Controlling Transgene Escape in GM Creeping Bentgrass

Trait improvement of turfgrass through genetic engineering is important to the turfgrass industry and the environment. However, the possible transgene escape to wild and non-transformed species raises ecological and commercial concerns. Male sterility provides an effective way for interrupting gene flow. We have designed and synthesized two chimeric gene constructs consisting of a rice tapetum-specific promoter (TAP) fused to either a ribonuclease gene barnase, or the antisense of a rice tapetum-specific gene rts. Both constructs were linked to the bar gene for selection by resistance to the herbicide glufosinate. Agrobacterium-mediated transformation of creeping bentgrass (cv Penn A-4) with both constructs resulted in herbicide-resistant transgenic plants that were also 100% pollen sterile. Mendelian segregation of herbicide resistance and male sterility was observed in T1 progeny derived from crosses with wild-type plants. Controlled self- and cross-pollination studies showed no gene transfer to non-transgenic plants from male-sterile transgenic plants. Thus, male sterility can serve as an important tool to control transgene escape in bentgrass, facilitating the application of genetic engineering in producing environmentally responsible turfgrass with enhanced traits. It also provides a tool to control gene flow in other perennial species using transgenic technology.     

Promoters of two anther-specific genes confer organ-specific gene expression in a stage-specific manner in transgenic systems

Differential screening of a stage-specific cDNA library of Indica rice has been used to identify two genes expressed in pre-pollination stage panicles, namely OSIPA and OSIPK coding for proteins similar to expansins/pollen allergens and calcium-dependent protein kinases (CDPK), respectively. Northern analysis and in situ hybridizations indicate that OSIPA expresses exclusively in pollen while OSIPK expresses in pollen as well as anther wall. Promoters of these two anther-specific genes show the presence of various cis-acting elements (GTGA and AGAAA) known to confer anther/pollen-specific gene expression. Organ/tissue-specific activity and strength of their regulatory regions have been determined in transgenic systems, i.e., tobacco and Arabidopsis. A unique temporal activity of these two promoters was observed during various developmental stages of anther/pollen. Promoter of OSIPA is active during the late stages of pollen development and remains active till the anthesis, whereas, OSIPK promoter is active to a low level in developing anther till the pollen matures. OSIPK promoter activity diminishes before anthesis. Both promoters show a potential to target expression of the gene of interest in developmental stage-specific manner and can help engineer pollen-specific traits like male-sterility in plants. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Accessions: OSIPA cDNA, AF220610; OSIPK cDNA, AF312920; OSIPA partial gene and upstream promoter region, AY166659; OSIPK gene-specific and upstream sequence, AY168440.     

The PsEND1 promoter: a novel tool to produce genetically engineered male-sterile plants by early anther ablation

PsEND1 is a pea anther-specific gene that displays very early expression in the anther primordium cells. Later on, PsEND1 expression becomes restricted to the epidermis, connective, endothecium and middle layer, but it is never observed in tapetal cells or microsporocytes. We fused the PsEND1 promoter region to the cytotoxic barnase gene to induce specific ablation of the cell layers where the PsEND1 is expressed and consequently to produce male-sterile plants. Expression of the chimaeric PsEND1::barnase gene in two Solanaceae (Nicotiana tabacum and Solanum lycopersicon) and two Brassicaceae (Arabidopsis thaliana and Brassica napus) species, impairs anther development from very early stages and produces complete male-sterile plants. The PsEND1::barnase gene is quite different to other chimaeric genes previously used in similar approaches to obtain male-sterile plants. The novelty resides in the use of the PsEND1 promoter, instead of a tapetum-specific promoter, to produce the ablation of specific cell lines during the first steps of the anther development. This chimaeric construct arrests the microsporogenesis before differentiation of the microspore mother cells and no viable pollen grains are produced. This strategy represents an excellent alternative to generate genetically engineered male-sterile plants, which have proved useful in breeding programmes for the production of hybrid seeds. The PsEND1 promoter also has high potential to prevent undesirable horizontal gene flow in many plant species.     

