Download Polyploidy Enhances F Pollen Sterility Loci

Polyploidy Enhances F1 Pollen Sterility Loci Interactions
That Increase Meiosis Abnormalities and Pollen
Sterility in Autotetraploid Rice1[OPEN]
Jinwen Wu 2, Muhammad Qasim Shahid 2, Lin Chen, Zhixiong Chen, Lan Wang, Xiangdong Liu*, and
Yonggen Lu*
State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, South China
Agricultural University, Guangzhou 510642, China
ORCID IDs: 0000-0002-6767-4355 (M.Q.S.); 0000-0003-1568-1745 (X.L.).
Intersubspecific autotetraploid rice (Oryza sativa ssp. indica 3 japonica) hybrids have greater biological and yield potentials than
diploid rice. However, the low fertility of intersubspecific autotetraploid hybrids, which is largely caused by high pollen abortion
rates, limits their commercial utility. To decipher the cytological and molecular mechanisms underlying allelic interactions in
autotetraploid rice, we developed an autotetraploid rice hybrid that was heterozygous (SiSj) at F1 pollen sterility loci (Sa, Sb,
and Sc) using near-isogenic lines. Cytological studies showed that the autotetraploid had higher percentages (.30%) of abnormal
chromosome behavior and aberrant meiocytes (.50%) during meiosis than did the diploid rice hybrid control. Analysis of gene
expression profiles revealed 1,888 genes that were differentially expressed between the autotetraploid and diploid hybrid lines at
the meiotic stage, among which 889 and 999 were up- and down-regulated, respectively. Of the 999 down-regulated genes, 940
were associated with the combined effect of polyploidy and pollen sterility loci interactions (IPE). Gene Ontology enrichment
analysis identified a prominent functional gene class consisting of seven genes related to photosystem I (Gene Ontology 0009522).
Moreover, 55 meiosis-related or meiosis stage-specific genes were associated with IPE in autotetraploid rice, including
Os02g0497500, which encodes a DNA repair-recombination protein, and Os02g0490000, which encodes a component of the
ubiquitin-proteasome pathway. These results suggest that polyploidy enhances epistatic interactions between alleles of pollen
sterility loci, thereby altering the expression profiles of important meiosis-related or meiosis stage-specific genes and resulting in
high pollen sterility.
Autotetraploid rice (Oryza sativa) is a useful germplasm
developed from diploid rice through colchicine-mediated
chromosome doubling. Intersubspecific autotetraploid
rice (ssp. indica 3 ssp. japonica) hybrids have biological
advantages over diploid hybrids, such as greater adaptability and yield potential (Tu et al., 2007; Shahid et al., 2011,
2012; Wu et al., 2013). However, because autotetraploid
1
This work was supported by The National Natural Science Foundation of China (grant nos. 31270352 and 31210103023 to X.L.), the
Guangdong Provincial Science and Technique Project (grant no.
2014A030304055 to X.L.), and the Guangdong Provincial Key Platform of University and Major Research Project (Natural Science)
Characteristic Innovation Project (grant no. Yue–JK2014 [65] to X.L.).
2
These authors contributed equally to the article.
* Address correspondence to [email protected] and yglu@scau.
edu.cn.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is:
Xiangdong Liu ([email protected]).
J.W. performed all of the experiments, analyzed the data, and
wrote the article; J.W. and M.Q.S. revised the article; M.Q.S. analyzed
the data and wrote the article; L.C. performed the quantitative realtime PCR; Z.C. and L.W. contributed to the data analysis; X.L. and
Y.L. conceived the project and revised the article.
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rice hybrids have low seed set, largely because of high
pollen abortion rates, it is challenging to produce hybrids with commercially favorable characteristics (He
et al., 2011a, 2011b). The ability to develop high-yielding
intersubspecific autotetraploid rice hybrids depends on
a detailed understanding of the cytological and molecular mechanisms underlying pollen abortion in these
lines (He et al., 2011b).
Autotetraploid rice hybrids contain four sets of homologous chromosomes that can undergo abnormal
pairing during meiosis. Therefore, abnormal chromosome behavior was considered to be an important cause
of pollen abortion in autotetraploid rice hybrids (Luan
et al., 2007, 2009; He et al., 2011a, 2011b). However, we
found that interactions between hybrid pollen sterility
loci, including Sa, Sb, and Sc, contributed greatly to low
pollen fertility in autotetraploid rice hybrids (Zhao
et al., 2006; He et al., 2011b). Intersubspecific autotetraploid hybrids carry two alleles at each locus, an ssp.
indica (Si) allele and a ssp. japonica (Sj) allele. Lines that
are heterozygous (SiSj) at each pollen sterility locus
produce partially sterile pollen, whereas those that are
homozygous (SiSi or SjSj) produce normal pollen (He
et al., 2011b). Allelic interactions between Sa, Sb, and Sc
sterility loci also account for much of the sterility in
diploid intersubspecific hybrids (Zhang and Lu, 1989,
1993; Zhang et al., 1994, 2006, Shahid et al., 2013a).
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Allelic Interaction and Gene Expression in Polyploid
However, it was unclear how polyploidy affected allelic
interactions between pollen sterility loci during pollen
mother cell (PMC) meiosis in intersubspecific autotetraploid rice hybrids.
Microarray and RNA sequencing (RNA-Seq)-based
transcriptome profiling are helpful tools for characterizing molecular aspects of male and female gametophyte development in plants, including differentially
expressed genes and stage-specific gene expression.
The complexity of gene expression during anther and
ovary development has been revealed using microarrays or RNA sequencing in diploid rice (Tang et al.,
2010; Aya et al., 2011; Deveshwar et al., 2011; Jin et al.,
2013; Pan et al., 2014), maize (Zea mays; DukowicSchulze et al., 2014), wheat (Triticum aestivum; Crismani
et al., 2006), and Arabidopsis (Arabidopsis thaliana; Honys
and Twell, 2003, 2004; Yang et al., 2011; Zhao et al., 2014).
Microarrays have been used to identify differentially
expressed genes in autotetraploid plants. For instance,
microarray analysis was used to detect numerous
subtle differences in gene expression between diploid
(2x) and tetraploid (4x) Rangpur lime (Citrus limonia;
Allario et al., 2011). Our laboratory carried out microarray analysis to assess genetic variation between autotetraploid (Taichung65-4x) and diploid (Taichung65)
rice during pollen development. We identified a total of
1,251 differentially expressed genes, some of which are
associated with meiosis and pollen development (Wu
et al., 2014). Moreover, RNA-seq-based transcriptome
profiling revealed that polyploidy is strongly associated with genome-wide disruption of gene expression
and ultimately, resulted in aberrant phenotypes in allopolyploid rice hybrids (Xu et al., 2014).
In this study, we used transcriptome profiling and
cytological observations to examine the effect of polyploidy on three pollen sterility loci, namely Sa, Sb, and
Sc, which resulted in high levels of pollen sterility in
intersubspecific autotetraploid rice hybrids. Three factors are known to cause pollen sterility in autotetraploid rice hybrids: interactions between Sa, Sb, and Sc
loci; polyploidy (tetraploidy); and the combined effect
of pollen sterility loci and polyploidy. We sought to
distinguish the effect of each factor on pollen sterility
and gene expression profiles using two near-isogenic
lines of diploid and autotetraploid rice and their F1 hybrids that are heterozygous at each of these loci (SaiSaj,
SbiSbj, and SciScj). We examined chromosome behavior
during meiosis and the process of microsporogenesis
and microgametogenesis in diploid and autotetraploid
hybrids. Furthermore, we identified genes associated
with meiosis that were differentially expressed between
autotetraploid and diploid rice hybrids, which is a key
stage for Sa, Sb, and Sc loci expression and epistatic interactions. We found that autotetraploid hybrids had a
distinctly higher percentage of cytological abnormalities
than diploid rice hybrids and that polyploidy amplified
epistatic interaction between F1 pollen sterility loci,
leading to abnormalities in gene expression profiles and
high levels of pollen sterility in intersubspecific autotetraploid rice hybrids.
RESULTS
Genotypes of Parental Lines and Hybrids at Sa, Sb, and Sc
Loci and Their Effect on Pollen Fertility
We determined the genotypes of the diploid and
autotetraploid rice hybrids at the Sa, Sb, and Sc pollen
sterility loci using closely linked molecular markers
(Supplemental Figs. S1 and S2; Table I). Because the
parental lines each have different alleles at Sa, Sb, and
Sc, the hybrids showed allelic interactions at the three
pollen sterility loci. We also determined the pollen fertility and seed setting of the two hybrids (Table I).
