Expression of recessive Aprt- mutations in mouse CAK cells resulting from chromosome loss and duplication

Karyotypes of recessive mutants at the autosomal adenine phosphoribosyltransferase (Aprt) locus in a clone of the near-diploid mouse CAK cell line have been analyzed. The Aprt located on chromosome 8. One copy of chromosome 8 was morphologically abnormal in the parental clone (CAK-B3-Toyr13) from which Aprt- mutants were isolated. Among 22 mutants, there were ten in which one copy of chromosome 8 had been lost. Four of these were monosomic, and in the others duplication of the remaining homolog had occurred. These findings indicate that newly induced recessive mutations in cultured mammalian cells can be expressed as the result of loss of one chromosome carrying a wild-type allele with or without duplication of the homolog carrying the mutant allele. Loss and duplication would not be detected in cell lines lacking morphologically marked chromosomes.     

Role of the mismatch repair gene, Msh6, in suppressing genome instability and radiation-induced mutations

Mismatch repair (MMR) is critical for preserving genomic integrity. Failure of this system can accelerate somatic mutation and increase the risk of developing cancer. MSH6, in complex with MSH2, is the MMR protein that mediates DNA repair through the recognition of 1- and 2-bp mismatches. To evaluate the effects of MSH6 deficiency on genomic stability we compared the frequency of in vivo loss of heterozygosity (LOH) between MSH6-proficient and deficient, 129S2xC57BL/6 F1 hybrid mice that were heterozygous for our reporter gene Aprt. We recovered mutant cells that had functionally lost APRT protein activity and categorized the spectrum of mutations responsible for the LOH events. We also measured the mutant frequency at the X-linked gene, Hprt, as a second reporter for point mutation. In Msh6-/-Aprt+/- mice, mutation frequency at Aprt was elevated in both T cells and fibroblasts by 2.5-fold and 5.7-fold, respectively, over Msh6+/+Aprt+/- littermate controls. While a modest increase in mitotic recombination (MR) was observed in MSH6-deficient fibroblasts compared to wild type controls, point mutation was the predominant mechanism leading to APRT deficiency in both cell types. Base substitution, consisting of multiple types of transitions, accounted for all of the point mutations identified within the Aprt coding region. We also assessed the role of MSH6 in preventing mutations caused by a common environmental mutagen, ionizing radiation (IR). In Msh6-/-Aprt+/- mice, 4Gy of X-irradiation induced a significant increase in point mutations at both Aprt and Hprt in T cells, but not in fibroblasts. These findings indicate that MutS alpha reduces spontaneous and IR-induced mutation in a cell type-dependant manner.     

Spontaneous and induced chromosomal damage and mutations in Bloom Syndrome mice

Bloom Syndrome (BS) is characterized by both cancer and genomic instability, including chromosomal aberrations, sister chromosome exchanges, and mutations. Since BS heterozygotes are much more frequent than homozygotes, the issue of the sensitivity of heterozygotes to cancer is an important one. This and many other questions concerning the effects of BLM (the gene responsible for the BS) are more easily studied in mice than in humans. To gain insight into genomic instability associated with loss of function of BLM, which codes for a DNA helicase, we compared frequencies of micronuclei, somatic mutations, and loss of heterozygosity (LOH) in Blmtm3Brd homozygous, heterozygous, and wild-type mice carrying a cII transgenic reporter gene. It should be noted that the Blmtm3Brd is inserted into the endogenous locus with a partial duplication of the gene, so some function of the locus may be retained. The cII reporter gene was introduced from the Big Blue mouse by crossing them with Blmtm3Brd mice. All measurements were made on F2 mice from this cross. The reticulocytes of Blmtm3Brd homozygous mice had more micronuclei than heterozygous or wild-type mice (4.5, 2.7, and 2.5 per thousand, respectively; P < 0.01) but heterozygotes did not differ significantly from wild-type. Unlike spontaneous chromosome damage, spontaneous mutant frequencies did not differ significantly among homozygous, heterozygous, and wild-type mice (3.2 x 10(-5), 3.1 x 10(-5), and 3.1 x 10(-5), respectively; P > 0.05). Mutation measurements were also made on mice that had been treated with ethyl-nitrosourea (ENU) because Bloom Syndrome cells are sensitive to ethylating agents. The ENU-induced mutation frequency in Blmtm3Brd homozygous, heterozygous, and wild mice were 54 x 10(-5), 35 x 10(-5), and 25 x 10(-5) mutants/plaques, respectively. ENU induced more mutations in Blmtm3Brd homozygous mice than in wild-type mice (P < 0.01), but not significantly more in heterozygous mice (P = 0.06). Spontaneous LOH did not differ significantly among the genotypes, but ENU treatment induced much more LOH in Blmtm3Brd homozygous mice, as measured by means of the Dlb-1 test of Vomiero-Highton and Heddle. Hence, these Blmtm3Brd mice resemble Bloom Syndrome except that they have normal frequencies of spontaneous mutation. The fact that these mice have elevated rates of both cancer and chromosomal aberrations (as shown by more micronuclei and LOH) but normal rates of spontaneous mutation, shows the greater importance of chromosomal events than mutations in the origin of their cancers.     

