Tagging genes for blast resistance in rice via linkage to RFLP markers

Summary Both Pi-2(t) and Pi-4(t) genes of rice confer complete resistance to the blast fungal pathogen Pyricularia oryzae Cav. As economically important plant genes, they have been recently characterized phenotypically, yet nothing is known about their classical linkage associations and gene products. We report here the isolation of DNA markers closely linked to these blast resistance genes in rice. The DNA markers were identified by testing 142 mapped rice genomic clones as hybridization probes against Southern blots, consisting of DNA from pairs of nearly isogenic lines (NILs) with or without the target genes. Chromosomal segments introgressed from donor genomes were distinguished by restriction fragment length polymorphisms (RFLPs) between the NILs. Linkage associations of the clones with Pi-2(t) and Pi4(t) were verified using F₃ segregating populations of known blast reaction. Cosegregation of the resistant genotype and donor-derived allele indicated the presence of linkage between the DNA marker and a blast resistance gene. RFLP analysis showed that Pi-2(t) is closely linked to a single-copy DNA clone RG64 on chromosome 6, with a distance of 2.8+1.4(SE) cMorgans. Another blast resistance gene, Pi-4(t), is 15.3+4.2(SE) cMorgans away from a DNA clone RG869 on chromosome 12. These chromosomal regions can now be examined with additional markers to define the precise locations of Pi-2(t) and Pi-4(t). Tightly linked DNA markers may facilitate early selection for blast resistance genes in breeding programs. These markers may also be useful to map new genes for resistance to blast isolates. They may ultimately lead to the cloning of those genes via chromosome walking. The gene tagging approach demonstrated in this paper may apply to other genes of interest for both monogenic and polygenic traits.     

Identification of RAPD markers linked to a major blast resistance gene in rice

Rice blast, caused byPyricularia grisea, is a major production constraint in many parts of the world. The Korean rice variety Tongil showed high levels of resistance for about six years when widely planted under highly disease-conducive conditions, before becoming susceptible. Tongil was found to carry a single dominant gene, designatedPi-10t, conferring resistance to isolate 106 of the blast pathogen from the Philippines. We report here the use of bulked segregant RAPD analysis for rapid identification of DNA markers linked toPi-10t. Pooled DNA extracts from five homozygous blast-resistant (RR) and five susceptible (rr) BC₃F₂ plants, derived from a CO39 × Tongil cross, were analyzed by RFLP using 83 polymorphic probes and by RAPD using 468 random oligomers. We identified two RAPD markers linked to thePi-10t locus: RRF6 (3.8 ± 1.2 cM) and RRH18 (2.9 ± 0.9 cM). Linkage of these markers withPi-10t was verified using an F2 population segregating forPi-10t. The two linked RAPD markers mapped 7 cM apart on chromosome 5. Chromosomal regions surrounding thePi-10t gene were examined with additional RFLP markers to define the segment introgressed from the donor genome.Pi-10t is likely to be a new blast-resistance locus, because no other known resistance gene has been mapped on chromosome 5. These tightly linked RAPD markers could facilitate early selection of thePi-10t locus in rice breeding programmes.     

Identification and fine mapping of <Emphasis Type="Italic">Pi33</Emphasis>, the rice resistance gene corresponding to the Magnaporthe grisea avirulence gene <Emphasis Type="Italic">ACE1</Emphasis>

Rice blast disease is a major constraint for rice breeding. Nevertheless, the genetic basis of resistance remains poorly understood for most rice varieties, and new resistance genes remain to be identified. We identified the resistance gene corresponding to the cloned avirulence gene ACE1 using pairs of isogenic strains of Magnaporthe grisea differing only by their ACE1 allele. This resistance gene was mapped on the short arm of rice chromosome 8 using progenies from the crosses IR64 (resistant) × Azucena (susceptible) and Azucena × Bala (resistant). The isogenic strains also permitted the detection of this resistance gene in several rice varieties, including the differential isogenic line C101LAC. Allelism tests permitted us to distinguish this gene from two other resistance genes [Pi11 and Pi-29(t)] that are present on the short arm of chromosome 8. Segregation analysis in F₂ populations was in agreement with the existence of a single dominant gene, designated as Pi33. Finally, Pi33 was finely mapped between two molecular markers of the rice genetic map that are separated by a distance of 1.6 cM. Detection of Pi33 in different semi-dwarf indica varieties indicated that this gene could originate from either one or a few varieties.Communicated by D.J. Mackill     