The cauliflower mosaic virus (CaMV) 35S promoter sequence alters the level and patterns of activity of adjacent tissue- and organ-specific gene promoters

Here we report the effect of the 35S promoter sequence on activities of the tissue- and organ-specific gene promoters in tobacco plants. In the absence of the 35S promoter sequence the AAP2 promoter is active only in vascular tissues as indicated by expression of the AAP2:GUS gene. With the 35S promoter sequence in the same T-plasmid, transgenic plants exhibit twofold to fivefold increase in AAP2 promoter activity and the promoter becomes active in all tissue types. Transgenic plants hosting the ovary-specific AGL5:iaaM gene (iaaM coding an auxin biosynthetic gene) showed a wild-type phenotype except production of seedless fruits, whereas plants hosting the AGL5:iaaM gene along with the 35S promoter sequence showed drastic morphological alterations. RT-PCR analysis confirms that the phenotype was caused by activation of the AGL5:iaaM gene in non-ovary organs including roots, stems and flowers. When the pollen-, ovule- and early embryo-specific PAB5:barnase gene (barnase coding a RNase gene) was transformed, the presence of 35S promoter sequence drastically reduced transformation efficiencies. However, the transformation efficiencies were restored in the absence of 35S promoter, indicating that the 35S promoter might activate the expression of PAB5:barnase in non-reproductive organs such as calli and shoot primordia. Furthermore, if the 35S promoter sequence was replaced with the NOS promoter sequence, no alteration in AAP2, AGL5 or PAB5 promoter activities was observed. Our results demonstrate that the 35S promoter sequence can convert an adjacent tissue- and organ-specific gene promoter into a globally active promoter. Xuelian Zheng and Wei Deng contributed equally to this work and are considered co-first authors.     

Molecular characterization of mature pollen-specific genes encoding novel small cysteine-rich proteins in rice (Oryza sativa L.)

In our previous cDNA microarray analysis, we identified 53 mature anther-specific genes, whose function was unknown, in rice. We reanalyzed these genes from the viewpoint of the specific amino acid motif. Out of 53 genes, three genes, Os-26, Os-32, and Os-169 (renamed as OsSCP1, OsSCP2, and OsSCP3), encoded cysteine-rich motif (Cys-X₃-Cys-X₁₃-Cys-X₃-Cys), indicating that they were novel small cysteine-rich proteins. From the search of specific elements in promoter regions, several pollen-specific elements were found. In order to determine whether three promoters were functional in pollen or not, the gene constructs with promoter regions fused to the β-glucuronidase gene were transformed into tobacco. Histochemical analysis showed that these promoters were active in the mature pollen grains and pollen tubes. Furthermore, OsSCP1 and OsSCP3 formed a multigene family tandemly in the rice genome. From the results, OsSCPs might have important roles in mature pollen development and pollen tube growth.     

Field trial detects incomplete barstar attenuation of vegetative cytotoxicity in Populus trees containing a poplar LEAFY promoter::barnase sterility transgene

We tested the efficacy of an attenuation system developed to preclude the deleterious effects of floral promoter::cytotoxin genes on vegetative growth of transgenic sterile plants. We tested the promoter (2.6 kb 5′ region) of the poplar LEAFY gene PTLF driving barstar, combined on the same T-DNA with barstar driven by either the CaMV 35S basal promoter +5 to −72 fragment (35SBP), 35SBP fused to the TMV omega element (35SBP omega), or the NOS promoter. The unattenuated pPTLF::barnase construct failed to give rise to any transgenic events, suggesting substantial non-reproductive expression from this promoter. The barstar-attenuated constructs enabled transformation, but the rate was reduced by nearly one-third. Four events (7% of attenuated events) had highly abnormal morphology, and were identified during the early phases of propagation; these events had significantly higher barnase:barstar expression ratios based on quantitative RT-PCR. A greenhouse study showed that phenotypically normal attenuated plants grew at the same rate as wild-type and barnase-lacking transgenic plants. A statistically significant positive linear association was found between relative growth rate (RGR) and barstar:barnase ratio in the attenuated events, and graphical analysis suggested a threshold for barstar attenuation of barnase, above which additional levels of barstar did not provide further attenuation. Surprisingly, the appearance and growth rate of the nearly all of the attenuated events were substantially reduced after one or two growing seasons in the field, and the extent of growth reduction was associated with barstar:barnase expression ratio. These results demonstrate the importance of field testing during early phases of research to identify pleiotropic effects of transgenic sterility genes in trees.     