Radiation-induced genetic instability in vivo depends on p53 status

In response to ionizing radiation and other agents that damage DNA, the p53 tumor suppressor protein activates multiple cellular processes including cell cycle checkpoints and programmed cell death. Although loss of p53 function is associated with radiation-induced genetic instability in cell lines, it is not clear if this relationship exists in vivo. To study the role of p53 in maintenance of genetic stability in normal tissues following irradiation, we have measured mutant frequencies at the adenine phosphoribosyltransferase (Aprt) and hypothanine-guanine phosphoribosyltransferase (Hprt) loci and examined mechanisms of loss of heterozygosity (LOH) in normal T cells of p53-deficient, Aprt heterozygous mice that were subjected to whole-body irradiation with a single dose of 4Gy X-rays. The radiation-induced mutant frequency at both the Aprt and Hprt loci was elevated in cells from mice with different p53 genotypes. The radiation-induced elevation of p53-/- mice was significantly greater than that of p53+/- or p53+/+ mice and was caused by several different kinds of mutational events at the both chromosomal and intragenic levels. Most significantly, interstitial deletion, which occurs rarely in unirradiated mice, became the most common mechanism leading to LOH in irradiated p53 null mice. These observations support the idea that absence or reduction of p53 expression enhances radiation-induced tumorigenesis by increasing genetic instability at various loci, such as those for tumor suppressor genes.     

Determination of loss of heterozygosity in frozen and paraffin embedded tumors by denaturating high-performance liquid chromatography (DHPLC)

Spontaneous renal cell carcinoma (RCC) occurs with a high frequency in Eker rats carrying a germline alteration of the tuberous sclerosis-2 (Tsc-2) tumor suppressor gene. To determine the frequency with which the wild-type allele of the Tsc-2 gene is lost in RCC and the ability of DHPLC to detect loss of heterozygosity (LOH) at this gene locus, fresh-frozen and paraffin-embedded formalin-fixed tumors from heterozygous Eker rats (Tsc-2(Ek/+)) were examined for LOH at the Tsc-2 locus. LOH was determined by quantitation of peak areas of PCR products specific for the mutant and wild-type Tsc-2 alleles. For normal DNA isolated from heterozygous animals, the allele ratio (AR) of mutant to wild-type PCR products was empirically determined to be 1.5+/-0.3 (n=30) and LOH was defined as >2 standard deviations away from this mean, i.e. any AR >2.1. Analysis of 15 spontaneous frozen RCC samples showed LOH in 10/15 samples (66%). Carcinogen-induced tumors exhibited an even higher frequency of LOH, with 6/6 paraffin-embedded, formalin-fixed tumors exhibiting LOH. 100% concordance was observed between the results obtained by DHPLC and traditional methodologies. Therefore, LOH appears to occur with a high frequency in both spontaneous and carcinogen-induced RCC in this animal model and DHPLC is a sensitive and high throughput methodology for detecting this type of genetic alteration.     

Allelic losses in mutations at the aprt locus of human lymphoblastoid cells.

We analyzed the nature of mutations at the autosomal locus coding for adenine phosphoribosyltransferase (aprt) in human cells to elucidate the process(es) governing mutagenesis at autosomal loci. A human lymphoblastoid cell line, WR10, was found to be heterozygous for mutated allele at the aprt locus, and was used for mutation analyses. By the use of a restriction fragment length polymorphism associated with the aprt locus in WR10 cells, the molecular characteristics of mutations arising spontaneously or induced by gamma-rays were investigated. Eighty-five percent (22/26) of the spontaneous mutant clones and 93% (64/69) of the gamma-ray-induced mutant clones resulted from loss of one of the two aprt alleles. Determination of the dosage of aprt genes in those mutants with allelic losses revealed that approximately half of them retained two copies of the mutated allele. These data suggest that the mutational events leading to APRT deficiency are analogous to those reported for tumor suppressor genes in malignancies.     

