Gene 739 (2020) 144517

Contents lists available at ScienceDirect

Gene
journal homepage: www.elsevier.com/locate/gene

Research paper

Transcriptome analysis of near-isogenic lines for glume hairiness of wheat T

a,b,1 a,b,1 a,b a,b a,b a,b a,b
Wei Luo , Jiajun Liu , Puyang Ding , Cong Li , Hang Liu , Yang Mu , Huaping Tang ,
Qiantao Jianga,b, Yaxi Liua,b, Guoyue Chena,b, Guangdeng Chenc, Yunfeng Jianga,b, Pengfei Qia,b,
⁎ ⁎
Youliang Zhenga,b, Yuming Weia,b, Chunji Liud, Xiujin Lana,b, , Jian Maa,b,
a
Triticeae Research Institute, Sichuan Agricultural University, Chengdu, Sichuan 611130, China
b
State Key Laboratory of Crop Gene Exploration and Utilization in Southwest China, Sichuan Agricultural University, Chengdu, Sichuan 611130, China
c
College of Resources, Sichuan Agricultural University, Chengdu, Sichuan 611130, China
d
Commonwealth Scientific and Industrial Research Organization Agriculture and Food, St Lucia, QLD 4067, Australia

A R T I C LE I N FO A B S T R A C T

Keywords: Hairiness, which is a phenotypic trait common among land plants, primarily affects the stem, leaf, and floral
Wheat organs. Plant hairiness is associated with complex functions. For example, glume hairiness in wheat is related to
Hairy glume the resistance to biotic and abiotic stresses, and may also influence human health. In the present study, two pairs
NILs of near-isogenic lines (NILs) for glume hairiness, which were derived from a cross between a Tibetan semi-wild
Transcriptome analysis
wheat accession (Triticum aestivum ssp. tibetanum Q1028) and a common wheat cultivar (T. aestivum ‘Zhengmai
9023’), underwent a glume transcriptome analysis. We detected 27,935 novel genes, of which 18,027 were
annotated. Additionally, 488 and 600 differentially expressed genes (DEGs) were detected in NIL1 and NIL2,
respectively, with 37 DEGs detected in both NIL pairs. Moreover, 987 and 1584 single nucleotide polymorphisms
(SNPs) were detected in NIL1 and NIL2, respectively, with 39 SNPs detected in both NIL pairs, of which most
were located in the Hairy glume (Hg) gene region on chromosome arm 1AS. The annotation of the DEGs with
gene ontology terms revealed that genes associated with hairiness in Arabidopsis and rice were similarly en-
riched. The possible functions of these genes related to glume hairiness were examined. The study results provide
useful information for identifying candidate genes and the fine-mapping of Hg in the wheat genome.

1. Introduction mechanical strength, insolubility, and narrow diameter (O'Neill et al.,

Trichomes are widely distributed on the surface of many land plant
species (i.e., hairiness or pubescence) (Lundqvist and Franckowiak,
2003; Moose et al., 2004; Doroshkov et al., 2015; Hamaoka et al.,
2017). Hairiness contributes to the resistance of plants to several biotic
and abiotic stresses (Roy et al., 1999), including brown planthopper
infestations of rice (Xu et al., 2002) and UV irradiation, while also
decreasing transpiration (Ishida et al., 2008). Recently, Lian et al.
(2019) discovered that glume hairiness may be associated with the
increased risk of developing esophageal squamous cell carcinoma
(ESCC) following the consumption of contaminated wheat flour. Car-
cinogenic mineral fibers exhibit three common characteristics, namely
1980). Glume hairiness is due to silica deposition (Hodson and
Sangster, 1988). Silica fibers possess the same three traits. Considering
its ecological importance and the fact it may be harmful to human
health, wheat glume hairiness should be characterized in more detail.
Howard and Howard (1915) reported a 3:1 separation ratio for
hairy and glabrous glumes in an F2 population of wheat, implying that
glume hairiness of wheat is regulated by a single dominant gene (Hg)
(Kadam, 1936). Sears (1954) determined that Hg is located on chro-
mosome 1A (formerly XIV) based on an analysis of common wheat
aneuploids. This finding has been confirmed in multiple investigations
(Kuspira and Unrau, 1960; Sheybani and Jenkins, 1961; Tsunewaki,
1961). Briggle and Sears (1966) suggested that Hg is located on the

Abbreviations: NILs, Near-isogenic lines; DEGs, differentially expressed genes; SNPs, single nucleotide polymorphisms, ESCC, esophageal squamous cell carcinoma;
Hg, hairy glume; GL, GLABROUS; TTG1, TRANSPARENT TESTA GLABRA; TRY, TRIPTYCHON; CPC, CAPRICE; RNA-seq, RNA sequencing; QTL, quantitative trait loci;
RIL, recombinant inbred line; dp, days post; IWGSC, International Wheat Genome Sequencing Consortium; Nr, NCBI non-redundant; Pfam, Protein family; COG,
Clusters of Orthologous Groups of proteins; KOG, euKaryotic Orthologous Groups; KEGG, Kyoto Encyclopedia of Genes and Genomes; GO, Gene Ontology; qPCR,
quantitative real-time polymerase chain reaction; HL6, HAIRY LEAF 6; Fhb1, Fusarium head blight
⁎
Corresponding authors at: Triticeae Research Institute, Sichuan Agricultural University, Chengdu, Sichuan 611130, China.
E-mail addresses: lanxiujin@163.com (X. Lan), jianma@sicau.edu.cn (J. Ma).
1
Contributed equally to this paper.

