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Genetics and Molecular Research 10 (4): 3435-3445 (2011)
Identication of neutral genes at pollen sterility
loci Sd and Se of cultivated rice (Oryza sativa)
with wild rice (O. rupogon) origin
B. Liu, J.Q. Li, X.D. Liu, M.Q. Shahid, L.G. Shi and Y.G. Lu
State Key Laboratory for Conservation and Utilization of Subtropical
Agro-Bioresources, College of Agriculture,
South China Agricultural University, Guangzhou, China
Corresponding authors: J.Q. Li / Y.G. Lu
E-mail: lijinquan@scau.edu.cn / yglu@scau.edu.cn
Genet. Mol. Res. 10 (4): 3435-3445 (2011)
Received April 19, 2011
Accepted September 8, 2011
Published October 31, 2011
DOI http://dx.doi.org/10.4238/2011.October.31.10
ABSTRACT. Pollen sterility is one of the main hindrances against the
utilization of strong intersubspecic (indica-japonica) heterosis in rice.
We looked for neutral alleles at known pollen sterility loci Sd and Se
that could overcome this pollen sterility characteristic. Taichung 65,
a typical japonica cultivar, and its near isogenic lines E7 and E8 for
pollen sterility loci Sd and Se were employed as tester lines for crossing
with 13 accessions of wild rice (O. rupogon). Pollen fertility and
genotypic segregations of the molecular markers tightly linked with Sd
and Se loci were analyzed in the paired F1s and F2 populations. One
accession of wild rice (GZW054) had high pollen fertility in the paired
F1s between Taichung 65 and E7 or E8. Genotypic segregations of the
molecular markers tightly linked with Sd and Se loci t the expected
Mendelian ratio (1:2:1), and non-signicances were shown among the
mean pollen fertilities with the maternal, parental, and heterozygous
genotypes of each molecular markers tightly linked with Sd and Se loci.
Evidentially, it indicated that the alleles of Sd and Se loci for GZW054
did not interact with those of Taichung 65 and its near isogenic lines,
and, thus were identied as neutral alleles Sdn and Sen. These neutral
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Genetics and Molecular Research 10 (4): 3435-3445 (2011)
B. Liu et al. 3436
genes could become important germplasm resources for overcoming
pollen sterility in indica-japonica hybrids, making utilization of strong
heterosis in such hybrids viable.
Key words: Rice (Oryza sativa); Wild rice (Oryza rupogon);
Pollen sterility; Neutral gene; Molecular marker
INTRODUCTION
Asian cultivated rice (Oryza sativa L.) can be classied into two distinct subspecies
i.e., indica and japonica (Kato, 1930). Inter-subspecic hybrids have signicant hybrid vigour
that provides greater increase in rice production (Yuan, 1987). However, the partial or com-
plete hybrid sterility hinders the utilization of strong heterosis between subspecies (Long et
al., 2008).
The mechanism of F1 sterility in subspecies is complex. There are different theories
about the hybrid sterility in rice, such as male gamete abortions (Zhang et al., 1993; Liu et al.,
2004), female gamete abortions (Yokoo, 1984; Liu et al., 2004) and reduced dehiscence of
anthers (Zhang et al., 2006). Among them, pollen and embryo sac sterility are the two most im-
portant factors which cause hybrid sterility and both of them have contributed almost equally
to spikelet fertility (Song et al., 2005). Wide compatibility and specic compatibility hypoth-
esis are the main theories for rice hybrid sterility. The former theory concerns female gamete
abortion (embryo sac sterility) and it is caused by an allelic interaction (S5i, S5j and S5n) at
S5 locus on chromosome 6 as well as other loci with minor effects including S-7, S-8, S-9,
f1, f3, f8, Sd1
n (t), Sd2
n (t) and S-p(t) (Wan and Ikehashi, 1997; Yi et al., 2001). S5 locus has been
successfully cloned (Chen et al. 2008). The latter one concerns male gamete abortion (pollen
sterility) and controlled by at least six loci (i.e., Sa, Sb, Sc, Sd, Se and Sf) (Zhang and Lu, 1989,
1993). Until now, the Sa gene has been successfully cloned and two adjacently located genes
(SaM and SaF) jointly controlled indica-japonica hybrids sterility. Typical japonica cultivars
contain SaM-SaF- and typical indica cultivars contain SaM+SaF+ alleles at this locus, and the
interaction between SaM+/SaM- lead to abortion of the male gametes carrying the SaM - allele
in the presence of SaF+ allele (Long et al., 2008). The Sb, Sc, and Sd loci have been nely
mapped (Yang et al., 2004; Li et al., 2006; Li et al., 2008), and Se locus has been preliminary
targeted (Zhu et al., 2008).