Analysis of Aprt deficient mutants of Friend erythroleukaemia cells

Cytogenetic analysis of mutants of the Friend Erythroleukaemia cell line deficient in adenine phosphoribosyl transferase (Aprt) was undertaken to ascertain whether non-disjunctional events were involved in the production of this mutation. All mutant clones examined were found to carry two copies of chromosome 8, the chromosome to which Aprt maps. Since the two homologues are distinguishable by silver staining, it was also clear that no mutants contained two copies of one homologue having lost the other. Treatment of mutants with 5-azacytidine failed to reactivate the Aprt locus.     

Mutagenic radioadaptation in a human lymphoblastoid cell line

We investigated the mutagenic radioadaptive response of human lymphoblastoid TK6 cells by pretreating them with a low dose (5 cGy) of X-rays followed by a high (2 Gy) dose 6h later. Pretreatment reduced the 2-Gy-induced mutation frequency (MF) of the thymidine kinase (TK) gene (18.3 x 10(-6)) to 62% of the original level (11.4 x 10(-6)). A loss of heterozygosity (LOH) detection analysis applied to the isolated TK(-) mutants revealed the mutational events as non-LOH (resulting mostly from a point mutation in the TK gene), hemizygous LOH (resulting from a chromosomal deletion), or homozygous LOH (resulting from homologous recombination (HR) between chromosomes). For non-LOH events, pretreatment decreased the frequency to 27% of the original level (from 7.1 x 10(-6) to 1.9 x 10(-6)). cDNAs prepared from the non-LOH mutants revealed that the decrease was due mainly to the repression of base substitutions. The frequency of hemizygous LOH events, however, was not significantly altered by pretreatment. Mapping analysis of chromosome 17 demonstrated that the distribution and the extent of hemizygous LOH events were also not significantly influenced by pretreatment. For homozygous LOH events, pretreatment reduced the frequency to 61% of the original level (from 5.1 x 10(-6) to 3.1 x 10(-6)), reflecting an enhancement in HR repair of DNA double-strand breaks. Our findings suggest that the radioadaptive response in TK6 cells follows mainly from mutations at the base-sequence level, not the chromosome level.     

Induction of mutations by bismuth-212 alpha particles at two genetic loci in human B-lymphoblasts.

The human lymphoblast cell line TK6 was exposed to the alpha-particle-emitting radon daughter 212Bi by adding DTPA-chelated 212Bi directly to the cell suspension. Cytotoxicity and mutagenicity at two genetic loci were measured, and the molecular nature of mutant clones was studied by Southern blot analysis. Induced mutant fractions were 2.5 x 10(-5)/Gy at the hprt locus and 3.75 x 10(-5)/Gy at the tk locus. Molecular analysis of HPRT- mutant DNAs showed a high frequency (69%) of clones with partial or full deletions of the hprt gene among radiation-induced mutants compared with spontaneous mutants (31%). Chi-squared analyses of mutational spectra show a significant difference (P < or = 0.005) between spontaneous mutants and alpha-particle-induced mutants. Comparison with published studies of accelerator-produced heavy-ion exposures of TK6 cells indicates that the induction of mutations at the hprt locus, and perhaps a subset of mutations at the tk locus, is a simple linear function of particle fluence regardless of the ion species or its LET.     

Minisatellites show rare and simple intra-allelic instability in the mouse germ line

Minisatellites provide very informative systems for analyzing processes of tandem repeat DNA turnover in humans. The mouse genome also contains authentic minisatellites, but none has yet been found to show high levels of instability. Indirect evidence using minisatellite variant repeat mapping by PCR in Mus musculus subspecies suggested that mouse minisatellites mutate at a rate below 10(-3) per gamete and mainly by intra-allelic events. This is in sharp contrast to the complex interallelic mutations observed at high frequency at some human loci. To define more directly the turnover mechanisms and rates of instability at one of the most variable mouse minisatellites (MMS80), we used size-enrichment small-pool PCR (SESP-PCR) to recover de novo mutant alleles from sperm DNA from homozygous BALB/cJ mice and from strain DHA heterozygotes. The sperm mutation rate at MMS80 was extremely low, at or below 5 x 10(-6) per sperm. Comparison of progenitor and mutant allele structures showed that these rare mutants had arisen by simple and primarily, if not exclusively, intra-allelic mutation events. These results suggest a fundamental difference in turnover mechanisms at minisatellites between mice and human.     