https://doi.org/10.1016/j.gene.2020.144517
Received 17 October 2019; Received in revised form 25 February 2020; Accepted 26 February 2020
Available online 27 February 2020
0378-1119/ © 2020 Elsevier B.V. All rights reserved.
W. Luo, et al.
short arm of chromosome 1A with a telocentric monosome. This gene
was subsequently located on a linkage map of chromosome 1AS in T.
monococcum (Dubcovsky and Dvorák 1995) and common wheat
(Khlestkina et al., 2006; Luo et al., 2016a). Dozens of genes, including
those encoding transcription factors, involved in trichome development
in Arabidopsis (Arabidopsis thaliana) have been reported, such as GLA-
BROUS 1 (GL1) (Oppenheimer et al., 1991), GL2 (Larkin et al., 1993),
GL3 (Patra et al., 2013), TRANSPARENT TESTA GLABRA 1 (TTG1)
(Walker et al., 1999), TTG2 (Johnson, 2002), TRIPTYCHON (TRY)
(Szymanski and Marks, 1998), and CAPRICE (CPC) (Wada et al., 1997).
Several genes for hairiness in rice were recently fine-mapped or cloned
(Li et al., 2010; Jun et al., 2011; Zeng et al., 2013; Sun et al., 2017).
These genes provide a foundation for investigating Hg, which may be
involved in a similar regulatory pathway for the initiation and devel-
opment of hairiness.
Advances in next-generation sequencing technology have enabled
researchers to conduct transcriptome analyses based on RNA sequen-
cing (RNA-seq). This approach has been widely used in diverse in-
vestigations of plants, including in studies of plant development (Zhu
et al., 2012; Ma et al., 2018), responses to biotic and abiotic stresses
(Ma et al., 2014; Zeng et al., 2016; Wang et al., 2017), and candidate
genes (Ma et al., 2014; Barrero et al., 2015). Specifically, analyzing the
transcriptomes of near-isogenic lines (NILs) is an effective method for
locating a gene of interest or quantitative trait loci (QTLs) (Kim et al.,
2011; Biselli et al., 2018). For example, Xiao et al. (2016) identified an
important candidate gene for starch biosynthesis in maize kernels using
a pair of NILs. Moreover, Habib et al. (2017) uncovered candidate genes
for QTLs associated with barley resistance to Fusarium pseudo-
graminearum using three pairs of NILs.
In the present study, two pairs of NILs for glume hairiness under-
went a glume transcriptome analysis. A total of 27,935 novel genes
were assembled, and single nucleotide polymorphisms (SNPs) and dif-
ferentially expressed genes (DEG) located in the Hg mapping region
were investigated. Genes associated with the initiation and develop-
ment of trichomes in Arabidopsis and rice were used for the functional
annotation of candidate genes with gene ontology terms.
2. Materials and methods
2.1. Materials
A recombinant inbred line (RIL) population was previously con-
structed from a cross between Tibetan semi-wild wheat (Triticum aes-
tivum ssp. tibetanum Shao) accession Q1028 (with a hairy glume phe-
notype) and common wheat (T. aestivum) ‘Zhengmai 9023′ (with
glabrous glumes) (Luo et al., 2016b). The NILs were constructed based
on the procedure described by Wang et al. (2019). The hairy and
glabrous glume phenotypes were separated in distinct lines in the F10
generation. All plants with hairy glumes from the two lines were cul-
tivated to select lines heterozygous for hairiness in the following gen-
eration. The selection was repeated three times. The hairy and glabrous
glume lines had a similar plant architecture and growth period. The
NILs were developed during the final selection. Specifically, the plants
with glabrous glumes were retained and the homozygous hairy plants
were identified based on the phenotype of the following generation.
Accordingly, seven pairs of NILs for glume hairiness were derived from
the RILs. The two pairs of NILs selected for the transcriptome analysis
were designated as NIL1 (Fig. 1) and NIL2.
The NILs were cultivated on the Wenjiang experimental farm of the
Triticeae Research Institute, Sichuan Agricultural University, China.
Each row was 1.5 m long, with inter-row and inter-plant spacings of 30
and 10 cm, respectively. Two plants of each line were randomly chosen
for sampling. The date of stage Z39 (i.e., the flag leaf collar was just
visible) (Zadoks et al., 1974) was recorded for every spike in the se-
lected plants. Glumes were sampled at 5, 10, and 15 days post-Z39
(dpZ39) (Fig. 2A), rapidly frozen in liquid nitrogen, and stored at
2
Gene 739 (2020) 144517
−80 °C until used.
2.2. RNA extraction, library construction, and sequencing
Total RNA was extracted from the sampled glumes with the Plant
RNA Kit (Omega, Norcross, GA, USA) and then treated with DNase I
(Takara, Dalian, China). The RNA samples were sent to BGI (Shenzhen,
China) for RNA quality testing and further processing. The mRNA was
isolated using magnetic beads coated with oligo(dT) and divided into
short fragments (200 bp) using fragmentation buffer. The cDNA was
synthesized using the fragments as templates and then purified and
resolved with EB buffer for the end-repair step and addition of a single
adenine (A) nucleotide. The short cDNA fragments were ligated to
adapters, after which suitable fragments were chosen as templates for a
PCR amplification. The fragments were sequenced with the Illumina
HiSeq™ 2000 platform.
2.3. Analysis of RNA-seq data
Clean RNA-seq data were obtained after the low-quality reads (i.e.,
shorter than 13 bp or containing more than 5% unknown nucleotides)
were removed from the raw data by Biomarker Technologies Co., Ltd,
Beijing, China (http://www.biomarker.com.cn). The Q20 (base quality
greater than 20 and an error probability of 0.01) and Q30 (base quality
greater than 30 and an error probability of 0.001) values as well as the
GC content and extent of sequence duplication of the clean data were
calculated. All downstream analyses used the high-quality clean data.
The clean reads were mapped to the International Wheat Genome
Sequencing Consortium (IWGSC) RefSeqv1.0 wheat reference genome
(IWGSC, 2018) with the HISAT2 program (Wen, 2017). Only reads with
a perfect match or one mismatch were analyzed further and annotated
based on the reference genome.
2.4. Gene annotation and analysis
The mapped reads were assembled using StringTie (Pertea et al.,
2015) and then compared with the annotated IWGSC RefSeqv1.0
genome (IWGSC, 2018) to search for new transcripts and new genes.
Genes were annotated following a BLAST search of the following da-
tabases (E-value cutoff of 10−5) (Zhu et al., 2014): NCBI non-redundant
(Nr) protein sequences (Pertea et al., 2015), Protein family (Pfam) (Finn
et al., 2014), Clusters of Orthologous Groups of proteins (COG) (Natale
et al., 2000), euKaryotic Orthologous Groups (KOG) (Koonin et al.,
2004), eggNOG (Huerta-Cepas et al., 2016), Swiss-Prot, a manually
annotated and reviewed protein sequence database (The UniProt,
2017), Kyoto Encyclopedia of Genes and Genomes (KEGG) (Hattori
et al., 2004), and Gene Ontology (GO) (Ashburner et al., 2000).
2.5. Differential expression analysis
Gene expression levels were estimated as the number of fragments
per kilobase of transcript per million fragments mapped (FPKM). The
genes that were differentially expressed in the NILs were analyzed with
the DESeq2 software package. The DEGs were identified based on the
following threshold: false discovery rate ≤0.01 and an absolute log2
fold-change ≤−1 or ≥1.
Because the genetic regulation of trichome initiation and develop-
ment has been extensively studied in Arabidopsis, we used the query
“trichome” to search for genes in The Arabidopsis Information Resource
database (http://www.arabidopsis.org) in October 2018. The identified
genes were annotated based on the GO database and then compared
with the DEGs detected in the two sets of wheat NILs.
2.6. SNP analysis
BioKanga (version 2.76.2) was used to analyze SNPs as described by
W. Luo, et al.
Fig. 1. Morphological characteristics of NIL1 plants. Plant morphology during the (A) pustulation period and (B) at maturity. The plants on the left and right have
glabrous and hairy glumes, respectively. Scale bar = 50 cm.
Ma et al. (2014). The SNPs were detected after aligning the clean reads
against the IWGSC RefSeqv1.0 reference genome, allowing a maximum
of two mismatches and at most two supplementary mismatches in the
first 12 bp of the reads. Candidate SNPs in the NILs were identified
using a custom function in the R software (Ma et al., 2014). The
function used two criteria to identify SNPs that were polymorphic be-
tween the hairy and glabrous lines in each set of NILs across replicates
and was able to detect the presence or absence of polymorphism. The
criteria for identifying SNPs were as follows: (a) the locus must be
homozygous in all of the replicates and the percentage of the dominant
nucleotide must be greater than 80%; and (b) each SNP should be
covered by at least three reads.
3
Gene 739 (2020) 144517
2.7. Validation of RNA-seq data
To validate the RNA-seq data, three DEGs were analyzed in a
quantitative real-time polymerase chain reaction (qPCR) assay with
gene-specific primers. The qPCR analysis was completed with SYBR
Premix ExTaq™ (Takara, Dalian, China) and the CFX96 Real-Time PCR
System (Bio-Rad, USA). Relative gene expression levels were calculated
according to the 2−ΔΔCt method, with the endogenous GAPDH gene
used as the reference control. Mean values and standard errors for three
independent experiments were calculated.
Fig. 2. Glume morphology of the NIL1
plants at various time-points. “P” and “G”
refer to pubescent and glabrous glumes, re-
spectively. The first day of stage Z39 is in-
dicated with “0 d”, and the numbers refer to
the days post-Z39. (A) Glume observed with
a stereomicroscope. Scale bar = 1 mm. (B)
Glume observed with an optical microscope.
Scale bar = 100 μm.
W. Luo, et al. Gene 739 (2020) 144517

3. Results

3.1. Glume sampling period

The glume hairiness phenotype developed before the heading stage

(i.e., before the spike emerged from the stem). Glume sampling at this
stage required the stripping of the flag leaf from the stem. Thus, an
observable phenotypic trait that could be used to indicate the devel-
opment of glume hairiness needed to be identified. We observed that
the Z39 growth stage coincided with glume hairiness development. At 5
dpZ39, glume hairiness was invisible to the naked eye, but was de-
tectable with a stereomicroscope. Glume hairiness was visible to the
naked eye at 10 dpZ39 (Fig. 2A). Additionally, hairiness was observable
with an optical microscope at 5 dpZ39 or even on the first day of the
Z39 stage (Fig. 2B); however, at this time-point, the spike was too small
to dissect the glume. Consequently, samples were collected at 5, 10, and
15 dpZ39 for the RNA-seq analysis.