Gene interaction at each pollen sterility locus could lead to partly abortive pollen.
Therefore, the alleles that do not interact with typical japonica (Sj) and indica (Si) alleles called
neutral alleles for pollen fertility (Sn) and they have signicant importance to overcome the
pollen sterility caused by each locus (Shi et al., 2009). A few neutral genes for certain pollen
sterility loci were identied in cultivated rice (Ding et al., 2003). O. rupogon Griff. Is the
ancestor of cultivated rice and has great genetic diversity and elite genes for rice breeding. O.
rupogon covers a large area in Gaozhou, Guangdong province. Earlier studies in our laboratory
indicated that they might have neutral genes for pollen fertility in wild rice (Li et al., 2007; Lian
et al., 2008). Using Taichung 65 and its near-isogenic line (NIL) at Sb locus as tester lines an
accession of O. rupogon GZW099 was identied to have the neutral gene for pollen fertility at
Sb locus (Shi et al., 2009). Sd and Se loci have important effect on pollen fertility in F1 between
indica and japonica (Li et al., 2008; Zhu et al., 2008). However, no neutral genes at Sd and Se
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Genetics and Molecular Research 10 (4): 3435-3445 (2011)
Identication of neutral genes in cultivated and wild rice
3437
loci have been identied either in cultivated rice or in wild rice.
An NIL of Taichung 65 (i.e., E7) for Sd locus was bred by successive backcrossing and
molecular assistant selection, using the japonica variety Taichung 65 (E1) as recipient parent
and the indica variety Dee-geowoo-gen as donor parents (Li et al., 2003, 2008). Another NIL
of Taichung 65 (i.e. E8) at Se locus was also bred by the similar manner, which is the BC4F3 of
Taichung 65 (recurrent parent) and the indica variety Guangluai-4 (donor parent) (Zhu et al.,
2008). Taichung 65 and its NILs at Sd and Se loci (i.e. E7 and E8) have a similar genetic back-
ground except some genotypic differentiation at Sd and Se locus, i.e. genotypes Sd
j Sd
j, Se
j Se
j
for E1, Sd
i Sd
i, Se
j Se
j for E7 and Sd
j Sd
j, Se
i Se
i for E8. The model of gene interaction for Sd and Se
locus tted the one-locus sporo-gametophytic interaction model (Zhang and Lu, 1989, 1993).
That is to say, indica and japonica varieties usually possess SiSi and SjSj alleles, respectively, at
a locus, and the gametes having Sj allele are partially aborted in the hybrid genotype SiSj.
To explore the neutral gene for pollen fertility at the Sd and Se loci in Gaozhou wild
rice, Taichung 65 and its NIL E7 and E8 were used as the genetic testers in this study. Thirteen
accessions of Gaozhou wild rice were selected to cross with the genetic testers. Analyses were
made on pollen fertility of F1 and F2 within the pairs of testcross and the genotypic segrega-
tion of the molecular markers linked tightly with these two loci to identify the neutral allele.
The objective of the present study was to identify the neutral genes for pollen fertility in O.
rupogon at Sd and Se locus and offer new germplasms for overcoming the pollen sterility in
indica-japonica hybrids.