Spontaneous loss of heterozygosity in diploid Saccharomyces cerevisiae cells

To obtain a broad perspective of the events leading to spontaneous loss of heterozygosity (LOH), we have characterized the genetic alterations that functionally inactivated the URA3 marker hemizygously or heterozygously situated either on chromosome III or chromosome V in diploid Saccharomyces cerevisiae cells. Analysis of chromosome structure in a large number of LOH clones by pulsed-field gel electrophoresis and PCR showed that chromosome loss, allelic recombination, and chromosome aberration were the major classes of genetic alterations leading to LOH. The frequencies of chromosome loss and chromosome aberration were significantly affected when the marker was located in different chromosomes, suggesting that chromosome-specific elements may affect the processes that led to these alterations. Aberrant-sized chromosomes were detected readily in approximately 8% of LOH events when the URA3 marker was placed in chromosome III. Molecular mechanisms underlying the chromosome aberrations were further investigated by studying the fate of two other genetic markers on chromosome III. Chromosome aberration caused by intrachromosomal rearrangements was predominantly due to a deletion between the MAT and HMR loci that occurred at a frequency of 3.1 x 10(-6). Another type of chromosome aberration, which occurred at a frequency slightly higher than that of the intrachromosomal deletion, appeared to be caused by interchromosomal rearrangement, including unequal crossing over between homologous chromatids and translocation with another chromosome.     

Further characterization of loss of heterozygosity enhanced by p53 abrogation in human lymphoblastoid TK6 cells: disappearance of endpoint hotspots

Loss of heterozygosity (LOH) is the predominant mechanism of spontaneous mutagenesis at the heterozygous thymindine kinase locus (tk) in TK6 cells. LOH events detected in spontaneous TK(-) mutants (110 clones from p53 wild-type cells TK6-20C and 117 clones from p53-abrogated cells TK6-E6) were analyzed using 13 microsatellite markers spanning the whole of chromosome 17. Our analysis indicated an approximately 60-fold higher frequency of terminal deletions in p53-abrogated cells TK6-E6 compared to p53 wild-type cells TK6-20C whereas frequencies of point mutations (non-LOH events), interstitial deletions, and crossing over events were found to increase only less than twofold by such p53 abrogation. We then made use of an additional 17 microsatellite markers which provided an average map-interval of 1.6Mb to map various LOH endpoints on the 45Mb portion of chromosome 17q corresponding to the maximum length of LOH tracts (i.e. from the distal marker D17S932 to the terminal end). There appeared to be four prominent peaks (I-IV) in the distribution of LOH endpoints/Mb of Tk6-20C cells that were not evident in p53-abrogated cells TK6-E6, where they appeared to be rather broadly distributed along the 15-20Mb length (D17S1807 to D17S1607) surrounding two of the peaks that we detected in TK6-20C cells (peaks II and III). We suggest that the chromosomal instability that is so evident in TK6-E6 cells may be due to DNA double-strand break repair occurring through non homologous end-joining rather than allelic recombination.     

Intervention of somatic mutational events in vivo by a germline defect at the adenine phosphoribosyltransferase locus

Both germline and somatic mutations are known to affect phenotypes of human cells in vivo. In previous studies, we cloned mutant peripheral blood T cells from germline heterozygous humans for adenine phosphoribosyltransferase (APRT) (EC 2.4.2.7) deficiency and found that approximately 1.3 × 10^–4 peripheral T cells had undergone in vivo somatic mutations. Loss of heterozygosity (LOH) was the major cause of the mutations at the APRT locus since approximately 80% of the mutant T cell clones exhibited loss of normal alleles. In the present study, we identified three heterozygous individuals for APRT deficiency (representing two separate families), in whom none of the somatic mutant cells exhibited LOH at the APRT locus. The germline mutant APRT alleles of these heterozygotes from two unrelated families had the same gross DNA abnormalities detectable by Southern blotting. None of the germline mutant APRT alleles so far reported had such gross DNA abnormalities. The data suggest that the germline mutation unique to these heterozygous individuals is associated with the abrogation of LOH in somatic cells. The absence of LOH at a different locus has already been reported in vitro in an established cell line but the present study describes the first such event in vivo in human individuals. Received: 10 May 1996     