Diploid hybrid (Taichung65 3 E245) F1 plants harbored
Si and Sj alleles at these three loci and had low pollen
fertility (30.51%) and seed setting (20.85%). However,
autotetraploid hybrid (Taichung65-4x 3 E245-4x) F1
plants, which had epistatic interactions between alleles
at the three pollen sterility loci, displayed very low pollen fertility (12.17%) and seed setting (3.56%; Table I).
These results indicate that interactions between pollen
sterility loci have a more dramatic effect on pollen fertility in the autotetraploid hybrid than in the diploid
hybrid.
Microsporogenesis and Microgametogenesis in Diploid
and Autotetraploid Rice Hybrids
We then used whole-mount eosin B-staining confocal
laser-scanning microscopy (WE-CLSM) to observe microsporogenesis and microgametogenesis in diploid
and autotetraploid rice hybrids. During microsporogenesis in the diploid hybrids, meiosis appeared similar to that in the parental lines of the diploid hybrids
Table I. Frequency of abnormal pollen and seed setting in the diploid and autotetraploid hybrids and Sa, Sb, and Sc loci genotypes
DF1 indicates the diploid hybrid (Taichung65 3 E245) that is heterozygous at the Sa, Sb, and Sc pollen sterility loci. TF1 indicates the autotetraploid
hybrid (Taichung65-4x 3 E245-4x) that is heterozygous at the Sa, Sb, and Sc pollen sterility loci.
Abnormal Pollen at the Mature Stage
Name
DF1
TF1
Genotype of Sa, Sb, and Sc Loci
Sai Saj Sbi Sbj Sci Scj
Sai Sai Saj Saj Sbi Sbi Sbj Sbj Sci Sci Scj Scj
Seed Setting
Pollen Fertility
% 6 SD
20.85 6 0.23 30.51 6 1.63
3.56 6 0.02 12.17 6 1.03
Sample Size
Asynchronous
Pollen
Abnormally
Shaped Pollen
Small Pollen
3,098
2,877
27.23 6 1.85
27.63 6 1.95
% 6 SD
32.93 6 3.27
56.52 6 1.03
1.88 6 0.46
4.22 6 0.16
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Figure 1. Cytological observation of pollen development in diploid and autotetraploid rice hybrids.
A to D, Normal pollen development in diploid rice.
A, Dyad stage. B, Tetrad stage. C, Late-stage microspore. D, Bicellular pollen stage. E to L, Abnormal
pollen development in diploid hybrids. E, Abnormal dyad stage. F and G, Abnormal tetrad stage
(arrows indicate abnormal tetrad cells). H to J, Examples of abnormal early-stage microspores. K,
Abnormal midstage bicellular pollen (arrow indicates small pollen). L, Mature pollen stage (arrows
indicate small pollen). M to X, Abnormal pollen
development in autotetraploid rice hybrids. M,
Abnormal dyad stage. N to P, Abnormal tetrad stage.
Q, Abnormal early-stage microspore (arrows). R,
Middle-stage abnormal microspore (arrow). S,
Abnormal late-stage microspore (arrows indicate
multiple apertures). T, Early-stage bicellular pollen (arrow indicates small pollen). U to X, Latestage bicellular pollen (arrows indicate small
pollen). Bars = 40 mm.
(Fig. 1, A and B). The diploid hybrids had few meiotic
abnormalities, such as dyad and tetrad degeneration
(Fig. 1, E–H). By contrast, the autotetraploid hybrids
exhibited high percentages of degenerated dyads and
tetrads (Fig. 1, M–O). After meiosis, microgametogenesis can be divided into seven stages, including
early microspore stage, middle microspore stage, late
microspore stage (Fig. 1C), early bicellular pollen stage,
middle bicellular pollen stage (Fig. 1D), late bicellular
pollen stage, and mature pollen stage. Some abnormal
microspores and bicellular pollen were found in diploid
hybrids during microgametogenesis (Fig. 1, I–L).
However, nearly 40% of the microspores and bicellular
pollen were abnormal in the autotetraploid hybrid
during microgametogenesis. For instance, some microspores were surrounded by a thick callose wall (i.e.
the callose remained after completion of meiosis; Fig.
1P), some exhibited cytoplasmic shrinkage (degeneration) at the early microspore stage (Fig. 1Q), some failed
to form a large vacuole (Fig. 1R), and a high percentage
of microspores had multiple apertures during the
middle and late microspore stages (Fig. 1S; Table II).
More than one-third (36.91%) of the microspores had
two to four apertures at the late microspore stage, and
most of the asynchronous development occurred during the bicellular pollen stage (Table II). The nuclei of
some microspores underwent synchronous divisions
during the late microspore stage, which resulted in
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Allelic Interaction and Gene Expression in Polyploid
Table II. Frequency of pollen with different numbers of aperture
DF1 indicates the diploid hybrid (Taichung65 3 E245) that is heterozygous at the Sa, Sb, and Sc pollen
sterility loci. TF1 indicates the autotetraploid hybrid (Taichung65-4x 3 E245-4x) that is heterozygous at the
Sa, Sb, and Sc pollen sterility loci.
Name
DF1
TF1
No.
Single Aperture
3,797
3,670
94.04 6 0.14
63.09 6 2.17
Two Aperture
%6
5.96 6 0.44
33.35 6 2.99
abnormal small pollen (Fig. 1, T–X). By the mature
pollen stage, 88.37% of the autotetraploid rice pollen
was abnormal (Table I).
Meiosis in Diploid and Autotetraploid Hybrids
Similar to normal diploid rice and consistent with
descriptions presented in Wu et al., 2014, meiosis in the
PMCs of the diploid rice hybrid could be divided into
the following nine developmental stages: prophase I,
consisting of leptotene, zygotene (Fig. 2A), pachytene
(Fig. 2, B and C), diplotene (Fig. 2D), and diakinesis
(Fig. 2E); metaphase I (Fig. 2F); anaphase I (Fig. 2G);
telophase I (Fig. 2H); prophase II (Fig. 2I); metaphase II
(Fig. 2J); anaphase II (Fig. 2K); telophase II (Fig. 2L); and
tetrad stage (Fig. 2M). A low percentage of abnormal
behavior, such as chromosome lagging (Fig. 2N) in
metaphase II, straggling in anaphase II (Fig. 2O), and
Three Aperture
Four Aperture
0.00
3.08 6 1.03
0.00
0.48 6 0.16
SD
asynchronous cell division (Fig. 2P), was observed in
PMCs of the diploid hybrid.
We also detected these nine developmental stages in
the meiotic PMCs of autotetraploid rice hybrids (Fig. 3,
A–N; leptotene is not shown). However, we observed
many abnormalities, including chromosome lagging at
metaphase I and metaphase II, chromosome straggling
at anaphase I and anaphase II, micronuclei formation at
telophase I and telophase II, and disordered spindles at
metaphase II, in the autotetraploid hybrid (Table III).
The main kinds of abnormalities were as follows.
Prophase I: At diakinesis, we observed quadrivalents, trivalents, and bivalents simultaneously in the
PMC (Fig. 3, D–F). The main chromosome configurations in the autotetraploid hybrid are shown in Table IV.
Forty-eight univalent chromosomes were present in
approximately 5% of PMCs at diakinesis (Fig. 3Q).
Metaphase I: Chromosome lagging was frequent at
this stage, with an average of 38.38% of cells having one
Figure 2. Chromosome behavior during PMC
meiosis in the diploid rice hybrid. A, Zygotene. B
and C, Pachytene. D, Diplotene. E, Diakinesis.
F, Metaphase I. G, Anaphase I. H, Telophase I. I,
Prophase II. J, Metaphase II. K, Anaphase II. L,
Telophase II. M, The tetrad stage. N, Abnormal
metaphase I (arrow indicates lagging chromosome).
O, Abnormal metaphase II (arrow indicates lagging
chromosome). P, Abnormal anaphase II (asynchronous
cell division). Bars = 10 mm.
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Wu et al.
Figure 3. Chromosome behavior during PMC meiosis in the autotetraploid rice hybrid. A, Zygotene.