Application of Arabidopsis AGAMOUS second intron for the engineered ablation of flower development in transgenic tobacco

To explore a new approach to generating reproductive sterility in transgenic plants, the barnase gene from Bacillus amyloliquefaciens was placed under the control of an 1853-bp nucleotide sequence from the 3′end of the second intron of Arabidopsis AGAMOUS and CaMV 35S (−60) minimal promoter [AG-I-35S (−60)::Barnase], and was introduced into tobacco through transformation mediated by Agrobacterium tumefaciens. All AG-I-35S (−60)::Barnase transgenic plants showed normal vegetative growth and 28% of the transgenic lines displayed complete ablation of flowering. Two transgenic lines, Bar-5 and Bar-15, were 98.1 and 98.4% sterile, respectively, as determined by seed production and germination. When controlled by AG-I-35S (−60) chimeric promoter, barnase mRNA was detected in the reproductive tissues of transgenic tobacco plants, but not in vegetative parts. This study presents the first application of an AG intron sequence in the engineered ablation of sexual reproduction in plants. The AG-I-35S (−60)::Barnase construct can be useful in diminishing pollen and seed formation in plants, providing a novel bisexual sterility strategy for interception of transgene escape and has other potentially commercial use for transgenic engineering.     

The use of a Spacer DNA fragment insulates the tissue-specific expression of a cytotoxic gene (barnase) and allows high-frequency generation of transgenic male sterile lines in Brassica juncea L.

Male-sterile lines were generated in oilseed mustard (Brassica juncea) with a cytotoxic gene (barnase) in conjunction with either of two tapetum-specific promoters, TA29 and A9. Several transformation vectors based on different promoter and marker gene combinations were developed and tested for their efficacy in generating agronomically viable male-sterile lines. Use of strong constitutive promoters (e.g. CaMV 35S or its double-enhancer variant) to express the marker gene (bar) in barnase constructs generated male-sterile plants at an extremely low frequency with most plants showing abnormalities in vegetative morphology, poor female fertility, low seed germination frequencies and/or distortion in segregation ratios of transgenes. Such abnormalities were considerably reduced on using weaker promoters (e.g. nos) to drive the marker gene (nptII) in barnase constructs and could therefore be attributed to leaky expression of the barnase gene under enhancing effects of strong constitutive promoters. We show that the use of a Spacer DNA fragment between the barnase gene (driven by a tapetum-specific promoter) and the CaMV 35S promoter-driven bar gene insulates tissue-specific expression of the barnase gene over all developmental stages of transgenic plants and significantly enhances recovery of agronomically viable male-sterile lines. All TA29-barnase male-sterile lines containing the Spacer DNA fragment exhibited normal morphology, growth and seed set on backcrossing as observed for wild-type plants. Around 75% of single-copy events tested further also showed proper segregation of the marker gene/male-sterile phenotype among backcross progeny. Constructs based on the use of Spacer DNA fragments as insulators could be successfully used to alleviate limitations associated with transformation of plant systems using cytotoxic genes for development of agronomically viable male-sterile lines in crop plants and for cell/tissue ablation studies in general.     

Tapetum-specific expression of theOsg6B promoter-β-glucuronidase gene in transgenic rice

The promoter of an anther tapetum-specific gene,Osg6B, was fused to a-glucuronidase (GUS) gene and introduced into rice byAgrobacterium-mediated gene transfer. Fluorometric and histochemical GUS assay showed that GUS was expressed exclusively within the tapetum of anthers from the uninucleate microspore stage (7 days before anthesis) to the tricellular pollen stage (3 days before anthesis). This is the first demonstration of an anther-specific promoter directing tapetum-specific expression in rice.Abbreviations GUS ßGlucuronidase     