High frequency induction of mitotic recombination by ionizing radiation in Mlh1 null mouse cells

Mitotic recombination in somatic cells involves crossover events between homologous autosomal chromosomes. This process can convert a cell with a heterozygous deficiency to one with a homozygous deficiency if a mutant allele is present on one of the two homologous autosomes. Thus mitotic recombination often represents the second mutational step in tumor suppressor gene inactivation. In this study we examined the frequency and spectrum of ionizing radiation (IR)-induced autosomal mutations affecting Aprt expression in a mouse kidney cell line null for the Mlh1 mismatch repair (MMR) gene. The mutant frequency results demonstrated high frequency induction of mutations by IR exposure and the spectral analysis revealed that most of this response was due to the induction of mitotic recombinational events. High frequency induction of mitotic recombination was not observed in a DNA repair-proficient cell line or in a cell line with an MMR-independent mutator phenotype. These results demonstrate that IR exposure can initiate a process leading to mitotic recombinational events and that MMR function suppresses these events from occurring.     

Further characterization of loss of heterozygosity enhanced by p53 abrogation in human lymphoblastoid TK6 cells: disappearance of endpoint hotspots

Loss of heterozygosity (LOH) is the predominant mechanism of spontaneous mutagenesis at the heterozygous thymindine kinase locus (tk) in TK6 cells. LOH events detected in spontaneous TK⁻ mutants (110 clones from p53 wild-type cells TK6-20C and 117 clones from p53-abrogated cells TK6-E6) were analyzed using 13 microsatellite markers spanning the whole of chromosome 17. Our analysis indicated an approximately 60-fold higher frequency of terminal deletions in p53-abrogated cells TK6-E6 compared to p53 wild-type cells TK6-20C whereas frequencies of point mutations (non-LOH events), interstitial deletions, and crossing over events were found to increase only less than twofold by such p53 abrogation. We then made use of an additional 17 microsatellite markers which provided an average map-interval of 1.6 Mb to map various LOH endpoints on the 45 Mb portion of chromosome 17q corresponding to the maximum length of LOH tracts (i.e. from the distal marker D17S932 to the terminal end). There appeared to be four prominent peaks (I–IV) in the distribution of LOH endpoints/Mb of Tk6-20C cells that were not evident in p53-abrogated cells TK6-E6, where they appeared to be rather broadly distributed along the 15–20 Mb length (D17S1807 to D17S1607) surrounding two of the peaks that we detected in TK6-20C cells (peaks II and III). We suggest that the chromosomal instability that is so evident in TK6-E6 cells may be due to DNA double-strand break repair occurring through non homologous end-joining rather than allelic recombination.     

Age-related accumulation of autosomal mutations in solid tissues of the mouse is gender and cell type specific

Most cancers in solid tissues increase with age and invariably contain causal mutations eliminating expression of one or more autosomal tumor suppressor genes. However, very little is known about the effect of age on autosomal mutations, often large in size, in cells of solid tissues. In this study, the frequency and spectrum of autosomal mutations were examined as a function of age for kidney epithelial cells and ear mesenchymal cells in B6D2F1 mice heterozygous for the selectable Aprt locus. Aprt mutant frequencies were found to increase with age in the kidneys of both male and female mice, but at all ages the mutant frequencies were approximately twice as high in the females, which in this strain have shorter lifespans than the males. An age-related increase in Aprt mutant frequencies was also observed for ear cells from female mice, but no significant increases in mutant frequencies were observed for the ear cells of male mice. A molecular analysis showed that the kidney and ear mutational spectra were distinct and that the age-related increases in mutant frequencies did not involve significant shifts in the mutational spectra. In total, the results demonstrate both gender and cell-type-specific patterns of autosomal mutational accumulation as a function of age in two solid tissues of the mouse.     