B, Pachytene. C, Diplotene. D to F, Diakinesis. G,
Metaphase I. H, Anaphase I. I, Telophase I. J, Dyad.
K, Prophase II. L, Metaphase II. M, Anaphase II. N,
The tetrad stage. O, Abnormal metaphase I (arrow
indicates lagging chromosome). P, Abnormal anaphase I (arrow indicates straggling chromosome).
Q, Pollen mother cell containing 48 univalent
chromosomes at diakinesis. R, Distorted spindle. S,
Abnormal anaphase II showing asynchronous cell
division. T, The triad stage. Bars = 10 mm.
or more instance of chromosome lagging (Fig. 3O;
Table III).
Anaphase I to telophase I: We found that 30.49% of
cells had one or more instance of chromosome straggling (i.e. some chromosomes remained at the center of
the cell, whereas others had already reached the poles;
Fig. 3P). Furthermore, 10.83% of cells contained micronuclei at telophase I (Table III).
Metaphase II: We found that 54.81% of cells at this
stage exhibited abnormal chromosome behavior, spindles, or division, including chromosome lagging (4.31%),
V-shaped spindles (12.51%), T-shaped spindles (11.28%),
misshapen spindles (1.38%), and asynchronous division (25.32%; Table V). The two spindles of the dyad
were arranged in different orientations in PMCs at
anaphase II (Fig. 3R).
Anaphase II: We found that 11.63% of chromosomes
exhibited straggling in anaphase II (Table VI). Moreover,
we observed dyad asynchrony, in which one dyad was at
metaphase II and the other was at anaphase II (Fig. 3S).
Telophase II to the tetrad stage: We observed triads
consisting of one large cell and two small cells (Fig. 3T).
Furthermore, the tetrads contained different numbers
of nucleoli, including one (14.65%), two (18.79%), three
(4.14%), and four double nucleoli (2.55%; Table VII).
Gene Expression Profile Changes and Gene Ontology
Analysis in Autotetraploid and Diploid Rice Hybrids
during Meiosis
Our cytological analysis showed that abnormalities
began to appear in PMC meiosis at the stage of dyad
formation in autotetraploid rice hybrids. Therefore, we
next performed transcriptome profiling using Affymetrix
GeneChips to investigate the impact of polyploidy and
interactions between pollen sterility loci on global patterns of gene expression in autotetraploid hybrids
during PMC meiosis. Microarray data revealed significant correlations (correlation coefficient . 0.95; three
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Allelic Interaction and Gene Expression in Polyploid
Table III. Frequency of cells exhibiting abnormal chromosome behavior during meiosis I in diploid and autotetraploid rice hybrids
DF1 indicates the diploid hybrid (Taichung65 3 E245) that is heterozygous at the Sa, Sb, and Sc pollen sterility loci. TF1 indicates the autotetraploid
hybrid (Taichung65-4x 3 E245-4x) that is heterozygous at the Sa, Sb, and Sc pollen sterility loci.
Name
Metaphase I
No.
Normal
Lagging
Anaphase I
Multipolar Spindle
No.
Normal
Straggling
93.52
63.41
%
6.48
30.49
%
DF1
TF1
444
297
97.97
59.93
2.03
38.38
0.00
1.68
216
328
replicates) in the expression profiles of diploid and autotetraploid hybrids (Supplemental Fig. S3; Supplemental
Table S1). Significant Analysis of Microarray software
identified 1,888 genes that were differentially expressed
(2-fold at P value , 0.05) between diploid and autotetraploid hybrids during meiosis, with 889 genes being
up-regulated in the autotetraploid relative to the diploid hybrid and 999 genes being down-regulated (Fig. 4;
Supplemental Table S2). We then categorized both the
up- and down-regulated genes using Cluster 3.0 software and found that these genes could be clustered into
subgroups based on expression levels (Supplemental
Fig. S4).
Next, we conducted Gene Ontology (GO) analysis to
annotate the genes that were differentially expressed
between the diploid and autotetraploid rice hybrids
during meiosis. We found that many of the differentially expressed genes were associated with multiorganism processes and transcriptional regulatory
functions, and there was significant variation between
putatively up-regulated and down-regulated genes related to these two categories. Notably, we found that
genes associated with the extracellular matrix and
metallochaperone were down-regulated in the autotetraploid cells (Fig. 5).
Among the 889 genes that were differentially upregulated in autotetraploid rice during early meiosis,
three prominent functional gene groups, including response to stress (GO: 0006950; 41 genes), cell wall macromolecule catabolic process (GO: 0016998; 6 genes), and
jasmonic acid stimulus (GO: 0009753; 6 genes), were
common in the biological process category (Fig. 5;
Supplemental Fig. S5). In the cellular component category, which contained a total of 180 genes, we identified one prominent functional gene group, which
mainly involved cytoplasmic membrane-bound vesicles
Telophase I
Bridge
No.
Normal
Micronucleus
%
0.00
6.10
246
257
97.96
89.17
2.43
10.83
(GO: 0016023; Supplemental Fig. S6). In the molecular
function category, we detected two prominent functional gene groups involved in Ser-type endopeptidase
inhibitor activity (GO: 0004867; 7 genes) and transcription factor activity (GO: 0003700; 27 genes;
Supplemental Fig. S7).
Among the 999 genes that were differentially downregulated in autotetraploid rice during meiosis, we
detected two prominent functional gene groups in the
biological process category that were associated with
starch metabolic processes (GO: 0005982) and fatty
acid biosynthetic processes (GO: 0006633; Fig. 5;
Supplemental Fig. S8) and consisted of 12 and 13 genes,
respectively. In the cellular component category, we
identified two prominent functional gene classes, namely
chloroplast (GO: 0009507), consisting of 36 genes, and PSI
(GO: 0009522), consisting of 7 genes (Supplemental Fig.
S9). In the molecular function category, we detected one
prominent functional gene class associated with catalytic
activity (GO: 0003824), and 212 genes were included in
this category (Supplemental Fig. S10).
Furthermore, we then used the David data tool
(National Institute of Allergy and Infectious Diseases,
National Institutes of Health) to identify genes that
were preferentially down-regulated in autotetraploid
hybrids and found that 999 differentially down-regulated
genes were mainly categorized into 17 clusters according
to k score, Similarity, and GO enrichment analyses.
Among these clusters of genes, clusters 1 and 2 consisted of photosynthetic-related genes, mainly associated with PSI and PSII. These results are consistent with
the AgriGo data (Supplemental Fig. S9). In addition,
clusters 10 and 11 were comprised of genes related to
transcription regulation and DNA binding. In cluster
15, genes were mainly associated with transcription
factors and pollen development (Supplemental Table S3).
Table IV. Meiotic chromosome configurations in diploid and autotetraploid hybrids
DF1 indicates the diploid hybrid (Taichung65 3 E245) that is heterozygous at the Sa, Sb, and Sc pollen sterility loci. TF1
indicates the autotetraploid hybrid (Taichung65-4x 3 E245-4x) that is heterozygous at the Sa, Sb, and Sc pollen sterility loci.
I to IV indicate univalent, bivalent, trivalent, and quadrivalent chromosomes, respectively.
Material Name/No. of Cells
DF1
127
167
TF1
168
210
Stage
Chromosome Configuration 6
SD
Diakinesis
Metaphase I
(0.74 6 0.38) I + (11.26 6 0.19) II
(0.23 6 0.04) I + (11.77 6 0.11) II
Diakinesis
Metaphase I
(0.50 6 0.10) I + (9.24 6 0.35) II + (0.64 6 0.06) III + (6.25 6 0.21) IV
(1.33 6 0.11) I + (15.79 6 0.36) II + (0.53 6 0.15) III + (3.74 6 0.19) IV
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Table V. Frequency of abnormal chromosome behavior at metaphase II in diploid and autotetraploid rice
hybrids
DF1 indicates the diploid hybrid (Taichung65 3 E245) that is heterozygous at the Sa, Sb, and Sc pollen
sterility loci. TF1 indicates the autotetraploid hybrid (Taichung65-4x 3 E245-4x) that is heterozygous at the
Sa, Sb, and Sc pollen sterility loci.
Name
DF1
TF1
No.