3.2. Analysis of RNA-seq data

The cDNA libraries constructed for the glumes harvested at 5, 10,

and 15 dpZ39 (with two biological replicates) were sequenced with the
Illumina HiSeq™ 2000 platform. About 146.37 Gb clean data were
obtained after the quality control step. Approximately 96.30%–97.02%
and 90.84%–92.29% of the clean data attained the Q20 and Q30
quality levels in each sample, respectively. The GC content of the
samples ranged from 54.00% to 56.51% (Table S1). The alignment ef-
ficiency (i.e., percentage between mapped reads and clean reads) of the
samples ranged from 88.75% to 91.23%. Additionally, 83.65%–85.93%
of the clean reads were unique mapped reads, whereas the remainder
(4.37%–6.31%) were multiple mapped reads (i.e., mapped more than
once on the reference genome) (Table S2).

3.3. Annotation of new genes

The mapped reads were compared with the annotated IWGSC

RefSeqv1.0 genome (IWGSC, 2018), after which 110,790 genes were
obtained. The low-quality sequences containing a single exon or en-
coding a short peptide chain (fewer than 50 amino acids) were elimi-
nated and 27,935 putative new genes were obtained after assembling
the reads with StringTie. Of these genes, 18,027 had sequences that
were similar to those in the eight searched databases (Fig. 3). Overall,
98.95% of the genes (17,837) were annotated according to the Nr da-
tabase, whereas only 8.45% of all genes were annotated based on the
COG database (Fig. 3).
3.4. Transcriptome differences between hairy and glabrous NILs
More DEGs were detected in NIL2 than in NIL1 at 5 and 10 dpZ39,
not only regarding the total number of DEGs, but also in terms of the
up-regulated or down-regulated DEGs (Fig. 4A). More interestingly,
there were more up-regulated DEGs than down-regulated DEGs at 5 and
Fig. 3. Annotated new genes based on a search of eight public databases.
Fig. 4. Differentially expressed genes (DEGs) in the two sets of NILs. (A)
Number of genes differentially expressed between the hairy and glabrous
glumes in the NILs at three time-points. (B) Venn diagrams for the DEGs de-
tected at three time-points in the two pairs of NILs. (C) Venn diagrams for the
DEGs between the two pairs of NILs.
10 dpZ39. However, the total number of DEGs and the number of down-
regulated DEGs were greater in NIL1 than in NIL2 at 15 dpZ39, whereas
there were fewer up-regulated DEGs in NIL1 than in NIL2 (Fig. 4A).
A total of 488 and 600 DEGs were detected in NIL1 and NIL2, re-
spectively (Fig. 4B). Furthermore, 53 and 62 DEGs were observed at the
three sampling time-points in NIL1 and NIL2, respectively (Fig. 4B).
Thirty-seven DEGs were detected in both sets of NILs (Fig. 4C). The
DEGs detected at the same time-point in the two pairs of NILs were also
analyzed (Fig. 5). Four DEGs were detected in both pairs of NILs at the 5
and 10 dpZ39 time-points, whereas 18 DEGs were detected in both NILs
at 15 dpZ39. Furthermore, 1 and 13 up-regulated DEGs overlapped in
the two NIL pairs at 10 and 15 dpZ39, respectively, but 2, 2, and 3
down-regulated DEGs were detected at 5, 10, and 15 dpZ39, respec-
tively (Fig. 5). Interestingly, the down-regulated gene
TraesCS1A01G010300 was detected in both NIL1 and NIL2 at all three
time-points (Fig. 5).
A total of 987 and 1582 SNPs were identified in NIL1 and NIL2,
respectively (Figs. S1 and S2; Table S3). In NIL1, the 987 SNPs were
distributed among 373 genes in the genome, and the majority of the
genes and SNPs were detected on chromosome 5B. In NIL2, the 1582
SNPs were assigned to 594 genes, and the majority of the genes were