MATERIAL AND METHODS
Plant materials
Taichung 65 (Sd
j Sd
j, Se
j Se
j), E7 (Sd
i Sd
i) and E8 (Se
i Se
i) were used as female parents,
which have same genetic background except at Sd and Se locus and was kindly provided by
professor Zhang GuiQuan. O. rupogon indigenous to Gaozhou, Guangdong province which
is conserved at the Oryza genus germplasm resources of South China Agricultural University
(SCAU), was used as the male parent. A total of 13 accessions of wild rice from Gaozhou
(GZW) were selected: GZW006, GZW009, GZW011, GZW013, GZW026, GZW054,
GZW060, GZW099, GZW101, GZW133, GZW135, GZW136, and GZW137 from 6 popula-
tions of 141 accessions. Crosses were made at SCAU from 2006 to 2007. The F1 plants were
sown at the same time in late season, 2007 at SCAU. The F2 plants derived from F1 were sown
in late season, 2007 at Sanya, Hainan province.
Pollen fertility analysis
Five spikelets that would open the following day in the top area of each spike of main til-
ler were selected and xed into Carnoy’s xative solution for 24 h. Then they were transferred to
70% alcohol. Three spikelets were selected randomly and dissected. Pollen grains were stained
with 1% iodine potassium iodide (I2-KI) solution. Microscopic observation of pollen was done
under Motic BA200 at 10 x 20. More than 300 pollen grains were scanned randomly on each
slide and pollen fertility was divided into four categories i.e., normal pollens, stained abortive
pollens, spherical abortive pollens and typical abortive pollens (Shahid et al., 2010).
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Genetics and Molecular Research 10 (4): 3435-3445 (2011)
B. Liu et al. 3438
DNA extraction and SSR analysis
The genomic DNA was extracted from fresh-frozen leaves of each plant as described
by Zheng et al. (1995) with minor modications.
We selected 5 and 4 polymorphic pair of SSR primers based on the ne mapping of
Sd and Se locus, and these primers were linked tightly with Sd and Se locus, respectively. The
genetic distance of PSM13, PSM91, PSM41, PSM42, and PSM43 from Sd locus was 0.4,
0.05, 4.8, 4.8 and 3.2 cM, respectively. The distance of PSM597, PSM448, and PSM461 from
Se locus was 4.75, 0.2 and 0.6 cM, respectively. PSM559 was inside the Se locus. PSM597 and
PSM559 are InDel markers, whereas the others are SSR markers. PCR and genotyping were
done according to Shi et al. (2009).
Identication method for the neutral gene at Sd and Se loci
Identication method for the neutral genes Sd and Se loci was modied from the
method of Shi et al. (2009). The genotype of candidate tested line was assumed to be Sd
xSd
x.
Firstly, the candidate tested line was crossed with both E1 and E7 (when Sd locus was de-
tected; or E8 when Se locus was detected) to make a pair of test combinations. Because E1 and
E7 (or E8) had the same genetic background and differed only at Sd (or Se) locus, the signicant
difference of pollen fertility in the same pair of test combination was due to the allelic interac-
tion at Sd (or Se) locus and the allelic interactions at the other loci were the same. Thus the
inuence of the genetic background was reduced. The interaction of the testcross at Sd (or Se)
locus would be one of following three conditions.
If Sd
xSd
x is Sd
iSd
i, the genotype of the F1 from the testcross between the candidate tested
line and E1 should be Sd
iSd
j. F1 pollen and the gametes of Sj would be partly abortive because
of the allelic interaction. Correspondingly, the genotypes of molecular markers tightly linked
with Sd locus in F2 generation would show a deviated distribution with reduced numbers of the
alleles from E1 (Sd
i gamete). However, the genotype of the F1 from the testcross between the
candidate tested line and E7 was Sd
iSd
i, and thus, the pollen fertility of F1 was normal due to no
allelic interaction. The corresponding molecular marker linked with Sd locus in F2 population
would segregate according to Mendelian ratio (1:2:1).
If Sd
xSd
x is Sd
jSd
j, the genotype of the F1 from the testcross between the candidate tested
line and E1 should be Sd
jSd
j and the F1 pollen was fertile because of no allelic interaction. The
corresponding molecular marker linked with Sd locus in its F2 population should follow the
Mendelian segregation (1:2:1). However, the genotype of the F1 from the testcross between
the candidate tested line and E7 should be Sd
jSd
i. F1 pollen and the gametes of Sj were partly
abortive because of an allelic interaction. Correspondingly, the genotypes of molecular marker
tightly linked with Sd locus in its F2 population would show deviated distribution with less
numbers of the alleles from candidate tested line (Sd
i gamete).