Normal
244
312
79.10
45.19
Lag
0.00
4.31
V Spindle
T Spindle
Chaos
Asynchronous Meiocytes
6.47
12.51
%
4.09
11.28
0.50
1.38
9.84
25.32
To confirm the results of our microarray analysis, we
validated the expression of nine genes (including four
genes belonging to the pollen allergen Ole e I family,
two belonging to the MYB transcription factor family,
two encoding members of the ubiquitin-proteasome
pathway, and one encoding a member of the WRKY
transcription factor family) in diploid and autotetraploid hybrids during PMCs meiosis by quantitative
reverse transcription (qRT)-PCR. The expression patterns of all of these genes as determined by qRT-PCR
were consistent with the microarray data (Fig. 6).
Genes Differentially Down-Regulated in Autotetraploid
Rice Were Associated with IPE (the Combined Effect of
Polyploidy and Pollen Sterility Loci Interactions)
It was previously reported that interactions between
pollen sterility loci (Sa, Sb, and Sc; IE) caused low pollen
fertility in intersubspecific diploid hybrids (Shahid
et al., 2013a). The following three possible factors could
affect gene expression in autotetraploid rice hybrids: (1)
polyploidy (PE), (2) interactions between Sa, Sb, and Sc
loci (IE), and (3) the combined effect of the polyploidy
and Sa, Sb and Sc loci interactions (IPE; Fig. 7). Reasoning that the down-regulation of important genes
may affect pollen development in the autotetraploid
hybrid, we evaluated the contribution of PE, IE, and IPE
to the expression of the 999 genes that were downregulated in autotetraploid PMCs. To accurately determine the effect of each group, we made the following
three types of comparisons.
Type I Comparison (PE Factor)
Comparison of the transcriptomic profiles of the
diploid and autotetraploid parents (i.e. E1-4x versus
E1-2x and E245-4x versus E245-2x) was mentioned as
PE (1) and PE (2), respectively (Fig. 7, A and B). We
previously showed that 125 genes were down-regulated
during PMC meiosis in autotetraploid rice (E1-4x) versus
diploid rice (E1-2x) and identified one prominent functional gene class with five genes associated with DNAdependent DNA replication (GO: 0006261) in the
biological process category. However, we did not
identify any significant GO terms in the cellular component category (Wu et al., 2014). In our study, we
identified 231 genes that were down-regulated in PMC
meiosis of autotetraploid rice (E245-4x) relative to the
diploid line (E245-2x) during PMC meiosis (Fig. 7B).
AgriGo analysis revealed that no significant GO terms
were in the biological process, cellular component, or
molecular function category.
Type II Comparison (IE Factor)
Comparison was made of transcriptomic profiles of
diploid parents and their hybrids (i.e. DF1 versus E1
and E245). E245 is a pollen-sterile isogenic line (PSIL)
of Taichung65 (E1, Sa j Sa j , Sb j Sb j , and ScjScj) at the
Sa, Sb, and Sc loci (i.e. harboring homozygous genotypes, SiSi, at these loci), and therefore, their hybrids
have the same genetic background as those of E1 and
E245, except for at the Sa, Sb, and Sc loci. Differences in
the transcriptomic profiles between the diploid hybrid
(DF1) and parental lines (i.e. E1-2x and E245-2x) may be
caused by IE. We identified a total of 591 and 735 genes
that were up- and down-regulated in DF1 versus E1-2x
and DF1 versus E245-2x, respectively (Fig. 7). AgriGo
analysis of the 735 down-regulated genes indicated
that there were no significant GO terms in the biological
process category. One prominent functional gene class
with 157 genes encoding cytoplasmic membranebounded vesicles (GO: 0016023) was found in the
Table VI. Frequency of cells exhibiting abnormal chromosome behavior at anaphase II in diploid and
autotetraploid rice hybrids
DF1 indicates the diploid hybrid (Taichung65 3 E245) that is heterozygous at the Sa, Sb, and Sc pollen
sterility loci. TF1 indicates the autotetraploid hybrid (Taichung65-4x 3 E245-4x) that is heterozygous at the
Sa, Sb, and Sc pollen sterility loci.
Materials
No.
Normal
Stragglers
V Spindle
DF1
TF1
213
246
95.14
50.93
0.00
11.63
2.86
6.81
T Spindle
Asynchronous Meiocytes
0.00
6.00
2.00
24.63
%
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Allelic Interaction and Gene Expression in Polyploid
Table VII. Frequency of cells exhibiting abnormal chromosome behavior at telophase II and the tetrad stage in diploid and autotetraploid rice
hybrids
DF1 indicates the diploid hybrid (Taichung65 3 E245) that is heterozygous at the Sa, Sb, and Sc pollen sterility loci. TF1 indicates the autotetraploid
hybrid (Taichung65-4x 3 E245-4x) that is heterozygous at the Sa, Sb, and Sc pollen sterility loci.
Telophase II
Tetrad
Materials
DF1
TF1
No.
Normal
Lag
Abnormal Tetrad
Micronucleus
No.
Normal
(One Nucleolus)
284
258
98.60
71.85
0.00
0.78
%
0.70
21.98
0.70
5.39
250
314
96.80
59.55
cellular component (Supplemental Fig. S11). In the
molecular function category, we identified two prominent functional gene classes, including 16 genes related
to sequence-specific DNA binding protein (GO: 00043565)
and 21 genes encoding transcription factors (GO: 0003700;
Supplemental Fig. S12).
Type III Comparison (IPE Factor)
Comparison was made of transcriptomic profiles
between the autotetraploid and corresponding diploid
rice hybrids (i.e. TF1 versus DF1). We identified a total of
889 and 999 genes that were up- and down-regulated in
TF1 versus DF1, respectively (Fig. 7, B and C). Furthermore, 940 of the down-regulated genes were specifically associated with IPE (Fig. 8; Supplemental Table
S4). We performed predicted protein-protein interaction analysis using STRING software and identified
interactions among these 940 genes (Supplemental Fig.
S13). AgriGo analysis of the 940 down-regulated genes
indicated one prominent functional gene class related to
PSI (GO: 0009522) that contained 7 genes (i.e. Os03g0592500,
Os04g0414700, Os08g0560900, Os12g0189400, Os03g0778100,
One Double
Nucleoli
Two Double
Nucleoli
Three Double
Nucleoli
Four Double
Nucleoli
3.20
14.65
%
0.00
18.79
0.00
4.14
0.00
2.55
Os06g0320500, and Os07g0435300) in the cellular component category that were not detected in the types
I and II comparisons. However, several genes, including Os03g0592500, Os04g0414700, Os12g0189400,
Os03g0778100, and Os06g0320500, were up-regulated
in type II comparison. Overall, these results indicate
that the effect of allelic interactions between the pollen
sterility loci (IE) is strikingly different from that of the
combined effect of polyploidy and pollen sterility loci
interactions (IPE). Two (Os06g0320500 and Os03g0592500)
of the seven genes affected the expression of
Os05g0496100, which was annotated as encoding the
translation initiation factor eukaryotic Initiation Factor-3
(eIF3) subunit. The above results suggest that IPE influences pollen development and the photosynthetic rate in
the autotetraploid rice hybrid.
We measured and compared the photosynthetic rates
of the diploid and autotetraploid rice and found that the
photosynthetic rate of autotetraploid hybrids was significantly lower than that of the diploid hybrids (Fig. 9A;
Supplemental Table S5). Furthermore, we confirmed the
differential gene expression of photosynthetic-related
genes (i.e. Os03g0592500, Os04g0414700, Os08g0560900,
Figure 4. Differentially expressed genes in diploid and autotetraploid rice hybrids. A, Scatter plot analysis of signal intensities for
all normalized genes on the Affymetrix microarray in diploid and autotetraploid rice hybrids. Red and green dots indicate genes
that are up- and down-regulated, respectively. DF 1 and TF 1 indicate the diploid (Taichung65 3 E245) and autotetraploid
(Taichung65-4x 3 E245-4x) hybrids that are heterozygous at the Sa, Sb, and Sc pollen sterility loci, respectively. B, Total number
of differentially expressed genes in diploid and autotetraploid rice hybrids. Genes were categorized as being down-regulated
or up-regulated if their transcript abundance decreased or increased, respectively, and the changes were statistically significant
(2-fold at P value , 0.05).
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Wu et al.
Figure 5. GO enrichment analysis of up- and down-regulated genes in diploid and autotetraploid rice hybrids during PMC
meiosis. Genes were divided into three categories: biological process, molecular function, and cellular component. The percentage of genes in each category was calculated for every GO term. GO functional classification analysis was performed using a
Plant GeneSet Enrichment Analysis Toolkit and AgriGO.
