebski et al. 1997), with minor modi-
fications. The genomic DNA of 23 individuals with ex-
tremely short awn (ESA) and 30 extremely long awn
(ELA) were selected as two bulked pools of the F2 popula-
tion. Isolated DNA was quantified using a Nanodrop
spectrophotometer (Thermo Scientific, Wilmington, USA).
Equal amounts of DNA from the ESA and ELA individuals
were mixed.
QTL-seq analysis
Total genomic DNA was extracted from two bulked
pools, and it was used to construct paired-end libraries with
an insert size of 151 bp using TruSeq Nano DNA Kit
(Illumina, San Diego, CA, USA). These libraries were
sequenced using the Illumina NGS platform at Macrogen
(Seoul, Korea). After sequencing, raw reads filtering was
performed by fastp program (Chen et al. 2018). This data
was aligned to the Nipponbare reference genome (IRGSP)
by using the BWA program (Li and Durbin 2009).
Samtools and GATK were used to clean the BAM file and
for SNP variation calling, respectively (Li et al. 2009;
McKenna et al. 2010). VCF file was filtered by vcftools
(Danecek et al. 2011) to obtain high quality genotype data.
The QTL-seq pipeline (QTL-seq version 2.1.3) (Takagi et
al. 2013; Sugihara et al. 2022) was used for mapping the
QTLs for Awn genes.
Prediction of candidate genes
To predict possible candidate genes associated with awn
length, the following strategies were employed. First, we
compared the DNA sequences of genes within the QTL
regions between the two parents using the whole-genome
DNA re-sequencing results to predict the candidate genes.
Second, comparing the results with known genes/QTLs for
awn length on 12 chromosomes in rice. Third, candidate
 Discovery of Genomic Regions for Awn Length ∙ 273

genes were reselected according to their functional anno-
tation from the rice genome database (http://rice.uga.edu/).
RESULTS
Evaluation of the awn length
The awn length of the two parental cultivars, RWG-45
and RWG-111 (Fig. 1A) along with their 197 F2 population
Fig. 1. Comparison of the awn phenotypes between RWG-
45 and RWG-111. (A) Phenotypic comparison of
seed arrays from mature spikelets of RWG-45 (left)
and RWG-111 (right). (B) Frequency distribution
of the awn length in the F2 population (RWG-45
× RWG-111).
(Fig. 1B), were evaluated two weeks after heeding. Sig-
nificant difference of the awn length was observed between
the two parents. The F2 population showed awn length
variation from 0.3 to 112.5 mm (Fig. 1B). Among the 197
F2 individuals, 23 extremely short awn and 30 extremely
long awn plants were selected to prepare the ESA-pool and
ELA-pool, respectively, which were then used for DNA
re-sequencing.
Whole-genome sequencing and SNP identification
We performed high-throughput genome sequencing using
four samples including RWG-45, RWG-111, ESA-pool,
ELA-pool and obtained a total of 238.9 million reads and
32.6 Gb of raw data (Table 1). After cleaning the data by
fastp, the average GC content was 42.310% and the Q30 of
all the samples reached more than 91%. The mapped ratios
between samples and the Nipponbare genome were
98.34%, 98.93%, 98.89%, and 98.94%, respectively. Most
samples except RWG-111 (81.92%) showed properly paired
ratio higher than 94% and unmapped ratio of all the sam-
ples was lower than 1.7%. The average genome-coverage
depth was 22X and the genome coverage was higher than
98%. These results suggest that the resequencing quality is
confirmed and they could be used for the following
analysis.
We obtained a total of 735,910 variants those were
including 583,569 SNPs and 152,341 indels from the sam-
ples. Among the 1,213.9 K annotations, 619.6 K, 51.4 K,
and 22.3 K were located at intergenic region, intron, and
exon, respectively. 10.2 K and 8.4 K of the annotations lo-
cated at the exon region were non-synonymous and syn-
onymous, respectively (Table 2).

Table 1. Quantity of genome sequence obtained for each sample.

Properly Genome
Sample Total GC AT Q20 Q30 Mapped Unmapped Average
Total base paired coverage
ID reads (%) (%) (%) (%) (%) (%) depth
(%) (%)
RWG-045 31,616,276 3,190,458,278 39.8 60.2 97.0 92.1 98.34 94.18 1.66 10X 98.33
RWG-111 36,820,718 3,679,271,766 41.9 58.1 97.1 92.4 98.93 81.92 1.06 11X 98.50
ESA-pool 84,862,982 12,814,310,282 43.7 56.3 96.5 91.1 98.89 95.79 1.04 34X 99.98
ELA-pool 85,611,904 12,927,397,504 43.9 56.1 96.8 91.6 98.94 95.92 1.09 33X 99.98
274 ∙ Plant Breed. Biotech. 2023 (December) 11(4):271~277