If Sd
xSd
x is Sd
nSd
n, the genotype of the F1 from the testcross between the candidate tested
line and E1 or E7 should be Sd
nSd
j or Sd
nSd
i. Allele Sd
n was compatible with both Sd
j and Sd
i, so
there was no allelic interaction between them. The F1s of them were fertile and would show
non-signicant differences between two crosses. The genetic segregation ratio of correspond-
ing molecular marker linked with Sd locus in both F2 population should be in accordance with
Mendelian ratio (1:2:1).
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Genetics and Molecular Research 10 (4): 3435-3445 (2011)
Identication of neutral genes in cultivated and wild rice
3439
The above method was used to identify the neutral gene for pollen fertility at Sd locus
and same method was used to detect neutral gene at Se locus.
RESULTS
Preliminary selection of materials carrying neutral gene
According to previous studies, a total of 13 wild rice accessions, which had high
pollen fertility, embryo sac fertility and seed set percentage or their F1s when crossed with
E1, were selected from 141 accessions of GZW. The average pollen fertility of F1s obtained
from the testcrosses of GZW006, GZW099, GZW094, GZW133 with E1 was signicantly
higher than the corresponding F1s between these wild rice and E7, showing that the genotype
at the Sd locus for the four accessions of wild rice was Sd
jSd
j. Similarly, average pollen fertil-
ity of E1×GZW094 and E1×GZW133 was signicantly higher than that of E8×GZW094 and
E8×GZW133, indicating Se
jSe
j genotype at Se locus.
Average pollen fertility for E1×GZW054, E7×GZW054 and E8×GZW054 was more
than 94% and their parents also showed high pollen fertility and seed set (>90%). Non sig-
nicant differences were found based on a Student’s-t-test in F1 generation (Figure 1, Table
1). These results indicated that GZW054 might posses the neutral genes for pollen fertility at
Sd and Se locus and further checked by molecular markers. Similarly, average pollen fertility
for E1×GZW013 and E8×GZW013 were high and non-signicant, indicating that GZW013
might possess neutral genes at Se locus.
Figure 1. Pollen fertility of different genotypes in E1, E7, E8, GZW054 and their F1s. 1. E1 (genotype Sd
jSd
j, Se
jSe
j);
2. E7 (genotype Sd
iSd
i); 3. E8 (genotype Se
iSe
i); 4. GZW054 (genotype Sd
nSd
n, Se
nSe
n); 5. F1 of E1×E7 (genotype Sd
iSd
j);
6. F1 of E1×E8 (Se
iSe
j); 7. F1 of E1×GZW054 (genotype Sd
jSd
n, Se
jSe
n); 8. F1 of E7×GZW054 (genotype Sd
iSd
n); 9. F1
of E8×GZW054 (genotype Se
jSe
n).
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Genetics and Molecular Research 10 (4): 3435-3445 (2011)
B. Liu et al. 3440
Molecular marker and pollen fertility analysis in paired F2
Pollen fertility of each F2 plant of three crosses (E1×GZW054, E7×GZW054 and
E8×GZW054) was observed and the results are as follows (Figure 2, 3):
A total of 119 F2 individuals were checked of E1×GZW054. The maximum, minimum
and average pollen fertility was 97.76, 0.29 and 87.36%, respectively (Figure 2). A total of 115
plants showed normal pollen fertility (>50%), and it constituted 96.64% of the total population.
Testcross Pollen fertility (%) (mean ± SE) t value P value
E1×GZW006 80.64 ± 5.30 2.328 0.067
E7×GZW006 59.77 ± 2.38
E1×GZW054 95.54 ± 1.37 0.679 0.546
E7×GZW054 94.05 ± 1.77
E1×GZW054 95.54 ± 1.37 -0.464 0.688
E8×GZW054 96.81 ± 0.08
E1×GZW099 89.28 ± 1.07 6.34 0.001
E7×GZW099 77.79 ± 0.56
E1×GZW013 74.95 ± 3.69 0.215 0.837
E8×GZW013 73.41 ± 4.88
E1×GZW094 89.85 37.564 0.017
E7×GZW094 69.03 ± 0.32
E1×GZW094 89.85 69.125 0.009
E8×GZW094 50.34 ± 0.33
E1×GZW133 87.79 ± 2.87 3.749 0.020
E7×GZW133 70.75 ± 2.42
E1×GZW133 87.79 ± 2.87 3.851 0.031
E8×GZW133 63.09
Table 1. Pollen fertility in F1s between different accession of Gaozhou wild rice and Taichung 65 (E1) and its
NILs (E7, E8).