Os12g0189400, Os03g0778100, and Os06g0320500) in
diploid and autotetraploid hybrids using real-time
qRT-PCR analysis (Fig. 9B).
detected in the types II and III comparisons, we constructed a Venn diagram showing genes that were
identified as being up-regulated in the IE and specifically
Analysis of Meiotic-Related Down-Regulated Genes
Associated with IPE in Autotetraploid Hybrid
Because we used transcriptomic profiling to analyze
the impact of polyploidy and interactions between
pollen sterility loci on global patterns of gene expression
in autotetraploid hybrids during PMC meiosis, we now
focused on meiosis-related and meiosis stage-specific
differentially down-regulated genes associated with
IPE. We compared the expression of these genes in
autotetraploid PMCs with microarray data reported for
diploid rice anther meiosis stage-specific expression
(Fujita et al., 2010; Deveshwar et al., 2011) and meiosisrelated expression (Fujita et al., 2010; Yant et al., 2013;
Wright et al., 2015). We identified 54 meiosis stagespecific genes and 1 meiosis-related gene (i.e.
Os02g0497500 [OsAffx.12275.1.S1_x_at] that encodes
the DNA repair-recombination protein [RAD50]) that
were strongly expressed in the anthers of diploid rice
hybrids during meiosis but weakly expressed in autotetraploid rice hybrids. All of the genes were putatively
down-regulated with at least a 2-fold change in expression and specifically associated with IPE.
To compare the differentially expressed genes associated with the meiosis-related or stage-specific expression
Figure 6. Confirmation of microarray data (gene expression profiles) by
real-time qRT-PCR in diploid and autotetraploid rice hybrids during
PMC meiosis. The bars denote the mean expression of nine genes
(relative expression patterns). Gene identification numbers from left to
right along the x axis are related to pollen allergen Ole e I family (four
genes), MYB transcription factor family (two genes), ubiquitin-proteasome
pathway (two genes), and WRKY transcription factor family (one gene).
The error bars indicate the 6SD of the average of three independent
replicates.
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Allelic Interaction and Gene Expression in Polyploid
Figure 7. Relationship between differentially expressed genes, pollen sterility loci, and polyploidy. A, Expression levels of differentially expressed genes in four pairwise comparisons: (1) E1-4x versus E1-2x, (2) E245-4x versus E245-2x (polyploidy effect),
(3) diploid hybrid (Taichung65 3 E245) that is heterozygous at the Sa, Sb, and Sc pollen sterility loci (DF1) versus E1-2x and E245-2x
(pollen sterility loci interactions), and (4) autotetraploid hybrid (Taichung65-4x 3 E245-4x) that is heterozygous at the Sa, Sb, and Sc
pollen sterility loci (TF1) versus DF1 (the effect of polyploidy, pollen sterility loci interactions, and the combined effect of polyploidy
and pollen sterility loci interactions). B, Effect of allelic interactions between the Sa, Sb, and Sc pollen sterility loci (IE), polyploidy
effect (PE), and the combined effect of polyploidy and pollen sterility loci interactions (IPE) on gene expression patterns during PMC
meiosis. C, Venn diagram represents the differentially expressed genes in the PMCs meiosis of the parental lines, the diploid hybrid,
and the autotetraploid hybrid that were putatively associated with PE (type I), IE (type II), and PE + IE + IPE (type III) factors.
down-regulated in IPE (Supplemental Fig. S14). We
found that 17 of 55 meiosis-related or stage-specific
genes were putatively down-regulated because of IPE.
These down-regulated genes displayed very low levels
of expression, and the remaining 38 genes were upregulated in response to IE but down-regulated in response to IPE. Of the 17 IPE-specific genes, 6 genes were
related to metabolism, encoding proteins, such as
GDSL-like lipase and polygalacturonase (Supplemental
Table S6). Os12g0578700 and Os10g0570700 encode a
zinc finger protein and ribosome-inactivating protein,
respectively. Two genes were associated with the
ubiquitin-proteasome pathway (Os02g0490000: U-box
domain-containing protein) and phenylpropanoid pathway (Os04g0310700: AMP-binding domain containing
protein; Supplemental Figs. S15 and S16). Two genes
(Os08g0496800 and Os09g0329000) encode a BURP
domain-containing protein. Os07g0144500 and
Os05g0496100 were associated with PLA IIIA/Patatinlike protein7 and the translation initiation factor eIF3
subunit, respectively. Three genes were annotated as
encoding an expressed protein.
Predicted Protein-Protein Interaction Analysis of MeiosisRelated or Stage-Specific Down-Regulated Genes
Associated with IPE
The products of 23 of the 55 genes found to be downregulated in response to IPE were predicted to undergo
protein-protein interactions (Supplemental Fig. S13, A–E).
We found that the DNA repair-recombination protein
(RAD50) encoded by the key meiosis-related gene,
Os02g0497500, interacted with BTB domain-containing
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Wu et al.
Figure 8. Venn diagram representing the overlap between downregulated genes associated with polyploidy effect (PE), effect of allelic
interactions between the Sa, Sb, and Sc pollen sterility loci (IE), and the
combined effect of polyploidy and pollen sterility loci interactions (IPE)
factors (http://bioinfogp.cnb.csic.es/tools/venny/; Oliveros, 2007).
protein (Os01g0893400) and the calmodulin-binding
domains of retrotransposon protein (Os03g0423850;
Fig. 10). Os05g0496100 encodes the translation initiation factor eIF3 subunit and interacted with
genes encoding chlorophyll A-B binding proteins
(i.e. Os01g0600900, Os08g0435900, Os07g0558400,
Os06g0320500, Os07g0562700, and Os03g0592500; Fig.
10). Os08g0440800 was annotated as an aldehyde
dehydrogenase and interacted with genes encoding
protoporphyrin-IX (Os01g0286600), 6-phosphofructokinase (Os06g0151900), and Fru-bisphosphate aldolase
isozyme (Os01g0905800; Fig. 10). Os01g0924933 encodes a transferase family protein that interacts with
glycerol-3-P acyltransferase, 3-ketoacyl-CoA synthase,
and cytochrome P450 (Fig. 10). Other important interactions were associated with DNA binding protein,
ubiquitin fusion degradation protein, and HOTHEAD
precursor. All of these interactions suggest that IPE has
a significant impact on the expression of 23 meiosisrelated or stage-specific genes and thereby, PMC meiosis and pollen development.
Coexpression and cis-Motif Analysis of Meiosis-Related or
Stage-Specific Down-Regulated Genes Associated with IPE
Coexpression analysis is a useful method to determine
interactions between meiotic-related and stage-specific
down-regulated genes associated with IPE. Coexpression analysis revealed that 8 of the 55 meiosisrelated genes were coexpressed with genes involved in
metabolism (Fig. 11A). Of these eight putatively coexpressed genes, five encoded proteins related to metabolism, including an aspartyl protease (Os04g0595000),
dihydroflavonol-4-reductase (Os01g0127500), stilbene synthase (Os10g0484800), polygalacturonase (Os03g0216800),
and chalcone and stilbene synthase (Os07g0411300).
Os07g0556800 and Os10g0570700 were annotated
as encoding ribosome-inactivating proteins, and
Os09g0329000 encodes a BURP domain containing
protein. In addition, we detected another 11 genes that
interact with the 8 specifically coexpressed genes
(Supplemental Table S4A). Among these 11 genes,
Os12g0242700 was annotated as encoding a 3-oxoacylreductase and chloroplast precursor; Os04g0573100
was annotated as encoding a HOTHEAD precursor,
and five genes were annotated as encoding a zinc finger
C-x8-C-x5-C-x3-H-type family protein, auxin response
factor, aquaporin protein, polygalacturonase, and
transporter. Furthermore, IPE prominently influenced 3
of the 11 genes that interacted with 8 specifically coexpressed genes and 12 of the 55 meiosis-related or stagespecific down-regulated genes (Fig. 11B).
To investigate whether certain promoter motifs were
involved in the transcriptional changes resulting from
IPE, we surveyed the sequence 1,500 bp upstream of
each meiosis-related and meiosis stage-specific gene for
cis-motifs using the Plant CARE database. According to
the microarray profiles of 55 meiosis-related or meiosis
stage-specific genes, 19 types of cis-motifs were detected
in the gene sequences. Interestingly, the Skn-1_motif,
which is required for endosperm development (Lescot
et al., 2002), was the main type of regulatory element
present in the promoter regions of these 55 genes
(Supplemental Table S7).