4
W. Luo, et al.
Fig. 5. Co-DEGs between the two sets of NILs at three time-points and the up-regulated and down-regulated genes in the hairy and glabrous glumes.
located on chromosome 2D (Fig. S2; Table S3). A total of 2530 SNPs
were identified in the two sets of NILs, and about 1.5% (39) of the SNPs
were detected at the same time-point (Fig. S1). Of these 39 SNPs, 32
were located on chromosome 1A, whereas the chromosomal location of
the remaining seven SNPs (localized to one gene) could not be de-
termined (Table S4). The 32 SNPs were concentrated in eight genes on
chromosome arm 1AS, of which most were localized to
TraesCS1A01G002300 (4), TraesCS1A01G002500 (11),
TraesCS1A01G002700 (11), and TraesCS1A01G007500 (2) (Table S4).
Moreover, 952 expressed genes with SNPs (GSNP) were detected, of
which 1.6% (15) were simultaneously detected in the two pairs of NILs
(Fig. S1), and 12 genes were located on chromosome 1A (Table S4).
These 15 genes included the nine genes mentioned above (i.e., eight
genes on chromosome 1AS and one gene that was not localized to a
chromosome) and an additional six genes with SNP sites that differed
between the two sets of NILs (Table S4).
3.5. Validation of DEGs
To validate the expression profiles determined with the RNA-seq
data, three DEGs with distinct changes in expression patterns were
randomly selected for a qPCR analysis. Details regarding the qPCR
primers are provided in Table S5. The expression patterns of the three
genes determined in the qPCR assay were essentially consistent with
those obtained from the RNA-seq data (Fig. S3).
5
Gene 739 (2020) 144517
3.6. Functional classification of DEGs
A total of 119 genes associated with trichome formation and de-
velopment were obtained from The Arabidopsis Information Resource
database (http://www.arabidopsis.org). These 119 genes and the DEGs
detected between the two pairs of NILs were functionally characterized
based on the annotations in the GO database. The genes were classified
in diverse GO categories (Fig. 6), and significant differences in the
number of genes classified in the GO categories were observed. In the
biological process category, many genes were classified in the sub-
categories biological regulation (GO:0065007), cellular component
organization or biogenesis (GO:0071840), cellular process
(GO:0009987), developmental process (GO:0032502), localization
(GO:0051179), metabolic process (GO:0008152), multicellular orga-
nismal process (GO:0032501), reproductive process (GO:0022414),
response to stimulus (GO:0050896), and single-organism process
(GO:0044699). For the cellular component category, the majority of
genes were classified in the cell (GO:0005623), cell part (GO:0044464),
macromolecular complex (GO:0032991), membrane (GO:0016020),
membrane part (GO:0044425), organelle (GO:0043226), and organelle
part (GO:0044422) subcategories. Regarding the molecular function
category, only the binding (GO:0005488) and catalytic activity
(GO:0003824) subcategories were prominently represented.
Most of the 37 DEGs detected in both sets of NILs (Fig. 4C; Table S6)
were classified into at least one of the 19 GO categories mentioned
above. The exceptions were TraesCS1B01G120500,
TraesCS3D01G155300, and six novel genes. For example,
W. Luo, et al.
Fig. 6. Functional annotation of the DEGs identified in this study and the trichome-related Arabidopsis thaliana genes based on GO terms.
TraesCS3B01G185300 and TraesCS2D01G182800 were classified into
12 and 10 of the 19 GO categories, respectively. Orthologs of the 37
genes in Arabidopsis, barley, and rice were searched for with the En-
semblPlants online tool (http://plants.ensembl.org).
4. Discussion
In the present study, two sets of NILs for glume hairiness, which
were derived from a cross between a Tibetan semi-wild wheat accession
and a common wheat cultivar, were used for a transcriptome analysis
during the development of glume hairiness. A total of 138,725 genes
were identified from the RNA-seq data, 20.1% of which were novel
genes. Additionally, 37 DEGs and 15 GSNPs were detected in both pairs
of NILs, and 18 DEGs exhibited the same regulatory trend in the two
sets of NILs. The data presented herein may be useful for future in-
vestigations of Hg.
4.1. Importance of hairiness in plants
The hairiness of plant surfaces reportedly has several protective
6
Gene 739 (2020) 144517
functions. For example, it can increase plant resistance to insect her-
bivory, provide protection against UV irradiation as well as freezing
and heat stresses, and decrease transpiration (Serna and Martin, 2006;
Ishida et al., 2008). Previous studies revealed that the effects of hairi-
ness in wheat are similar to those in other plant species. For example,
the hairiness of leaves improves the water-retention capacity and in-
fluences transpiration in wheat (Doroshkov et al., 2014; Doroshkov
et al., 2015). Pshenichnikova et al. (2018) reported that leaf hairiness in
wheat is a unique trait that exhibits adaptive plasticity under drought
conditions. Moreover, leaf hairiness in wheat is positively associated
with the resistance to the Hessian fly (Roberts et al., 1979) and the
cereal leaf beetle (Schillinger & Gallun, 1968).
There is relatively little available information regarding the func-
tional analysis of glume hairiness in wheat. Maes et al. (2001) reported
that wheat plants with hairy glumes are better protected from frost
damage at anthesis than plants that produce glabrous glumes. However,
glume hairiness has been linked to increased risks of developing ESCC
(Lian et al., 2019). In rice, the overexpression of HAIRY LEAF 6 (HL6),
which is involved in initiating leaf hairiness, decreases the grain yield
(Sun et al., 2017). Additionally, the hairiness of plants may produce
W. Luo, et al.
Fig. 7. Genetic map for Hg (Luo et al., 2016a) and the physical maps for the GSNPs and DEGs on chromosome 1A.
dust that can damage human skin and pollute the environment during
the harvesting and processing of rice (Zeng et al., 2013). The results of
these studies indicate that glume hairiness is a complex trait that po-
sitively and negatively affects wheat production (i.e., it may increase
the stress resistance of crops, while adversely affecting growers).
4.2. Utility of NILs for evaluating the effect of glume hairiness
Near-isogenic lines are an important resource for evaluating the
effects of a particular trait/gene without disrupting other genes (Chen
et al., 2014). For example, NILs for QTLs regarding Fusarium crown rot
resistance may eliminate the effects of plant height and growth rate in
barley (Habib et al., 2016). Thus, the potential resistance to biotic and
abiotic stresses due to glume hairiness may be investigated in future
studies.
A common method for developing NILs involves the application of
flanking markers to select heterozygous lines from backcross genera-
tions (Zhou et al., 2005) or early generations of the RIL population
(Wang et al., 2019). The method used in the current study differs
somewhat from those used previously, with two minor differences.
Specifically, molecular markers were not used to select heterozygous
lines. Because hairy glumes are due to a dominant gene (Kuspira and