Figure 2. Frequency distribution of pollen fertility in 119 individuals of E1×GZW054 in F2 population.
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Genetics and Molecular Research 10 (4): 3435-3445 (2011)
Identication of neutral genes in cultivated and wild rice
3441
Similarly, the average pollen fertility of E7×GZW054 in F2 generation was 86.40%
(Table 2), and it ranged from 2 to 100% in 174 samples, and a total of 166 plants showed nor-, and a total of 166 plants showed nor-and a total of 166 plants showed nor-
mal pollen fertility (>50%), and it accounted for 95.40% of the total population.
Testcross Molecular Genotypes Number of plant Pollen fertility Chi-squared test for Analysis of variance
makers (%) (mean ± SE) genotypes for pollen fertility
χ
2 value P value F value P value
E1×GZW054 PSM13 1 (Sd
j Sd
j) 34 90.58 ± 1.06 0.078 0.962 0.847 0.432
2 (Sd
j Sd
n) 72 87.51 ± 2.12
3 (Sd
n Sd
n) 35 84.81 ± 4.52
PSM91 1 (Sd
j Sd
j) 32 90.48 ± 1.63 2.751 0.349 2.751 0.068
2 (Sd
j Sd
n) 86 88.37 ± 1.73
3 (Sd
n Sd
n) 38 81.21 ± 4.55
E7×GZW054 PSM41 1 (Sd
i Sd
i) 55 87.94 ± 2.45 2.941 0.230 0.381 0.893
2 (Sd
i Sd
n) 89 87.29 ± 1.89
3 (Sd
n Sd
n) 58 83.80 ± 3.00
PSM42 1 (Sd
i Sd
i) 37 83.15 ± 5.18 5.856 0.054 0.645 0.526
2 (Sd
i Sd
n) 103 87.50 ± 1.43
3 (Sd
n Sd
n) 61 86.50 ± 2.39
PSM43 1 (Sd
i Sd
i) 59 83.00 ± 3.31 2.682 0.262 1.527 0.220
2 (Sd
i Sd
n) 90 87.15 ± 1.71
3 (Sd
n Sd
n) 52 89.14 ± 1.91
Table 2. Genotypic distribution of the molecular markers and their corresponding pollen fertility in the F2
populations of E1×GZW054 and E7×GZW054.
Figure 3. Pollen fertility of different genotypes in F2s of E1, E7, E8 and GZW054. 1. F2 of E1×GZW054 (genotype
Sd
j Sd
j); 2. F2 of E1×GZW054 (genotype Sd
j Sd
n); 3. F2 of E1×GZW054 (genotype Sd
n Sd
n); 4. F2 of E7×GZW054
(genotype Sd
i Sd
i); 5. F2 of E7×GZW054 (genotype Sd
i Sd
n); 6. F2 of E7×GZW054 (genotype Sd
n Sd
n); 7. F2 of
E1×GZW054 (genotype Se
j Se
j); 8. F2 of E1×GZW054 (genotype Se
j Se
n); 9. F2 of E1×GZW054 (genotype Se
n
Se
n); 10. F2 of E8×GZW054 (genotype Se
i Se
i); 11. F2 of E8×GZW054 (genotype Se
i Se
n); 12. F2 of E8×GZW054
(genotype Se
n Se
n).
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Genetics and Molecular Research 10 (4): 3435-3445 (2011)
B. Liu et al. 3442
Figure 4. Genotypes of PSM13 in the F2 population of E1×GZW054. 1-3 indicate maternal genotype (E1),
heterozygous genotype and paternal genotype (GZW054) in F2 population, respectively.