DISCUSSION
PSILs and Their Hybrids Revealed Epistatic Interactions
between Sa, Sb, and Sc Loci in Autotetraploid Rice Hybrids
Partial pollen sterility and embryo sac abortions affect seed setting of intersubspecific rice hybrids and
limit the commercial production of hybrids (Zhang and
Lu, 1989; Zhang et al., 2006; Ji et al., 2012; Shahid et al.,
2013a). Six loci, including Sa, Sb, Sc, Sd, Se, and Sf, have
been found to regulate intersubspecific rice hybrid
pollen sterility (Zhang and Lu, 1989, 1993; Zhang et al.,
1994). At all of these loci, the ssp. indica and ssp. japonica
rice varieties carried the SiSi and SjSj alleles, respectively. Each locus interaction (SiSj) caused partial pollen
sterility in intersubspecific hybrids (Zhang and Lu,
1989). Transmission electron microscopy revealed that
Sa allelic interactions caused microspore abnormalities
at the middle microspore stage and finally, produced
empty abortive pollen; Sb allelic interactions resulted
in asynchronous development of microspores at the
middle microspore stage, producing stainable abortive
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Allelic Interaction and Gene Expression in Polyploid
Figure 9. Comparison of photosynthetic data in diploid rice hybrids (DF1s) and autotetraploid rice hybrids (TF1s). A, Comparison
of photosynthetic rates between the DF1s and TF1s. B, Confirmation of the gene expression profiles of photosynthetic-related
genes by real-time qRT-PCR in DF1s and TF1s; y axis denotes the mean expression of six genes (relative expression patterns). The
error bars indicate the 6SD of the average of three independent replicates.
pollen, and Sc allelic interactions mainly led to the
nondissolution of the generative cell wall and finally,
produced stainable abortive pollen (Zhang et al., 2006).
In this study, we used Taichung65 and its PSIL to
generate a diploid hybrid with Sa, Sb, and Sc loci interactions. We observed abnormal chromosome behavior
(e.g. chromosome lagging at metaphases I and II) in a
low percentage of PMCs during meiosis, suggesting that
interactions between alleles at the Sa, Sb, and Sc loci
(SaiSaj SbiSbj SciScj) may have an effect on the stage of
meiosis I in diploid hybrids.
Intersubspecific autotetraploid rice hybrids are robust plants with the potential to increase rice yield but
suffer from low seed set (He et al., 2011b; Shahid et al.,
2013b; Wu et al., 2013). We developed over 1,000
combinations of autotetraploid rice in our laboratory
during 2002 to 2010 and found that partial pollen
sterility is a major cause of low seed setting in intersubspecific autotetraploid rice hybrids. Furthermore,
we found that interactions between different alleles of
hybrid pollen sterility loci (Sa, Sb, and Sc) caused low
pollen fertility in autotetraploid rice hybrids (Zhao
et al., 2006; He et al., 2011b; Wu et al., 2013). It took
4 years to generate the autotetraploid spp. japonica rice,
Taichung65-4x, and its PSILs (PSILs-4x) in our laboratory. We self-crossed PSILs-4x for more than 20 generations in our laboratory and confirmed that their
genotypes are stable and the same as those of diploid
rice at the Sa, Sb, and Sc loci. In this study, we used
Taichung65-4x and PSILs-4x to develop an autotetraploid rice hybrid that was heterozygous at Sa, Sb, and
Sc loci. More than 30% of PMCs exhibited abnormal
chromosome behavior, such as chromosome lagging in
metaphase I and metaphase II, chromosome straggling
in anaphase I and anaphase II, and micronuclei formation in telophase II and during meiosis in the autotetraploid rice hybrids. These results suggest that
interactions at the Sa, Sb, and Sc loci (i.e. SaiSaj SbiSbj
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Figure 10. The protein-protein interaction network of meiosis-related and stage-specific genes
associated with the IPE factor. The protein-protein
interaction subnetwork was constructed using
four important meiosis-related and stage-specific
genes (red) and other genes that interact with these
genes (blue).
SciScj) may affect the early stage of prophase I (leptotene) in autotetraploid rice hybrids and that epistatic
effects between alleles at these loci were more pronounced in autotetraploid than diploid rice hybrids.
The epistatic interactions increased pollen sterility,
resulting in much lower fertility in the autotetraploid
rice hybrid than in the diploid hybrid.
Although numerous studies have used a variety of
cytological techniques to examine pollen development
in rice, no live cytological observations of microsporocytes and developing microspores have been reported
for autotetraploid rice hybrids using WE-CLSM (Feng
et al., 2001; Lu et al., 2002; Zhang et al., 2005, 2006). To
observe microsporocyte meiosis and microspore development, we used WE-CLSM and found that interactions between three pairs of Si and Sj alleles in
autotetraploid hybrids resulted in abnormalities in
microsporocytes, such as abnormal dyads and a high
percentage of abnormal tetrads. These results were
consistent with our observations of aberrant chromosomes in PMCs.
Polyploidy Enhanced Epistatic Interactions between Pollen
Sterility Loci That Led to Distinct Alterations in
Transcriptome Profiles and Resulted in High Pollen
Sterility in Autotetraploid Rice Hybrids
Hybridization and whole-genome duplication alter
specific locus or genome-wide gene expression patterns
(transcriptomic shock) in many allopolyploids, although the extent of the impact varies in different
species (Hegarty et al., 2006; Flagel et al., 2008; Buggs
et al., 2011). Xu et al. (2014) used RNA-seq-based transcriptome profiling to analyze the impact of hybridization and whole-genome duplication in reciprocal
hybrids. Xu et al. (2014) developed allopolyploids from
rice japonica and indica ssp. and showed genome-wide
changes in gene expression, including biased parental
expression in the hybrids. Xu et al. (2014) speculated that
the emerging property of whole-genome doubling had
repercussions that reverberate throughout the transcriptome, ultimately generating altered phenotypes.
Here, we focused on the impact of polyploidy on
interactions between hybrid pollen sterility loci (Sa, Sb,
and Sc) and conducted global transcriptomic profiling
of Taichung65, its PSILs, and their reciprocal F1 diploid
and tetraploid hybrids. We explored genome-wide alterations in gene expression caused by polyploidy and
Sa, Sb, and Sc loci interactions during meiosis, a key
stage in pollen development. It used to be technically
challenging to isolate meiotic cells at a specific stage
for RNA-seq analysis. Bright-field microscopy, 4’,6diamidino-2-phenylindole (DAPI) fluorescence staining, and laser capture of individual cells have made it
possible to dissect these cells (Fujita et al., 2010; Tang
et al., 2010; Yang et al., 2011). Using DAPI fluorescence
staining and WE-CLSM techniques, we were able to
obtain PMCs at precise stages of meiosis and identify
genes that were differentially expressed during these
stages in rice (Feng et al., 2001; Lu et al., 2002; Zhang
et al., 2005, 2006; He et al., 2011a, 2011b; Wu et al., 2014).
Theoretically, the fact that the PSILs have the same
genetic background, except at the Sa, Sb, and Sc loci,
eliminates the effect of different parental genomes, thus
providing insight into the contribution of IE, PE, and
IPE but not hybridization of the whole genome on
genome-wide expression patterns.
We detected 1,888 genes that were differentially
expressed during meiosis, which included 889 upregulated and 999 down-regulated genes. We found
that 940 of the genes were down-regulated specifically
because of IPE. The 940 IPE genes included many genes
associated with pollen development and 7 genes that
were putatively associated with PSI and displayed
significant down-regulation during PMC meiosis.