Retrograde regulation of nuclear gene expression in CW-CMS of rice

The CW-cytoplasmic male sterility (CMS) line has the cytoplasm of Oryza rufipogon Griff, and mature pollen is morphologically normal under an optical microscope but lacks the ability to germinate; restorer gene Rf17 has been identified as restoring this ability. The difference between nuclear gene expression in mature anthers was compared for the CW-CMS line, [cms-CW] rf17rf17, and a maintainer line with normal cytoplasm of Oryza sativa L., [normal] rf17rf17. Using a 22-k rice oligoarray we detected 58 genes that were up-regulated more than threefold in the CW-CMS line. Expression in other organs was further investigated for 20 genes using RT-PCR. Five genes, including genes for alternative oxidase, were found to be preferentially expressed in [cms-CW] rf17rf17 but not in [normal] rf17rf17 or [cms-CW] Rf17Rf17. Such [cms-CW] rf17rf17-specific gene expression was only observed in mature anthers but not in leaves, stems, or roots, indicating the presence of anther-specific mitochondrial retrograde regulation of nuclear gene expression, and that Rf17 has a role in restoring the ectopic gene expression. We also used a proteomic approach to discover the retrograde regulated proteins and identified six proteins that were accumulated differently. These results reveal organ-specific induced mitochondrial retrograde pathways affecting nuclear gene expression possibly related to CMS. Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Antisense inhibition of a nuclear gene,BrDAD1, in Brassica causes male sterility that is restorable with jasmonic acid treatment

Male sterility is widely used for the production of hybrid seeds, but the use of genic male sterility is rather limited because of difficulty in maintaining homozygous male sterile plants. Recently, the DEFECTIVE IN ANTHER DEHISCENCE 1 (DAD1) gene, which encodes a phospholipase A1 involved in the first step of the jasmonic acid (JA) biosynthesis pathway, was isolated from a male sterile Arabidopsis mutant. To utilize this gene in Brassica crops, we characterized the BrDAD1 gene, the putative ortholog of DAD1 in Brassica rapa. Out of 25 plants transformed with an antisense gene constructed from the BrDAD1, 3 plants showed a defect of anther dehiscence at the flower bud opening stage and produced inviable pollen. One of the three showed male sterility only, but the other two showed a delay or a lack of flower opening in addition to male sterility. The male sterile and flower-opening phenotypes were rescued by the application of JA as well as linolenic acid. Furthermore, all these characteristics were inherited to the next generation. The present results demonstrate a novel control system for hybrid seed production by the use of nuclear genes.     

Retransformation of a male sterile barnase line with the barstar gene as an efficient alternative method to identify male sterile–restorer combinations for heterosis breeding

We report in this study, an improved method for identifying male sterile–restorer combinations using the barnase–barstar system of pollination control for heterosis breeding in crop plants, as an alternative to the conventional line × tester cross method. In this strategy, a transgenic male sterile barnase line was retransformed with appropriate barstar constructs. Double transformants carrying both the barnase and barstar genes were identified and screened for their male fertility status. Using this strategy, 66–90% of fertile retransformants (restored events) were obtained in Brassica juncea using two different barstar constructs. Restored events were analysed for their pollen viability and copy number of the barstar gene. Around 90% of the restored events showed high pollen viability and ∼30% contained single copy integrations of the barstar gene. These observations were significantly different from those made in our earlier studies using line (barnase) × tester (barstar) crosses, wherein only two viable male sterile–restorer combinations were identified by screening 88 different cross-combinations. The retransformation strategy not only generated several independent restorers for a given male sterile line from a single transformation experiment but also identified potential restorers in the T₀ generation itself leading to significant savings in time, cost and labour. Single copy restored plants with high pollen viability were selfed to segregate male sterile (barnase) and restorer (barstar) lines in the T₁ progeny which could subsequently be diversified into appropriate combiners for heterosis breeding. This strategy will be particularly useful for crop plants where poor transformation frequencies and/or lengthy transformation protocols are a major limitation.     

Characterization of a rice pollen-specific gene and its expression

Pollen development requires a large number of genes expressed in both sporophytic and gametophytic tissues. We have isolated a pollen-specific gene, PS1, from rice. PS1 is a unique gene in the rice genome and encodes a 164 amino acid long protein. RNA blot analysis shows that PS1 mRNAs accumulate specifically in rice anthers. When introduced into rice tissues by microprojectile bombardment, the PS1 promoter drives expression of a marker gene, β-glucuronidase, specifically in rice pollen. The PS1 gene and the deduced amino acid sequence of the PS1 protein share significant levels of homology with another monocot pollen-specific gene—the maize Zm-13 gene and its deduced protein, respectively. PS1 also shows some homology with the dicot tomato anther-specific gene LAT-52. Interestingly, the structure of the PS1 gene is more similar to that of the LAT-52 gene than to Zm-13. The coding regions of both PS1 and LAT-52 are interrupted by a single intron, and the positions of the introns are conserved in these genes. Moreover, there is considerable sequence homology in the introns of the PS1 and LAT-52 genes in regions immediately upstream of the 3' splice sites. The upstream regulatory sequences of the PS1 gene show several regions of homology with other pollen- or anther-specific genes from a number of plant species. The conservation of coding sequences of PS1 from rice, Zm-I3 from maize, and LAT-52 from tomato suggests a functional conservation of their gene products. Similarities in the regulatory regions of PS1 and other anther- or pollen-specific genes among monocotyledonous and dicotyledonous species indicate that at least some regulatory features controlling gene expression in male reproductive tissues are conserved. This is supported by the preservation of pollen-specific expression from the rice PS1 promoter when it is introduced into tobacco plants by Agrobacterium Ti plasmid-mediated transformation.     