Table 2. SNPs identified among two parents and two mixed QTL-seq analysis and sequence comparison of the
pools. candidate genes
Type Number Ratio (%)
Two major peaks on chromosome 4 and 8 were iden-
SNP 583,569 79.30
tified for awn length and named as qAwn-4 and qAwn-8,
MNP 0 0
INS 75,284 10.23
respectively (Fig. 2). The qAwn-4 was spanning 7.5-Mb
DEL 77,057 10.47 (12.8-20.3 Mb) intervals on chromosome 4 and the accu-
3’UTR 11,772 0.97 racy of this QTL was ascertained by a valid 99% Δ(SNP-
5’UTR 8,661 0.71 index) significance level (Fig. 2A, Table 3). The other
Downstream 205,221 16.93
QTL, qAwn-8, was spanning 4.9-Mb (22.3-27.2 Mb) inter-
Exon 22,380 14.52
Intergenic 619,687 51.12 vals on chromosome 8 and the accuracy of this QTL was
Intron 51,482 4.25 ascertained by a valid 95% Δ(SNP-index) significance
Splice site acceptor 75 0.01
Splice site donor 81 0.01
Splice site region 1,360 0.11
Table 3. QTLs associated with awn development identi-
Transcript 79,433 6.55
fied using QTL-seq.
Upstream 212,115 17.50
Missense 10,208 54.32 QTL name Chr. Start (Mb) End (Mb) Peak
Nonsense 160 0.85 qAwn-4 4 12.8 20.3 ‒0.6010
Silent 8,425 44.83 qAwn-8 8 22.3 27.2 ‒0.4153

Fig. 2. Single nucleotide polymorphism (SNP)-index charts. (A and B) SNP-index charts of awnless-pool (green), awned-
pool (orange), and corresponding Δ(SNP-index) plots (blue) with 95-99% confidence interval borders of RWG-45
× RWG-111 for chromosome 4 (A) and chromosome 8 (B). Average values of Δ(SNP-index) are plotted with
a 2 Mb sliding window and a 50 kb increment.

level (Fig. 2B, Table 3). The qAwn-4 coding region har-
bored 1,352 SNPs and 302 indels, and the qAwn-8 coding
region harbored 880 SNPs and 254 indels (data not shown).
Genomic sequence comparison was made between the
two parents based on the genes that were previously reported
on the identified QTL regions. The An-1 (Os04g0350700),
a major gene that regulates awn development, was located
in the qAwn-4 region. One SNP was identified in the second
exon between awnless and awned plants in Os04g0350700
(Fig. 3). Another major gene RAE2 (Os08g0485500) was
located in the qAwn-8 region. However, no nucleotide
variants were found between awnless and awned plants
indicating the existence of a novel gene contributing to awn
length (data not shown). Further analysis will be performed
to narrow down the candidate region and to discover the
novel genes.
DISCUSSION
Morpho-physiological traits of wild species have been
modified to meet human needs during crop domestication.
Wild rice typically exhibits long awns that help in seed
dispersal and provide protection from predators under nat-
ural conditions. However, in agriculture, long awns are
inconvenient for pre-harvesting and post-harvesting because
of its structure. Hence, rice domestication has led to culti-
vated rice with no or short awns. Recently, studies have
been conducted to elucidate the genetic mechanisms under-
Fig. 3. Sequence variation in an-1, awn-4, and Awn-4. The common variations among Nipponbare, RWG-45, and RWG-111
are indicated in this figure. Black bars represent introns and grey boxes represent coding regions. Bar = 1 kb.
Discovery of Genomic Regions for Awn Length ∙ 275
lying the rice awn development. Although several genes/
QTLs associated with awn development have been detected,
only a few major QTLs have been cloned and characterized
at the molecular level.
To identify the novel QTLs for awn length, two DNA
pools with extreme phenotypic difference were used to
perform QTL-seq analysis. We used the Δ(SNP-index)
algorithm approach to map QTL regions at the 95% or 99%
significance level. Two highly significant peaks (qAwn-4
and qAwn-8) were detected on chromosome 4 and 8, with
the former mapped between 12.8-20.3 Mb and the latter
between 22.3-27.2 Mb (Fig. 2).
QTLs and genes for awn length on chromosome 4 and 8
have been previously reported. The An-1 encoding a bHLH
transcription factor was identified from wild rice (O.
rufipogon) and it regulates long awn formation (Luo et al.
2013). This gene was detected in qAwn-4 region. In
chromosome 8, the location of qAwn-8 in the present study
was containing the previously reported gene, RAE2 that
encodes one member of the epidermal patterning factor-like
protein (EFPL) family regulating awn formation (Bessho-
Uehara et al. 2016; Jin et al. 2016; Zhang et al. 2019).
Comparison between the genomic sequences of the two
parents revealed that one SNP difference in the coding
region of An-1 (Fig. 3), in which +244-bp (A ＞ G) changes
the amino acid sequence, suggesting that this SNP might be
involved in awn formation and elongation. However, no
SNPs were found between the sequence of the two parents
in the coding region of RAE2 suggesting that a novel gene
276 ∙ Plant Breed. Biotech. 2023 (December) 11(4):271~277
associated to awn length might exist in the interval of
qAwn-8 which warrants further exploration. Therefore,
further discovery of the novel genes will provide insights
into the genetic architecture of awn length.
ACKNOWLEDGEMENTS
This work was supported by the Rural Development
Administration, Republic of Korea (RS-2022-RD010201).
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