These seven genes also showed a low level of expression in leaves, which resulted in a reduction in photosynthetic rate in autotetraploid rice versus the diploid
hybrid. However, we were unable to detect such a
phenomenon in genes associated with IE and PE. By
contrast, Fu et al. (1999) found that photosynthetic rate
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Allelic Interaction and Gene Expression in Polyploid
Figure 11. The coexpression and protein-protein
interaction network of meiosis-related and stagespecific genes associated with the IPE factor. A,
The coexpression subnetwork was constructed
using eight meiosis-related and stage-specific
genes (blue circles and red labels). B, Proteinprotein interaction network of 3 genes (green) that
interact with 8 specifically coexpressed genes and
12 of 55 meiosis-related and stage-specific genes
(red labels) that were found to be down-regulated
and associated with the IPE factor.
was significantly higher in autotetraploid rice than in
diploid rice. Net photosynthetic rate decreased significantly, especially under severe drought stress conditions, in diploid rice, whereas drought did not have a
significant effect on net photosynthetic rate in autotetraploid lines (Yang et al., 2014). The results of our study
suggest that polyploidy enhances the interaction between Sa, Sb, and Sc loci, which reduces the photosynthetic rate in autotetraploid rice hybrids compared
with the corresponding diploid rice hybrids.
Meiosis is a crucial process in plant reproduction.
Twenty-eight rice meiotic genes have been characterized to date (Luo et al., 2014), and more than 300 meiosis stage-specific genes have been identified in rice
anthers (Fujita et al., 2010; Jin et al., 2013). Here, our
main focus was on differentially down-regulated
meiosis-related or stage-specific genes associated with
the IPE factor. We found that 54 meiosis stage-specific
genes were putatively down-regulated during various
stages of meiosis. Among the meiosis stage-specific
genes, 16 down-regulated genes were specifically related to IPE. Therefore, IPE might be a major reason for
the low pollen fertility of autotetraploid rice hybrids.
Among the down-regulated genes associated with
the IPE factor, we detected two genes, Os05g0496100
and Os02g0490000, related to pollen development.
Os05g0496100 regulates the translation of proteins that
function in the cell cycle (Pyronnet and Sonenberg,
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Wu et al.
2001) and forms the 48S complex through high-affinity
binding to eIF4G in mammalian eIF3 (LeFebvre et al.,
2006). Protein-protein interaction analysis indicated
significant interactions between the translation initiation factor eIF3 subunit (encoded by Os05g0496100)
and chlorophyll A-B binding protein encoded by six
genes (Os01g0600900, Os08g0435900, Os07g0558400,
Os06g0320500, Os07g0562700, and Os03g0592500).
This interaction suggests that down-regulation of
Os05g0496100 might have a strong effect on the regulation of starch biosynthesis genes in response to levels
of photoassimilates (Morita et al., 2015). Here,
Os05g0496100 may cause a significant reduction in the
photosynthetic rate of the autotetraploid in comparison
with the corresponding diploid rice. Os02g0490000
was annotated as a U-box domain-containing protein,
which possessed E3 ubiquitin-protein ligase activity
and was involved in the ubiquitination pathway. The
ubiquitination pathway is known to function in signaling, transcription, DNA repair, cell viability, and
endosomal trafficking (Bader and Steller, 2009; Li et al.,
2012). Os02g0490000 is required for normal pollen development in kiwifruit (Actinidia deliciosa; Scoccianti
et al., 1999). Thus, we speculated that down-regulation
of Os02g0490000 may affect pollen development of
autotetraploid rice hybrids.
Moreover, another important down-regulated gene,
Os02g0497500, was found to encode a DNA repairrecombination protein (RAD50; Gherbi et al., 2001).
RAD50 plays important roles in meiosis, chromosomal
recombination, mitosis, telomere maintenance, and
cellular DNA damage responses and forms a protein
complex with Mre11 and Xrs2/Nbs1 (Mre11/RAD50/
Xrs2; Gallego and White, 2001; Gherbi et al., 2001;
Daoudal-Cotterell et al., 2002; Vannier et al., 2006).
Absence of RAD50 stimulates homologous recombination, and disruption of RAD50 leads to male sterility
in Arabidopsis (Gallego et al., 2001; Gherbi et al., 2001;
Bleuyard et al., 2004). The down-regulation of RAD50
observed in our study may increase homologous
recombination and thereby, promote abnormal chromosome pairing, resulting in the formation of quadrivalents
and trivalents during meiosis in autotetraploid rice hybrids.
All of the above putatively down-regulated genes
were associated with anther development and the
photosystem. Therefore, down-regulation of their expression would affect PMC meiosis and result in low
pollen fertility in the autotetraploid rice hybrid. We
have revealed the different expression patterns of some
key genes related to pollen development in autotetraploid rice. Such a combined study is important for
understanding locus/gene function(s). These results
provide a genome-wide view of the complexity of
multigene interactions during early meiosis and insight
into the intricate molecular mechanism underlying the
effect of polyploidy on allelic interactions between Sa,
Sb, and Sc loci that result in pollen sterility of autotetraploid hybrids. Future work should aim to develop
new autotetraploid rice lines harboring neutral or
widely compatible pollen fertility genes, which do no
interact with each other, to overcome pollen sterility
and develop fertile autotetraploid rice hybrids.
MATERIALS AND METHODS
Plant Materials
Two hybrids, an autotetraploid F1 hybrid (Taichung65-4x 3 E245-4x) and a
diploid F1 hybrid (Taichung65 3 E245), that are heterozygous, SiSj, at the Sa, Sb,
and Sc loci were used in this study. Taichung65-4x and E245-4x were developed
from Taichung65 (E1) and E245 by colchicine-mediated chromosome doubling
and self-crossed for more than 20 generations in our laboratory. E245 is a PSIL
of Taichung65 (E1), which has the same genetic background as E1, except at the
Sa, Sb, and Sc loci. The genotypes of all hybrids are shown in Table I. Diploid F1
hybrids (Taichung65 3 E245) were used as the control for comparisons with
autotetraploid F1 hybrids (Taichung65-4x 3 E245-4x). Taichung65 (E1), E245,
Taichung65-4x, and E245-4x were also used as controls to evaluate the effect of
allelic interactions between the Sa, Sb, and Sc pollen sterility loci (IE), the effect
of polyploidy (PE), and the combined effect of polyploidy and pollen sterility
loci interactions (IPE) on pollen fertility. We evaluated PE for two groups of
parental lines (i.e. Taichung65-4x [E1-4x] versus Taichung65 [E1] and E245-4x
versus E245; Fig. 7, A and B). All plants were grown under natural conditions at
the experimental farm of South China Agricultural University in 2013, and
standard practices were followed.
Validation of Parental and Hybrid Genotypes at the Sa, Sb,
and Sc Loci
Polymorphic pairs of primers were selected from published markers at the Sa,
Sb, and Sc loci for genotype confirmation (Shahid et al., 2013a). The Sa gene has
been cloned, and Sb and Sc have been fine mapped; therefore, closely linked
markers to these genes were used. Two single-nucleotide polymorphism
markers (G02-148 and G02-69) at the Sa locus, two markers (A07-55 and A07-130)
at the Sb locus, and two Simple Sequence Repeat markers (P24-85.7 and
P24-100.7) at the Sc locus were selected (Shahid et al., 2013a).
Classification of Chromosome Behavior
Spikelets were collected from the shoots of rice (Oryza sativa) plants with 22
to 2 cm between their flag leaf cushion and the second to last leaf cushion and
fixed in Carnoy solution (ethanol:acetic acid [3:1, v/v]) for at least 24 h. The
samples were washed three times using 90% (v/v) ethanol and then stored in
70% (v/v) ethanol at 4°C. Anthers were removed from the floret using forceps
and a dissecting needle and placed in a drop of 1% (w/v) acetocarmine on a
glass slide. After 3 to 5 min, the glass slide was covered with a coverslip and
examined under a microscope (Motic BA200). Meiotic stages were classified
and explained according to the works by He et al. (2011b) and Wu et al. (2014).
Characterization of Microsporogenesis
and Microgametogenesis
WE-CLSM was used to observe microsporogenesis and microgametogenesis in diploid and autotetraploid rice hybrids. Spikelets were collected from
the shoots of rice plants with 24 to 20 cm between their flag leaf cushion and the
second to last leaf cushion and kept in a petri dish with moist filter paper.
Anthers were removed from the floret using forceps and a dissecting needle and
placed in a drop of 10 mg L21 eosin B (C20H6N2O9Br2Na2; Mr 624.1; a tissue stain
for cell granules and nucleoli) solution (dissolved in 4% [w/v] Suc) on a glass slide.
After 10 min, the glass slide was covered with a coverslip and scanned under a
Leica SPE Laser-Scanning Confocal Microscope (Leica Microsystems). The excitation wavelength was 543 nm, and emission light was detected between 550
and 630 nm (Shahid et al., 2010; Wu et al., 2014). Pollen fertility was evaluated
according to the method by Shahid et al. (2013a).