Assessing hybrid sterility in <Emphasis Type="Italic">Oryza glaberrima</Emphasis> × <Emphasis Type="Italic">O. sativa</Emphasis> hybrid progenies by PCR marker analysis and crossing with wide compatibility varieties

Interspecific crossing of the African indigenous rice Oryza glaberrima with Oryza sativa cultivars is hindered by crossing barriers causing 100% spikelet sterility in F₁ hybrids. Since hybrids are partially female fertile, fertility can be restored by back crossing (BC) to a recurrent male parent. Distinct genetic models on spikelet sterility have been developed predicting, e.g., the existence of a gamete eliminator and/or a pollen killer. Linkage of sterility to the waxy starch synthase gene and the chromogen gene C, both located on chromosome 6, have been demonstrated. We selected a segregating BC₂F₃ population of semi-sterile O. glaberrima × O. sativa indica hybrid progenies for analyses with PCR markers located at the respective chromosome-6 region. These analyses revealed that semi-sterile plants were heterozygous for a marker (OSR25) located in the waxy promoter, whereas fertile progenies were homozygous for the O. glaberrima allele. Adjacent markers showed no linkage to spikelet sterility. Semi-sterility of hybrid progenies was maintained at least until the F₄ progeny generation, suggesting the existence of a pollen killer in this plant material. Monitoring of reproductive plant development showed that spikelet sterility was at least partially due to an arrest of pollen development at the microspore stage. In order to address the question whether genes responsible for F₁ sterility in intraspecific hybrids (O. sativa indica × japonica) also cause spikelet sterility in interspecific hybrids, crossings with wide compatibility varieties (WCV) were performed. WCV accessions possess "neutral" S-loci (Sⁿ) improving fertility in intraspecific hybrids. This experiment showed that the tested Sⁿ-loci had no fertility restoring effect in F₁ interspecific hybrids. Pollen development was completely arrested at the microspore stage and grains were never obtained after selfing. This suggests that distinct or additional S-loci are responsible for sterility of O. glaberrima × O. sativa hybrids.Communicated by H.C. Becker     

Anther-specific expression of mutated melon ethylene receptor gene Cm-ERS1/H70A affected tapetum degeneration and pollen grain production in transgenic tobacco plants

To develop a new system for inducible male sterility without any modification of the floral architecture in tobacco plants, a mutated ethylene receptor gene Cm-ERS1/H70A was fused either to the tobacco Nin88 promoter known to function mainly in the tapetum and microspore or to the CaMV 35S promoter known to be a constitutive promoter. The fusion genes pNin88::Cm-ERS1/H70A and p35S::Cm-ERS1/H70A were introduced in tobacco plants, which generated two independent transformants. Transformants with 35S::Cm-ERS1/H70A produced less normal pollen and had modified floral architecture while those with Nin88::Cm-ERS1/H70A produced less normal pollen without modification of floral architecture. Histological observations of anthers at stage 2 showed that tapetum degeneration in NH70A #8 and H70A #2 transformants occurred later than in wild types, strongly indicating that the expression of the mutated gene was involved in this delay. These results suggest that the tapetum-specific expression of a mutated ethylene receptor gene is a potential strategy for inducing male sterility in transgenic plants.     