Figure 5. Genotypes of PSM91 in the F2 population of E1×GZW054. 1-3 indicate maternal genotype (E1),
heterozygous genotype and paternal genotype (GZW054) in F2 population, respectively.
Testcross Molecular Genotypes Number of plant Pollen fertility Chi-squared test for Analysis of variance
makers (%) (mean ± SE) genotypes for pollen fertility
χ
2 value P value F value P value
E1×GZW054 PSM597 1 (Se
j Se
j) 37 88.86 ± 2.06 1.450 0.484 0.239 0.788
2 (Se
j Se
n) 70 86.97 ± 2.37
3 (Se
n Se
n) 44 86.13 ± 3.02
PSM448 1 (Se
j Se
j) 43 89.72 ± 1.82 2.703 0.259 1.104 0.335
2 (Se
j Se
n) 82 87.38 ± 1.94
3 (Se
n Se
n) 30 83.14 ± 4.85
E8×GZW054 PSM461 1 (Se
i Se
i) 38 87.17 ± 2.63 2.000 0.368 1.756 0.178
2 (Se
i Se
n) 56 82.68 ± 2.32
3 (Se
n Se
n) 26 78.89 ± 3.91
PSM559 1 (Se
i Se
i) 31 80.81 ± 4.88 0.024 0.988 0.107 0.898
2 (Se
i Se
n) 62 82.84 ± 2.29
3 (Se
n Se
n) 32 82.42 ± 2.77
Table 3. Genotypic distribution of the molecular markers and their corresponding pollen fertility in the F2
populations of E1×GZW054 and E8×GZW054.
The highly polymorphic molecular markers between E1 and GZW054, i.e. PSM13 and
PSM91 at Sd locus were selected. The distribution of three genotypes (maternal, parental, and
heterozygous) in their F2 is shown in Figure 4, Figure 5 and Table 2. The segregation ratio of the
three genotypes for both markers in the F2 population followed the expected Mendelian ratio
(1:2:1) based on the chi-squared test results (P = 0.962 and 0.349, respectively), and average
pollen fertility corresponding to the three genotypes in the F2 population showed non-signi-
cant difference based on the analysis of variance (P = 0.432 and 0.068, respectively). Therefore,
it indicated that the alleles of GZW054 had no interaction with those of E1 at Sd locus.
A total of 106 plants of E8×GZW054 were observed and their average pollen fertil-
ity was 82.36% (Table 3). The minimum and maximum pollen fertility was 4.4 and 98.79%,
respectively. A total of 99 plants showed normal pollen fertility (>50%), and it was 93.40% of
the population.
Furthermore, the SSR markers PSM41, PSM42 and PSM448 were selected for trac-
ing the genotypic distribution in the F2 of E7×GZW054 because of high polymorphism be-
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Genetics and Molecular Research 10 (4): 3435-3445 (2011)
Identication of neutral genes in cultivated and wild rice
3443
tween them. It showed that the distribution of the three genotypes in the F2 of E7×GZW054
tted the Mendelian ratio of 1:2:1 using a chi-squared test (P = 0.230, 0.054 and 0.262, re-
spectively), and there were non-signicant difference in average pollen fertility correspond-
ing to the three genotypes based on the analysis of variance (P = 0.893, 0.526 and 0.220,
respectively) (Table 2). It suggested that there was no interaction between the alleles of E7
and GZW054 at Sd locus.
According to the method of identifying neutral alleles, it was conrmed from the
above results that the alleles of GZW054 are compatible to those of E1 (i.e. SjSj) and E7
(i.e. SiSi) at Sd locus and thus, we concluded that GZW054 has a neutral gene at Sd locus
(i.e. Sd
nSd
n).
Similarly, the molecular markers PSM597 and PSM448 were highly polymorphic
between E1×GZW054 as well as PSM461 and PSM559 for E8×GZW054 at Se locus. We
obtained similar results to Sd locus, i.e. the segregation ratio of the three genotypes in their
F2 population tted the expected Mendelian ratio (1:2:1) (Table 3) based on a chi-squared
test, and the average pollen fertility corresponding to the three genotypes in their F2 popula-
tion showed non-signicant difference by the analysis of variance for both crosses (Table 3).