Unrau, 1960; Sheybani and Jenkins, 1961; Tsunewaki, 1961) and have
been used as a morphological marker (Luo et al., 2016a), the hetero-
zygous lines were identified by analyzing the next generation. Ad-
ditionally, selections were initiated at an advanced generation. The
separated plants from the F10 generation were examined, and another
three selections were completed to develop NILs.
7
Gene 739 (2020) 144517
4.3. Importance of exploring novel genes
Investigations of the pangenome have become an important part of
wheat research, particularly for studying the gene presence–absence
variation among different cultivars. In this study, 138,725 genes were
obtained from the RNA-seq data, of which 20.1% (27,935) were pre-
dicted to be new genes. Dong et al. (2015) reported that 42.1% of the
unique transcripts revealed during a transcriptome sequencing analysis
of wheat cultivar ‘Xiaoyan 81′ had not previously been detected. In an
earlier pangenome analysis of ‘Chinese Spring’ and 18 other wheat
cultivars, 35.7% of the predicted genes were present or absent de-
pending on the variety (Montenegro et al., 2017). Additionally, the 19
cultivars were missing 4.7%–13.3% of the predicted genes. The novel
genes predicted in the current study may be useful for future analyses of
the wheat pangenome.
Candidate genes for Hg may not be isolated based on sequence
alignments with the ‘Chinese Spring’ wheat genome if Hg is absent from
the reference genome. For example, Rawat et al. (2016) reported that
there is no active copy of the major gene for Fusarium head blight re-
sistance (Fhb1) in the ‘Chinese Spring’ genome, and the gene was iso-
lated through positional cloning in the common wheat cultivar ‘Sumai
3′. Li et al. (2017) described five novel genes as candidate drought-
responsive genes regulated by hydrogen sulfide. Therefore, in the pre-
sent study, we sought to uncover novel genes from RNA-seq data for the
subsequent exploration of candidate genes associated with the forma-
tion and development of glume hairiness in wheat.
In this study, seven novel genes were detected in both sets of NILs
(Table S6). Specifically, Traes_newGene_43186 was classified into nine
trichome-related GO categories (Table S6), implying this gene might be
W. Luo, et al.
involved in the formation and development of glume hairiness.
4.4. Candidate genes for Hg
Transcriptome analyses of NILs by RNA-seq have been widely used
to examine the transcript profiles of genes or QTLs in diverse plant
species (Moriconi et al., 2012; Zhu et al., 2012; Xiao et al., 2016; Zhang
et al., 2018). Additionally, RNA-seq is an effective method for mapping
or detecting candidate genes. For example, by analyzing the tran-
scriptomes of multiple sets of NILs, Ma et al. (2014) detected several
genes associated with the QTLs for Fusarium crown rot resistance lo-
cated on chromosome arm 3BL in wheat.
The Hg gene was mapped to the interval between the simple se-
quence repeat markers Xsaufc2 and Xgwm136 (Luo et al., 2016a). The
physical positions of these markers are 1.37 and 6.42 Mb, respectively,
based on the IWGSC RefSeqv1.0 reference genome (IWGSC, 2018). In
the present study, 37 DEGs and 15 GSNPs (i.e., 52 genes) were detected
in both sets of NILs. Of these genes, 5 DEGs and 12 GSNPs were loca-
lized to chromosome 1A, with 3 DEGs and 4 GSNPs detected within the
first 6 Mb of chromosome 1AS (Fig. 7). Because the expression of
downstream genes may be influenced by the sequence (Ma et al., 2014)
or expression-level changes (Liu et al., 2017) of the upstream genes, a
candidate gene for Hg may be among or affected by the above-men-
tioned 52 genes. The 18 DEGs with the same regulatory trend in the two
pairs of NILs should be examined in greater detail in future studies.
Of the 32 SNPs detected in both sets of NILs that were located on
chromosome 1A, 28 were localized to four genes distributed in the first
6 Mb of chromosome arm 1AS (Table S4). Thus, these SNPs may be
relevant for designing PCR-based markers for the fine-mapping of Hg.
Analyzing orthologs is another way to functionally characterize genes.
Although TraesCS1A01G018700 is not located within the 6 Mb region,
it may be important for the glume trichome development pathway. Its
orthologs in Arabidopsis, AT3G11570 and AT5G06230, encode the
TRICHOME BIREFRINGENCE-LIKE protein. A mutation to this protein
reportedly results in a decrease in the number of trichomes on leaves
(Nita 2005).
We also searched for orthologs of the 37 DEGs detected in both NIL
pairs. As expected, the DEGs or their orthologs are related to the re-
sistance to biotic and abiotic stresses. For example,
TraesCS1A01G008100 (down-regulated at 10 dpZ39) is the functional
gene for powdery mildew resistance gene Pm3 (https://www.ncbi.nlm.
nih.gov/nucleotide/JF739341.1?report=genbank&log$=nucltop&
blast_rank=2&RID=0JJBNZ1X014). Additionally, Os10g0382100,
which is a rice ortholog of TraesCS1A01G336400 (up-regulated at 15
dpZ39), is a candidate gene related to low-temperature tolerance
during germination (Wang et al., 2018).
Three of the 37 DEGs identified in both pairs of NILs were localized
to the mapping region of chromosome 1AS (Fig. 7). Of these three
genes, TraesCS1A01G010300 expression was down-regulated at the
three sampling time-points, whereas TraesCS1A01G008100 and
TraesCS1A01G008200 expression levels were down-regulated at 10 and
15 dpZ39, respectively. Moreover, Os05g0103500 and HOR-
VU1Hr1G000940 are orthologs of TraesCS1A01G008200 and
TraesCS1A01G010300 in rice and barley, respectively (http://plants.
ensembl.org/index.html). A recent study indicated HOR-
VU1Hr1G000940 is a candidate gene for the barley leaf rust resistance
gene RphMBR102 (Fazlikhani et al., 2019). Similarly, Os05g0103500 re-
portedly contributes to biotic stress resistance (Silvar et al., 2013). On
the basis of these previous studies, our data suggest that hairiness may
influence wheat resistance to biotic and abiotic stresses.
Only two of the orthologs for the 52 genes, AT3G11570 and
AT5G06230, are associated with the development of hairiness in
Arabidopsis (Table S4; Table S6). This may have been due to the dis-
tinct differences in the development of the wheat glume and the Ara-
bidopsis and rice plant structures. Additionally, most of the orthologs
reportedly control the trichome development on the leaves and stems of
8
Gene 739 (2020) 144517
Arabidopsis and rice.
4.5. GO categories associated with Hg
An examination of gene ontologies facilitates cross-species com-
parisons of gene products (Horn et al., 2005). Genes that encode similar
products may be structurally similar. For example, many resistance
genes in diverse species, including Arabidopsis, rice, and wheat, contain
a leucine-rich repeat nucleotide-binding site sequence (Pan et al.,
2000).
Given that hairiness has been widely investigated in Arabidopsis,
119 genes for this trait were selected from the Arabidopsis genome for a
GO-based functional analysis of the wheat glume RNA-seq data (Fig. 6).
Many of the Arabidopsis and wheat genes were classified in 19 GO
categories (Fig. 6). These categories are reportedly related to trichomes