Tk+/- mouse model for detecting in vivo mutation in an endogenous, autosomal gene

Tk+/- transgenic mice were created using an embryonic stem cell line in which one allele of the endogenous thymidine kinase (Tk) gene was inactivated by targeted homologous recombination. Breeding Tk+/- parents produced viable Tk-/- knockout (KO) mice. Splenic lymphocytes from KO mice were used in reconstruction experiments for determining the conditions necessary for recovering Tk somatic cell mutants from Tk+/- mice. The cloning efficiency of KO lymphocytes was not affected by the toxic thymidine analogues 5-bromo-2'-deoxyuridine (BrdUrd) or trifluorothymidine (TFT), or by BrdUrd in the presence of lymphocytes from Tk+/- animals; however, it was easier to identify clones resistant to BrdUrd than to TFT when Tk+/- cells were present. Tk+/- mice were treated with vehicle or 100 mg/kg of N-ethyl-N-nitrosourea (ENU), and after 4 months, the frequency of Tk mutant lymphocytes was measured by resistance to BrdUrd. The frequency of Tk mutants was 22+/-5.9x10-6 in control animals and 80+/-31x10-6 in treated mice. In comparison, the frequency of Hprt mutant lymphocytes, as measured by resistance to 6-thioguanine, was 2.0+/-1.2x10-6 in control animals and 84+/-28x10-6 in the ENU-treated mice. Analysis of BrdUrd-resistant lymphocyte clones derived from the ENU-treated animals revealed point mutations in the non-targeted Tk allele. These results indicate that the selection of BrdUrd-resistant lymphocytes from Tk+/- mice may be used for assessing in vivo mutation in an endogenous, autosomal gene.     

Transposon mutagenesis of Trypanosoma brucei identifies glycosylation mutants resistant to concanavalin A

We have engineered Trypanosoma brucei with a novel mariner transposition system that allows large populations of mutant cells to be generated and screened. As a proof of principle, we isolated and characterized two independent clones that were resistant to the cytotoxic action of concanavalin A. In both clones, the transposon had integrated into the locus encoding a homologue of human ALG12, which encodes a dolichyl-P-Man: Man(7)GlcNAc(2)-PP-dolichyl-alpha6-mannosyltransferase. Conventional knock-out of ALG12 in a wild-type background gave an identical phenotype to the mariner mutants, and biochemical analysis confirmed that they have the same defect in the N-linked oligosaccharide synthesis pathway. To our surprise, both mariner mutants were homozygous; the second allele appeared to have undergone gene conversion by the mariner-targeted allele. Subsequent experiments showed that the frequency of gene conversion at the ALG12 locus, in the absence of selection, was 0.25%. As we approach the completion of the trypanosome genome project, transposon mutagenesis provides an important addition to the repertoire of genetic tools for T. brucei.     

Development of a STS marker assay for detecting loss of heterozygosity in rice hybrids.

The RAPD (random amplified polymorphic DNA) markers OPE15750 and OPE15300 were affected by loss of heterozygosity (LOH) in rice hybrids AMR x 'M202' and AMR x 'L202'. The markers were mapped to the same locus at or near the centromere of rice chromosome 2. The two RAPD products were cloned, sequenced, and found to have lengths of 734 bp and 297 bp, respectively. The 297-bp sequence shares a 98% homology with one end of the 734-bp sequence, accounting for the cross-hybridization previously observed between the two clones. Based on the DNA sequence of the 734-bp fragment, a pair of STS (sequence-tagged site) primers was designed and tested. Rice plants homozygous for either OPE15734 or OPE15297 all produced PCR fragments of the same length, 482 bp. However, the two PCR products were discernible by digestion with the restriction enzyme XbaI prior to gel electrophoresis. The STS product from plants homozygous for OPE15734 was cut into two fragments of 239 and 240 bp, which appeared as one single band in an agarose gel; whereas the STS product from plants homozygous for OPE15297 was not cut by XbaI and was characterized by a 482-bp band in the agarose gel. These STS primers were tested in rice hybrids and F2 progenies derived from the hybrids of AMR x 'M202' and AMR x 'L202'. Homozygosity for OPE15297 was confirmed for all F2 panicle rows derived from AMR x 'M202'. In contrast, F2 panicle rows of AMR x 'L202' exhibited two different segregation patterns (genotypes), i.e., either uniformly homozygous for the 240-bp marker (OPE15734/OPE15734) or segregating for the 482- and 240-bp markers (OPE15734/OPE15297). This STS-marker system provides a robust assay for detecting the occurrence of LOH in rice hybrid progenies.