Expression of sunflower cytoplasmic male sterility-associated open reading frame, orf H522 induces male sterility in transgenic tobacco plants

Sterility in the universally exploited PET1-CMS system of sunflower is associated with the expression of orfH522, a novel mitochondrial gene. Definitive evidence that ORFH522 is directly responsible for male sterility is lacking. To test the hypothesis that ORFH522 is sufficient to induce male sterility, a set of chimeric constructs were developed. The cDNA of orfH522 was cloned in-frame with yeast coxIV pre-sequence, and was expressed under tapetum-specific promoter TA29 (construct designated as TCON). For developing control vectors, orfH522 was cloned without the transit peptide under TA29 promoter (TON) or orfH522 was cloned with or without transit peptide under the constitutive CaMV35S promoter (SCOP and SOP). Among several independent transformants obtained with each of the gene cassettes, one third of the transgenics (6/17) with TCON were completely male sterile while more than 10 independent transformants obtained with each of the control vectors were fertile. The male sterile plants were morphologically similar to fertile plants, but had anthers that remained below the stigmatic surface at anthesis. RT-PCR analysis of the sterile plants confirmed the anther-specific expression of orfH522 and bright-field microscopy demonstrated ablation of the tapetal cell layer. Premature DNA fragmentation and programmed cell death was observed at meiosis stage in the anthers of sterile plants. Stable transmission of induced male sterility trait was confirmed in test cross progeny. This constitutes the first report at demonstrating the induction of male sterility by introducing orfH522 gene that could be useful for genetic engineering of male sterility. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterization and functional analysis of a pollen-specific gene st901 in Solanum tuberosum

A pollen-specific gene, sb401, which was isolated from a cDNA library of in vitro geminated pollen of the diploid potato species Solanum berthaultii, belongs to the class of genes expressed late during pollen development. Using sb401 as a probe, a pollen-specific gene st901 was isolated from the genomic library of a potato species Solanum tuberosum cv. Desiree. Sequencing and RT-PCR analysis showed that the st901 genomic gene is 2,889 bp long, contains three exons and two introns, and encodes a putative polypeptide of 217 residues. The predicted protein sequence contains four imperfect repeated motifs of V–V–E–K–K–N/E–E; the core sequence of the repeats (K–K–N/E–E) resembles a microtubule-binding domain of the microtubule-associated protein MAP1B from mouse. The examination of a promoter–reporter construct in transgenic potato plants revealed that the st901 is expressed exclusively in mature pollen grains, which is consistent with the results of Northern blot and RT-PCR. For analysis of the function of st901, transgenic plants harboring antisense copies of st901 cDNA driven by a native st901 promoter were generated. Suppression of st901 gene in potato resulted in aberrant pollen at maturation and pollen viability of transgenic plants ranged from 4.4 to 14.8%, while that of control plants were more than 90%. These results strongly suggest that st901 has an essential role in pollen development.The st901 gene sequence has been deposited in GenBank under accession number AY526087. Accession number for SB401 is X95984.1     

Fine mapping of S32(t), a new gene causing hybrid embryo sac sterility in a Chinese landrace rice (Oryza sativa L.)

Ketan Nangka, the donor of wide compatibility genes, showed sterility when crossed to Tuanguzao, a landrace rice from Yunnan province, China. Genetic and cytological analyses revealed that the semi-sterility was primarily caused by partial abortion of the embryo sac. Genome-wide analysis of the linkage map constructed from the backcross population of Tuanguzao/Ketan Nangka//Ketan Nangka identified two independent loci responsible for the hybrid sterility located on chromosomes 2 and 5, which explained 18.6 and 20.1% of phenotypic variance, respectively. The gene on chromosome 5 mapped to the previously reported sterility gene S31(t), while the gene on chromosome 2, a new hybrid sterility gene, was tentatively designated as S32(t). The BC₁F₂ was developed for further confirmation and fine mapping of S32(t). The gene S32(t) was precisely mapped to the same region as that detected in the BC₁F₁ but its position was narrowed down to an interval of about 1.9 cM between markers RM236 and RM12475. By assaying the recombinant events in the BC₁F₂, S32(t) was further narrowed down to a 64 kb region on the same PAC clone. Sequence analysis of this fragment revealed seven predicted open reading frames, four of which encoded known proteins and three encoded putative proteins. Further analyses showed that wide-compatibility variety Dular had neutral alleles at loci S31(t) and S32(t) that can overcome the sterilities caused by these two genes. These results are useful for map-based cloning of S32(t) and for marker-assisted transferring of the neutral allele in hybrid rice breeding.