in other plant species (Trikka et al., 2015). For example, in an earlier
transcriptome analysis of the isolated trichomes of Greek sage (Salvia
fruticosa Mill.), the assembled genes were classified in two biological
process categories (GO:0009987, 32%; GO:0008152, 31%), three cel-
lular component categories (GO:0005623, 29%; GO:0044464, 29%;
GO:0043226, 23%), and two molecular function categories
(GO:0003824, 40%; GO:0005488, 39%) (Chatzopoulou et al., 2010).
These categories are likely to include candidate genes for hairiness in
wheat. Furthermore, most of the 37 DEGs detected in both sets of NILs
were classified into 19 GO categories. These genes may be useful for
future genetic investigations of wheat glume hairiness. Because of their
importance in rice, some genes related to leaf or glume hairiness have
been fine-mapped or cloned. The GO terms for these genes (Table S7)
were obtained from the Rice Genome Annotation Project database
(http://rice.plantbiology.msu.edu) (Kawahara et al., 2013), including
the GO terms for BLANKET LEAF (Hamaoka et al., 2017), GLABROUS
RICE 1 (Li et al., 2012), and HL6 (Sun et al., 2017). Specifically,
GO:0005634 was assigned to all of these (candidate) genes (Table S7).
However, GO:0005634 and other enriched GO terms for rice hairiness
genes were not among the 19 most enriched GO categories in the pre-
sent study. Moreover, only a few genes detected in the wheat glume
transcriptome were annotated with these GO terms. For example, 17
genes were annotated with GO:0005634, including a novel gene (data
not shown). The rice and wheat genes classified in the same GO cate-
gories might be functionally similar. Therefore, the genes associated
with the hairy glume phenotype in rice may be useful for identifying the
wheat Hg gene.
5. Conclusions
We conducted a transcriptome analysis for glume hairiness in wheat
with two sets of NILs. A total of 27,935 novel genes were annotated,
and 39 SNPs and 37 DEGs were detected in both sets of NILs.
Additionally, determining the GO categories enriched among the DEGs
associated with hairiness in rice and Arabidopsis might expedite the
identification of a candidate gene for Hg in wheat. The data generated
in the current study provide the foundation for detecting and cloning
the candidate gene for Hg in wheat.
CRediT authorship contribution statement
W. Luo: Formal analysis, Writing - Original draft preparation. J.J.
Liu: Investigation. P.Y. Ding: Investigation. C. Li: Investigation. H.
Liu: Investigation. Y. Mu: Resources. H.P. Tang: Resources. Q.T.
Jiang: Data Curation, Visualization. Y.X. Liu: Data Curation,
Visualization. G.Y. Chen: Data Curation, Visualization. G.D. Chen:
Data Curation, Visualization. Y.F. Jiang: Data Curation, Visualization.
P.F. Qi: Data Curation, Visualization. Y.L. Zheng: Supervision. Y.M.
Wei: Supervision. C.J. Liu: Writing - Review & Editing. X.J. Lan:
Funding acquisition. J. Ma: Conceptualization, Project administration.
W. Luo, et al.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgments
This work was supported by the National Natural Science
Foundation of China (31970243 and 31971937), the Key Research and
Development Program of Sichuan Province (2018NZDZX0002), and the
Key Projects of Scientific and Technological Activities for Overseas
Students of Sichuan Province. We thank Robert McKenzie, PhD, from
Liwen Bianji, Edanz Group China (www.liwenbianji.cn/ac), for editing
the English text of a draft of this manuscript.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.gene.2020.144517.
References
Ashburner, M., Ball, C.A., Blake, J.A., Botstein, D., Butler, H., Cherry, J.M., Davis, A.P.,
Dolinski, K., Dwight, S.S., Eppig, J.T., Harris, M.A., Hill, D.P., Issel-Tarver, L.,
Kasarskis, A., Lewis, S., Matese, J.C., Richardson, J.E., Ringwald, M., Rubin, G.M.,
Sherlock, G., 2000. Gene ontology: tool for the unification of biology. Nat. Genet.
25, 25.
Barrero, J.M., Cavanagh, C., Verbyla, K.L., Tibbits, J.F., Verbyla, A.P., Huang, B.E.,
Rosewarne, G.M., Stephen, S., Wang, P., Whan, A., Rigault, P., Hayden, M.J., Gubler,
F., 2015. Transcriptomic analysis of wheat near-isogenic lines identifies PM19-A1
and A2 as candidates for a major dormancy QTL. Genome Biol. 16, 93.
Biselli, C., Bagnaresi, P., Faccioli, P., Hu, X., Balcerzak, M., Mattera, M.G., Yan, Z.,
Ouellet, T., Cattivelli, L., Vale, G., 2018. Comparative transcriptome profiles of near-
isogenic hexaploid wheat lines differing for effective alleles at the 2DL FHB resistance
QTL. Front. Plant Sci. 9, 37.
Briggle, L.W., Sears, E.R., 1966. Linkage of resistance to Erysiphe graminis f sp. tritici (Pm3)
and Hairy Glume (Hg) on chromosome 1A of Wheat1. Crop Sci. 6, 559–561.
Chatzopoulou, F.M., Makris, A.M., Argiriou, A., Degenhardt, J., Kanellis, A.K., 2010. EST
analysis and annotation of transcripts derived from a trichome-specific cDNA library
from Salvia fruticosa. Plant Cell Rep 29, 523–534.
Chen, G., Yan, W., Liu, Y., Wei, Y., Zhou, M., Zheng, Y.L., Manners, J.M., Liu, C., 2014.
The non-gibberellic acid-responsive semi-dwarfing gene uzu affects Fusarium crown
rot resistance in barley. BMC Plant Biol. 14, 22.
Dong, L., Liu, H., Zhang, J., Yang, S., Kong, G., Chu, J.S., Chen, N., Wang, D., 2015.
Single-molecule real-time transcript sequencing facilitates common wheat genome
annotation and grain transcriptome research. BMC Genomics 16, 1039.
Doroshkov, A.V., Afonnikov, D.A., Pshenichnikova, T.A., 2014. Genetic analysis of leaf
pubescence in isogenic lines of bread wheat Novosibirskaya 67. Russ. J. Genet. 50,
153–160.
Doroshkov, A.V., Afonnikov, D.A., Dobrovolskaya, O.B., Pshenichnikova, T.A., 2015.
Interactions between leaf pubescence genes in bread wheat as assessed by high
throughput phenotyping. Euphytica 207, 491–500.
Dubcovsky, J., Dvorák, J., 1995. Ribosomal RNA multigene loci: nomads of the Triticeae
genomes. Genetics 140, 1367–1377.
Fazlikhani, L., Keilwagen, J., Kopahnke, D., Deising, H., Ordon, F., Perovic, D., 2019.
High Resolution Mapping of RphMBR1012 Conferring Resistance to Puccinia hordei in
Barley (Hordeum vulgare L.). Front. Plant Sci 10, 640.
Finn, R.D., Bateman, A., Clements, J., Coggill, P., Eberhardt, R.Y., Eddy, S.R., Heger, A.,
Hetherington, K., Holm, L., Mistry, J., Sonnhammer, E.L., Tate, J., Punta, M., 2014.
Pfam: the protein families database. Nucleic Acids Res. 42, D222–D230.
Habib, A., Shabala, S., Shabala, L., Zhou, M., Liu, C., 2016. Near-isogenic lines developed
for a major QTL on chromosome arm 4HL conferring Fusarium crown rot resistance
in barley. Euphytica 209, 555–563.
Habib, A., Powell, J.J., Stiller, J., Liu, M., Shabala, S., Zhou, M., Gardiner, D.M., Liu, C.,
2017. A multiple near isogenic line (multi-NIL) RNA-seq approach to identify can-
didate genes underpinning QTL. Theor. Appl. Genet. 131, 613–624.
Hamaoka, N., Yasui, H., Yamagata, Y., Inoue, Y., Furuya, N., Araki, T., Ueno, O.,
Yoshimura, A., 2017. A hairy-leaf gene, BLANKET LEAF, of wild Oryza nivara in-
creases photosynthetic water use efficiency in rice. Rice 10, 20.
Hattori, M., Kanehisa, M., Kawashima, S., Goto, S., Okuno, Y., 2004. The KEGG resource
for deciphering the genome. Nucleic Acids Res. 32, D277–D280.