Tissue Collection and RNA Extraction
Anthers of the two hybrids and their parental lines were collected during the
meiotic stage of pollen development, and DAPI fluorescence staining was used
to confirm the developmental stage. After collection, samples were frozen
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Allelic Interaction and Gene Expression in Polyploid
immediately in liquid nitrogen and stored at 280°C until used for RNA extraction. All samples were collected in biological triplicate. Total RNA was
isolated following the RNA isolation protocol from Invitrogen Trizol Reagent.
RNA quality was assessed by formaldehyde agarose gel electrophoresis and
quantitated by spectrophotometry (Wu et al., 2014).
Affymetrix Gene Chip Microarrays and Data Analysis
To identify differences in gene expression between the diploid and autotetraploid F1 hybrids at the meiotic stage, Affymetrix microarrays were used.
Two micrograms of total RNA isolated from anthers was used to generate
biotin-labeled complementary RNA targets, and then fragmented complementary RNA targets were hybridized to GeneChip arrays. The GeneChip
arrays were washed, stained, and scanned according to the Affymetrix gene
chip protocol. The quantified data were analyzed using GeneChip Operating
software (GCOS 1.4). The scanned images were first assessed by visual inspection and then analyzed to generate raw data files saved as cell intensity
values (CEL) files using default settings of GCOS 1.4. An invariant set normalization procedure was used to normalize the different arrays using a DNA
chip analyzer (Wu et al., 2014).
GO Annotation and Pathway Analysis
All microarray data were analyzed using Significant Analysis of Microarray software. Genes with a fold change of $2 or #0.5 were subjected to
Student’s t test analysis, and genes with P values of #0.05 were chosen for
further analysis. Cluster analysis was performed using Cluster 3.0 software. GO
analysis was performed for the functional categorization of differentially
expressed genes using the Plant GeneSet Enrichment Analysis Toolkit (http://
structuralbiology.cau.edu.cn/PlantGSEA/) and AgriGO tool (http://bioinfo.
cau.edu.cn/agriGO/). Pathway analysis was performed using MapMan
software and David analysis tools (http://david.abcc.ncifcrf.gov/home.jsp;
Huang et al., 2009a, 2009b). Predicted protein-protein interactions were analyzed using STRING software (http://www.string-db.org/). Annotations for
differentially expressed genes were retrieved from the Rice Genome Annotation
Project (http://rice.plantbiology.msu.edu/). Gene identifications were converted using the identification converter (http://rapdb.dna.affrc.go.jp/tools/
converter/run) and bioDBnet (http://biodbnet.abcc.ncifcrf.gov/).
Coexpression and cis-Motif Analysis of Genes Associated
with IPE
To determine predicted gene functions and gene regulatory networks of
differentially down-regulated genes that were putatively associated with IPE,
coexpression analysis was performed using the RiceFREND database (http://
ricefrend.dna.affrc.go.jp/; Stuart et al., 2003; Sato et al., 2013). To determine the
potential functional relevance of the down-regulated genes that are involved in
meiosis or expressed at specific stages of meiosis, the PlantCARE database
(http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) was used to
search for transcriptional cis-elements.
qRT-PCR
To verify the microarray results, 15 candidate genes from the microarray
analysis were validated by qRT-PCR using the same RNA samples as used in
the microarray analysis. Fifteen gene-specific primer pairs were designed based
on the target gene sequences using Primer Premier5.0 and Oligo6.0 software.
Primers were designed to obtain a product size of between 80 and 200 bp
(Supplemental Table S8). Complementary DNA (cDNA) was synthesized from
1 mg of total RNA using oligo(dT)18 primers and a Transcriptor First Strand
cDNA Synthesis Kit (Roche). qRT-PCR was performed using SYBR Green (BioRad) in a LightCycler480 System (Roche). The final reaction volume for qRTPCR was 20 mL, and each reaction contained 2 mL of cDNA, 10 mM forward and
reverse primer, and 10 mL (23) Advanced SYBR Green Supermix (Bio-RAD).
The following amplification program was used: denaturation at 95°C for 30 s
and 40 cycles of amplification (95°C for 5 s followed by 58°C for 20 s). Melting
curve analysis was performed from 65°C to 95°C, with 5-s increments of 0.5°C.
The rice Ubiquitin gene was used as an internal control. The relative expression
levels were calculated as 22(DCt of treatment 2 DCt) of control (Livak and Schmittgen,
2001), where Ct represents threshold cycle. Each PCR, including the control
reaction, was performed in triplicate (Wu et al., 2014).
Measurement of Photosynthetic Rate
An Li-6400XT Portable Photosynthesis System (LI-COR) was used to
measure the photosynthetic rate of autotetraploid and diploid rice. All
measurements were performed at 26°C (air temperature) with an ambient
CO2 concentration of about 350 mmol mol21 and a relative air humidity of
about 75%. Each calculation was the mean of 20 replicates (Yang et al., 2014).
Supplemental Data
The following supplemental materials are available.
Supplemental Figure S1. PCR amplification of genomic DNA of diploid
and autotetraploid rice hybrids using the P24-85.7 marker.
Supplemental Figure S2. PCR amplification of genomic DNA of diploid
and autotetraploid rice hybrids using the A07-55 marker.
Supplemental Figure S3. Clustering of microarray data of diploid and
autotetraploid hybrids during meiosis.
Supplemental Figure S4. Expression patterns of differentially expressed
genes in diploid and autotetraploid rice hybrids during meiosis.
Supplemental Figure S5. GO enrichment analysis of differentially upregulated genes of the biological process category in autotetraploid
rice hybrid.
Supplemental Figure S6. GO enrichment analysis of differentially upregulated genes of the cellular component category in autotetraploid
rice hybrid.
Supplemental Figure S7. GO enrichment analysis of differentially upregulated genes of the molecular function category in autotetraploid
rice hybrid.
Supplemental Figure S8. GO enrichment analysis of putatively downregulated genes of the biological process category in autotetraploid
rice hybrid.
Supplemental Figure S9. GO enrichment analysis of differentially downregulated genes of the cellular component category in autotetraploid rice
hybrid.
Supplemental Figure S10. GO enrichment analysis of differentially downregulated genes of the molecular function category in autotetraploid rice
hybrid.
Supplemental Figure S11. GO enrichment analysis of differentially downregulated genes of the cellular component category associated with Sa,
Sb, and Sc loci interaction (IE).
Supplemental Figure S12. GO enrichment analysis of differentially downregulated genes of the molecular function category associated with Sa,
Sb, and Sc loci interaction (IE).
Supplemental Figure S13. Predicted protein-protein interaction network of
differentially down-regulated genes (940) associated with IPE (combined
effect of polyploidy and pollen sterility loci interaction) in the autotetraploid rice hybrid during meiosis.
Supplemental Figure S14. Venn diagram showing differentially expressed
up- and down-regulated genes associated with IE and IPE.
Supplemental Figure S15. Ubiquitin-proteasome pathway analysis of
55 down-regulated genes.
Supplemental Figure S16. Phenylpropanoid pathway analysis of 55 downregulated genes.
Supplemental Table S1. The correlation coefficients of microarray data of
diploid and autotetraploid rice hybrids during meiosis.
Supplemental Table S2. Differentially up- and down-regulated genes in
diploid and autotetraploid rice hybrids during meiosis.
Supplemental Table S3. Groups of 940 genes classified by the David analysis tool.
Supplemental Table S4. Product(s) or function(s) of genes associated with
IPE.
Plant Physiol. Vol. 169, 2015
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Wu et al.
Supplemental Table S5. Photosynthetic rate and variance analysis between autotetraploid and diploid rice hybrids.
Supplemental Table S6. Product(s) or function(s) of meiosis-related and
stage-specific genes associated with IPE.
Supplemental Table S7. The number of cis-motifs found upstream of the
genomic sequences corresponding to the meiosis-related and stagespecific genes.
Supplemental Table S8. List of primers used for qRT-PCR analysis.
ACKNOWLEDGMENTS
We thank Guiquan Zhang for donating Taichung65 and E245; Hongbin
Wang for helpful advice and discussions; and Dr. Meiyang Duan, Dunzhou
Mo, Minyi Chen, and Shuhong Yu for technical assistance.
Received May 26, 2015; accepted October 27, 2015; published October 28, 2015.
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