Therefore, the results indicted that GZW054 has a neutral gene at Se locus.
DISCUSSION
Hybrid sterility is common between the subspecies of rice. On one hand, it drives
speciation and evolution; on the other hand, it blocks favorable genes/traits intercross as com-
bining elite genes/traits together in breeding is needed. Wide Compatibility Gene theory (Ike-
hashi and Araki, 1984) and Specic Compatibility Hypothesis (Zhang and Lu, 1989, 1993)
was used to overcome the embryo sac sterility and pollen sterility, respectively. It can increase
spikelets fertility of indica-japonica hybrids by introducing neutral genes into subspecies. It is
well known that wide-compatibility gene S5
n proposed by Ikehashi and Araki (1984), offered a
bright prospect to overcome the sterility in indica-japonica hybrids. Recently, a compatibility
gene Sa
n for pollen sterility was proposed by Long et al. (2008). The rapid development of mo-
lecular marker techniques and genome sequencing has made it easier to identify neutral genes.
A total of 10 wild rice accessions were identied carrying S5
n allele by functional molecular
markers (Yang et al., 2009). However, sometimes molecular markers are not related to the
gene, so phenotype identication by testcross is also necessary to obtain accurate results. Shi
et al. (2009) proposed a new method to identify neutral genes by use of testcross and molecu-
lar markers together. This is an accurate method to identify neutral alleles and was applied in
the present study.
Wild rice (O. rupogon) is considered as an ancestor of cultivated rice and has a num-
ber of useful genes. Wild rice is resistant to pests and diseases, it can grow well in poor soil
even in polluted water and it has many useful genes to improve rice varieties. Some earlier
studies indicated that there might be neutral gene in wild rice. For example, high intra-specic
hybrid fertility was found when wild rice was crossed with cultivated rice (Oka, 1964). There
is a highly signicant correlation between pollen fertility and spikelets fertility of the F1s
between wild rice and cultivated rice, and wild rice has afnity with cultivated rice (Li et
al., 2007). Gaozhou wild rice has abundant genetic diversity and high pollen fertility (>85%)
which was achieved when crossed with other cultivars (Lian et al., 2008). These results indi-
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Genetics and Molecular Research 10 (4): 3435-3445 (2011)
B. Liu et al. 3444
cated that wild rice might have neutral genes for pollen fertility, which can overcome the pol-
len sterility of intra-specic hybrids. The present study furthermore conrmed that Gaozhou
wild rice had neutral alleles for pollen fertility at Sd and Se locus.
In our case, we used Taichung 65, a typical japonica cultivar and its NILs for pollen
sterility locus as testers and combine traditional method (testcross) and molecular marker
analysis together to detect neutral gene in wild rice. The materials used for testers were bred
using Taichung 65 as a recipient parent and indica variety Dee-geowoo-gen, Guangluai-4 as
donor parent through successive backcrossing (Li et al., 2003, 2008; Zhu et al., 2008). Mo-
lecular markers were used to check the polymorphism between Taichung 65 and NILs and
similar genetic background was found between them except at Sd and Se loci (Li et al., 2008).
Therefore, the results of present study are more precise than a common variety used as tester.
The pollen fertility of F1 hybrid depends on allelic interaction at each pollen steril-
ity locus. The neutral alleles did not have an interaction with both Sj and Si alleles. Therefore,
neutral alleles at each locus have potential to overcome the F1 pollen sterility associated with
the locus. Exploitation and utilization of neutral alleles are of signicant importance. At pres-
ent, Sa
n was proposed by Long et al. (2008), Sb
n was detected by Shi et al. (2009), and Sd
n and
Se
n were found by our study. By pyramiding these novel neutral genes into elite cultivated
rice varieties, varieties having strong compatibility either with indica or japonica would be
expected. Thus, it provides an effective way and important germplasm to overcome the hybrid
pollen sterility of F1 between indica and japonica and enhance the utilization of the strong
heterosis between subspecies.
ACKNOWLEDGMENTS
Research supported by the Joint Funds of the National Natural Science Foundation
of China and Guangdong Province (Grant #U0631003) and Natural Science Foundation of
Guangdong Province (Grant #5300831).
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