Hodson, M.J., Sangster, A.G., 1988. Silica deposition in the inflorescence bracts of wheat
(Triticum aestivum). I. Scanning electron microscopy and light microscopy. Can. J.
Bot. 66, 829–838.
Horn, R., Lecouls, A.-C., Callahan, A., Dandekar, A., Garay, L., McCord, P., Howad, W.,
Chan, H., Verde, I., Main, D., Jung, S., Georgi, L., Forrest, S., Mook, J.,
Zhebentyayeva, T., Yu, Y., Kim, H.R., Jesudurai, C., Sosinski, B., Arús, P., Baird, V.,
Parfitt, D., Reighard, G., Scorza, R., Tomkins, J., Wing, R., Abbott, A.G., 2005.
9
Gene 739 (2020) 144517
Candidate gene database and transcript map for peach, a model species for fruit trees.
Theor. Appl. Genet. 110, 1419–1428.
Howard, A., Howard, G., 1915. On the inheritance of some characters in wheat. II. India
Dept. Agr. Mem. Bot. Ser 7, 273–285.
Huerta-Cepas, J., Szklarczyk, D., Forslund, K., Cook, H., Heller, D., Walter, M.C., Rattei,
T., Mende, D.R., Sunagawa, S., Kuhn, M., Jensen, L.J., von Mering, C., Bork, P., 2016.
eggNOG 4.5: a hierarchical orthology framework with improved functional annota-
tions for eukaryotic, prokaryotic and viral sequences. Nucleic Acids Res. 44,
D286–D293.
Ishida, T., Kurata, T., Okada, K., Wada, T., 2008. A genetic regulatory network in the
development of trichomes and root hairs. Annu. Rev. Plant Biol. 59, 365–386.
IWGSC, 2018. Shifting the limits in wheat research and breeding using a fully annotated
reference genome. Science 361, eaar7191.
Johnson, C.S., 2002. TRANSPARENT TESTA GLABRA2, a trichome and seed coat devel-
opment gene of arabidopsis, encodes a WRKY transcription factor. Plant Cell 14,
1359–1375.
Jun, H., Qizhao, W., Haowei, F., Dianxing, W., Qingyao, S., 2011. Fine mapping and
candiate gene analysis of glabrous lead and hull gene (gl1) in rice (Oryza sativa L.). J.
Nucl. Agr. Sci 25, 1088–1093.
Kadam, B., 1936. Genetics of the Bansi wheat of the Bombay-Deccan and a synthetic
Khapli—Part I, Proceedings of the Indian Academy of Sciences-Section B. Springer,
pp. 357–369.
Kawahara, Y., de la Bastide, M., Hamilton, J.P., Kanamori, H., McCombie, W.R., Ouyang,
S., Schwartz, D.C., Tanaka, T., Wu, J., Zhou, S., Childs, K.L., Davidson, R.M., Lin, H.,
Quesada-Ocampo, L., Vaillancourt, B., Sakai, H., Lee, S.S., Kim, J., Numa, H., Itoh, T.,
Buell, C.R., Matsumoto, T., 2013. Improvement of the Oryza sativa Nipponbare re-
ference genome using next generation sequence and optical map data. Rice 6, 4.
Khlestkina, E.K., Pshenichnikova, T.A., Roder, M.S., Salina, E.A., Arbuzova, V.S., Borner,
A., 2006. Comparative mapping of genes for glume colouration and pubescence in
hexaploid wheat (Triticum aestivum L.). Theor. Appl. Genet. 113, 801–807.
Kim, K.H., Kang, Y.J., Kim, D.H., Yoon, M.Y., Moon, J.K., Kim, M.Y., Van, K., Lee, S.H.,
2011. RNA-Seq analysis of a soybean near-isogenic line carrying bacterial leaf pus-
tule-resistant and -susceptible alleles. DNA Res. 18, 483–497.
Koonin, E.V., Fedorova, N.D., Jackson, J.D., Jacobs, A.R., Krylov, D.M., Makarova, K.S.,
Mazumder, R., Mekhedov, S.L., Nikolskaya, A.N., Rao, B.S., Rogozin, I.B., Smirnov,
S., Sorokin, A.V., Sverdlov, A.V., Vasudevan, S., Wolf, Y.I., Yin, J.J., Natale,
D.A.J.G.B., 2004. A comprehensive evolutionary classification of proteins encoded in
complete eukaryotic genomes. Genome Biol. 5, R7.
Kuspira, J., Unrau, J., 1960. Determination of gene-chromosome associations and es-
tablishment of chromosome markers by aneuploid analysis in common wheat I. F2
analysis of glume pubescence, spike density and culm color. Can. J. Genet. Cytol. 2,
301–310.
Larkin, J.C., Oppenheimer, D.G., Pollock, S., Marks, M.D., 1993. Arabidopsis GLABROUS1
gene requires downstream sequences for function. Plant Cell 5, 1739–1748.
Li, H., Li, M., Wei, X., Zhang, X., Xue, R., Zhao, Y., Zhao, H., 2017. Transcriptome analysis
of drought-responsive genes regulated by hydrogen sulfide in wheat (Triticum aes-
tivum L.) leaves. Mol. Genet. Genomics 292, 1091–1110.
Li, W., Wu, J., Weng, S., Zhang, D., Zhang, Y., Shi, C., 2010. Characterization and fine
mapping of the glabrous leaf and hull mutants (gl1) in rice (Oryza sativa L.). Plant Cell
Rep. 29, 617–627.
Li, J., Yuan, Y., Lu, Z., Yang, L., Gao, R., Lu, J., Li, J., Xiong, G., 2012. Glabrous Rice 1,
encoding a homeodomain protein, regulates trichome development in rice. Rice
5, 32.
Lian, C., Zuo, X., Tian, L., 2019. A possible role of biogenic silica in esophageal cancer in
North China? Environ. Sci. Pollut. Res. Int 26, 8340–8343.
Liu, J., Chen, J., Zheng, X., Wu, F., Lin, Q., Heng, Y., Tian, P., Cheng, Z., Yu, X., Zhou, K.,
Zhang, X., Guo, X., Wang, J., Wang, H., Wan, J., 2017. GW5 acts in the brassinos-
teroid signalling pathway to regulate grain width and weight in rice. Nat. Plants 3,
17043.
Lundqvist, U., Franckowiak, J.D., 2003. Chapter 5 – Diversity of barley mutants. In: von
Bothmer, R., van Hintum, T., Knüpffer, H., Sato, K. (Eds.), Developments in Plant
Genetics and Breeding. Elsevier, pp. 77–96.
Luo, W., Ma, J., Zhou, X.H., Sun, M., Kong, X.C., Wei, Y.M., Jiang, Y.F., Qi, P.F., Jiang,
Q.T., Liu, Y.X., 2016b. Identification of quantitative trait loci controlling agronomic
traits indicates breeding potential of Tibetan semiwild wheat (Triticum aestivum ssp.
tibetanum). Crop Sci. 56, 2410–2420.
Luo, W., Ma, J., Zhou, X.H., Jiang, Y.F., Sun, M., Yang, Y.J., Kong, X.C., Qi, P.F., Jiang,
Q.T., Liu, Y.X., 2016a. Genetic analysis of glume hairiness (Hg) gene in bread wheat
(Triticum aestivum L.). Genet. Resour. Crop Eviron. 63, 763–769.
Ma, L., Ma, S.W., Deng, Q., Yuan, Y., Wei, Z., Jia, H., Ma, Z., 2018. Identification of wheat
inflorescence development-related genes using a comparative transcriptomics ap-
proach. Int. J. Genomics 2018, 6897032.
Ma, J., Stiller, J., Zhao, Q., Feng, Q., Cavanagh, C., Wang, P., Gardiner, D., Choulet, F.,
Feuillet, C., Zheng, Y.L., Wei, Y., Yan, G., Han, B., Manners, J.M., Liu, C., 2014.
Transcriptome and allele specificity associated with a 3BL locus for fusarium crown
rot resistance in bread wheat. PLoS ONE 9, e113309.
Maes, B., Trethowan, R.M., Reynolds, M.P., Ginkel, M.V., Skovmand, B., 2001. The in-
fluence of glume pubescence on spikelet temperature of wheat under freezing con-
ditions. Funct. Plant Biol. 28, 141–148.
Montenegro, J.D., Golicz, A.A., Bayer, P.E., Hurgobin, B., Lee, H., Chan, C.K.K., Visendi,
P., Lai, K., Doležel, J., Batley, J., Edwards, D., 2017. The pangenome of hexaploid
bread wheat. Plant J. 90, 1007–1013.
Moose, S.P., Lauter, N., Carlson, S.R., 2004. The maize macrohairless1 locus specifically
promotes leaf blade macrohair initiation and responds to factors regulating leaf
identity. Genetics 166, 1451–1461.
Moriconi, J.I., Buet, A., Simontacchi, M., Santa-Maria, G.E., 2012. Near-isogenic wheat
W. Luo, et al.
lines carrying altered function alleles of the Rht-1 genes exhibit differential responses
to potassium deprivation. Plant Sci. 185–186, 199–207.
Natale, D.A., Koonin, E.V., Galperin, M.Y., Tatusov, R.L., 2000. The COG database: a tool
for genome-scale analysis of protein functions and evolution. Nucleic Acids Res. 28,
33–36.
Nita, A.S., 2005. Genetic Mapping and Molecular Characterization of tbr1 Mutant in
Arabidopsis Thaliana. PhD Thesis. University of Potsdam, Potsdam, Germany.
O'Neill, C.H., Hodges, G.M., Riddle, P.N., Jordan, P.W., Newman, R.H., Flood, R.J.,
Toulson, E.C., 1980. A fine fibrous silica contaminant of flour in the high oesophageal
cancer area of North-East Iran. Int. J. Cancer 26, 617–628.
Oppenheimer, D.G., Herman, P.L., Sivakumaran, S., Esch, J., Marks, M.D., 1991. A myb
gene required for leaf trichome differentiation in Arabidopsis is expressed in stipules.
Cell 67, 483–493.
Pan, Q., Wendel, J., Fluhr, R., 2000. Divergent evolution of plant NBS-LRR resistance
gene homologues in dicot and cereal genomes. J. Mol. Evol 50, 203–213.
Patra, B., Pattanaik, S., Yuan, L., 2013. Ubiquitin protein ligase 3 mediates the protea-
somal degradation of GLABROUS 3 and ENHANCER OF GLABROUS 3, regulators of
trichome development and flavonoid biosynthesis in Arabidopsis. Plant J. 74,
435–447.
Pertea, M., Pertea, G.M., Antonescu, C.M., Chang, T.C., Mendell, J.T., Salzberg, S.L.,
2015. StringTie enables improved reconstruction of a transcriptome from RNA-seq
reads. Nat. Biotechnol. 33, 290–295.
Pshenichnikova, T.A., Doroshkov, A.V., Osipova, S.V., Permyakov, A.V., Permyakova,
M.D., Efimov, V.M., Afonnikov, D.A., 2018. Quantitative characteristics of pub-
escence in wheat (Triticum aestivum L.) are associated with photosynthetic parameters
under conditions of normal and limited water supply. Planta 249, 839–847.
Rawat, N., Pumphrey, M.O., Liu, S., Zhang, X., Tiwari, V.K., Ando, K., Trick, H.N., Bockus,
W.W., Akhunov, E., Anderson, J.A., Gill, B.S., 2016. Wheat Fhb1 encodes a chimeric
lectin with agglutinin domains and a pore-forming toxin-like domain conferring re-
sistance to Fusarium head blight. Nat. Genet. 48, 1576–1580.
Roberts, J.J., Gallun, R.L., Patterson, F.L., Foster, J.E., 1979. Effects of wheat leaf pub-
escence on the Hessian fly. J. Econ. Entomol. 72, 211–214.
Roy, B.A., Stanton, M.L., Eppley, S.M., 1999. Effects of environmental stress on leaf hair
density and consequences for selection. J. Evolution. Biol 12, 1089–1103.
Schillinger Jr., J.A., Gallun, R.L., 1968. Leaf pubescence of wheat as a deterrent to the
cereal leaf beetle, Oulema melanopus. Ann. Entomol. Soc. Am 61, 900–903.
Sears, E.R., 1954. The aneuploids of common wheat. Mo. Agric. Exp. Stn. Res. Bull 572,
1–59.
Serna, L., Martin, C., 2006. Trichomes: different regulatory networks lead to convergent
structures. Trends. Plant Sci 11, 274–280.
Sheybani, H.A., Jenkins, B.C., 1961. The inheritance of glume pubescence in some durum
varieties. Can. J. Genet. Cytol. 3, 23–25.
Silvar, C., Perovic, D., Kopahnke, D., Casas, A.M., Igartua, E., Ordon, F., 2013.
Contribution of serbian and spanish landraces to disease resistance in barley. The
international conference newenviro: New approaches for assessment and improve-
ment of environmental status in Balkan region: Interactions between organisms and
environment, Sremska Kamenica, Serbia, pp. 29–35.
Sun, W., Gao, D., Xiong, Y., Tang, X., Xiao, X., Wang, C., Yu, S., 2017. Hairy Leaf 6, an
AP2/ERF transcription factor, interacts with OsWOX3B and regulates trichome for-
mation in rice. Mol. Plant 10, 1417–1433.
Szymanski, D.B., Marks, M.D., 1998. GLABROUS1 overexpression and TRIPTYCHON alter
the cell cycle and trichome cell fate in Arabidopsis. Plant Cell 10, 2047–2062.
The UniProt, C., 2017. UniProt: the universal protein knowledgebase. Nucleic Acids Res.
10
Gene 739 (2020) 144517
45, D158–D169.
Trikka, F.A., Nikolaidis, A., Ignea, C., Tsaballa, A., Tziveleka, L.A., Ioannou, E., Roussis,
V., Stea, E.A., Božić, D., Argiriou, A., Kanellis, A.K., Kampranis, S.C., Makris, A.M.,
2015. Combined metabolome and transcriptome profiling provides new insights into
diterpene biosynthesis in S. pomifera glandular trichomes. BMC Genomics 16, 935.
Tsunewaki, K., 1961. Monosomic and conventional gene analysis in common what. IV.
glume hairiness and ear density. Jap. J. Genet. 36, 55–62.
Wada, T., Tachibana, T., Shimura, Y., Okada, K., 1997. Epidermal cell differentiation in
Arabidopsis determined by a Myb homolog. CPC Sci. 277, 1113–1116.
Walker, A.R., Davison, P.A., Bolognesi-Winfield, A.C., James, C.M., Srinivasan, N.,
Blundell, T.L., Esch, J.J., Marks, M.D., Gray, J.C., 1999. The TRANSPARENT TESTA
GLABRA1 locus, which regulates trichome differentiation and anthocyanin bio-
synthesis in Arabidopsis, encodes a WD40 repeat protein. Plant Cell 11, 1337–1349.
Wang, W., Hao, Q., Wang, W., Li, Q., Wang, W., 2017. The genetic characteristics in
cytology and plant physiology of two wheat (Triticum aestivum) near isogenic lines
with different freezing tolerances. Plant Cell Rep. 36, 1801–1814.
Wang, H., Lee, A.R., Park, S.Y., Jin, S.H., Lee, J., Ham, T.H., Park, Y., Zhao, W.G., Kwon,
S.K., 2018. Genome-wide association study reveals candidate genes related to low
temperature tolerance in rice (Oryza sativa) during germination. 3 Biotech. 8, 235.
Wang, Z., Shi, H., Yu, S., Zhou, W., Li, J., Liu, S., Deng, M., Ma, J., Wei, Y., Zheng, Y., Liu,
Y., 2019. Comprehensive transcriptomics, proteomics, and metabolomics analyses of
the mechanisms regulating tiller production in low-tillering wheat. Theor. Appl.
Genet. 132, 2181–2193.
Wen, G., 2017. A Simple Process of RNA-Sequence Analyses by Hisat2, Htseq and DESeq2.
In: Proceedings of the 2017 International Conference on Biomedical Engineering and
Bioinformatics, pp. 11–15.
Xiao, Y., Thatcher, S., Wang, M., Wang, T., Beatty, M., Zastrow-Hayes, G., Li, L., Li, J., Li,
B., Yang, X., 2016. Transcriptome analysis of near-isogenic lines provides molecular
insights into starch biosynthesis in maize kernel. J. Integr. Plant Biol 58, 713–723.
Xu, X.F., Mei, H.W., Luo, L.J., Cheng, X.N., Li, Z.K., 2002. RFLP-facilitated investigation
of the quantitative resistance of rice to brown planthopper (Nilaparvata lugens).
Theor. Appl. Genet. 104, 248–253.
Zadoks, J.C., Chang, T.T., Konzak, C.F., 1974. A decimal code for the growth stages of
cereals. Weed Res 14, 415–421.
Zeng, X., Bai, L., Wei, Z., Yuan, H., Wang, Y., Xu, Q., Tang, Y., Nyima, T., 2016.
Transcriptome analysis revealed the drought-responsive genes in Tibetan hulless
barley. BMC Genomics 17, 386.
Zeng, Y., Zhu, Y., Lian, L., Xie, H., Zhang, J., Xie, H., 2013. Genetic analysis and fine
mapping of the pubescence gene GL6 in rice (Oryza sativa L.). Chinese Sci. Bull. 58,
2992–2999.
Zhang, W., Sun, P., He, Q., Shu, F., Deng, H., 2018. Transcriptome analysis of near-iso-
genic line provides novel insights into genes associated with panicle traits regulation
in rice. PLoS One 13, e0199077.
Zhou, R., Zhu, Z., Kong, X., Huo, N., Tian, Q., Li, P., Jin, C., Dong, Y., Jia, J., 2005.
Development of wheat near-isogenic lines for powdery mildew resistance. Theor.
Appl. Genet. 110, 640–648.
Zhu, Y., Cao, Z., Xu, F., Huang, Y., Chen, M., Guo, W., Zhou, W., Zhu, J., Meng, J., Zou, J.,
Jiang, L., 2012. Analysis of gene expression profiles of two near-isogenic lines dif-
fering at a QTL region affecting oil content at high temperatures during seed ma-
turation in oilseed rape (Brassica napus L.). Theor. Appl. Genet. 124, 515–531.
Zhu, S., Tang, S., Tang, Q., Liu, T., 2014. Genome-wide transcriptional changes of ramie
(Boehmeria nivea L. Gaud) in response to root-lesion nematode infection. Gene 552,
67–74.