Molecular breeding: marker-assisted selection combined with biolistic transformation for blast and bacterial blight resistance in Indica rice (cv. CO39)

Based on blast pathogen population dynamics and lineage exclusion assays, we found that the major blast resistance genes Pi-1 and Piz-5 confer resistance against most Magnaporthe grisea lineages. Near-isogenic rice lines C101LAC and C101A51 carrying these two major genes for blast resistance in the background of a most blast-susceptible genotype were used for developing the pyramids. The closely linked RFLP marker RZ536 and NBS-LRR r10 marker for Pi-1 and a PCR-based SAP marker RG64 for Piz-5 were used to identify the genes in the parents and in marker-assisted breeding of the pyramided populations. To achieve multiple resistance against blast and blight in this cultivar, these blast-resistant pyramids were transformed with the cloned bacterial blight resistance gene Xa21 known to confer resistance to all races of Xanthomonas oryzae pv. oryzae (Xoo). Bioassays with six independent transformants showed that transgenic CO39 plants were resistant to both pathogens, M. grisea and Xoo. We report here the stacking of three major genes (Pi-1 + Piz-5 + Xa21) into rice using two different approaches of molecular breeding: marker-assisted selection (MAS) and genetic transformation.     

Molecular mapping of genes for resistance to rice blast (Pyricularia grisea Sacc.)

Two dominant genes conferring complete resistance to specific isolates of the rice blast fungus, Pyricularia grisea Sacc., were located on the molecular map of rice in this study. Pi-l(t) is a blast resistance gene derived from the cultivar LAC23. Its map location was determined using a pair of nearly isogenic lines (NILs) and a B₆F₃ segregating population from which the isoline was derived. RFLP analysis showed that Pi-l(t) is located near the end of chromosome 11, linked to RZ536 at a distance of 14.0±4.5 centiMorgans (cM). A second gene, derived from the cultivar Apura, was mapped using a rice doubled-haploid (DH) population. This gene was located on chromosome 12, flanked by RG457 and RG869, at a distance of 13.5+-4.3 cM and 17.7+-4.5 cM, respectively. The newly mapped gene on chromosome 12 may be allelic or closely linked toPi-ta. (=Pi-4(t)), a gene derived from Tetep that was previously reported to be linked to RG869 at a distance of 15.4±4.7 cM. The usefulness of markers linked to blast resistance genes will be discussed in the context of breeding for durable blast resistance.     

利用微卫星标记鉴定水稻的稻瘟病抗性

应用水稻稻瘟病抗性基因Pid(t)紧密连锁的微卫星标记RM262对含有该抗病基因的品种地谷与感病品种江南香糯和8987的杂交F2群体进行遗传分析和抗性鉴定,结果表明,RM262的PCR扩增物在抗、感品种之间的多态性较好;在2个F2群体中,RM262和抗病基因间的重组率分别为5.74%和8.17%,应用该标记的抗性纯合和杂合带型选择抗性植株,其准确率可达98%以上。此外,还就分子标记辅助育种进行了讨论。     

Molecular mapping of rice blast resistance gene <Emphasis Type="Italic">Pi-1</Emphasis>(<Emphasis Type="Italic">t</Emphasis>) in the elite <Emphasis Type="Italic">indica</Emphasis> variety Samba mahsuri

Samba mahsuri (BPT 5204) is a cultivar of the medium slender grain indica variety of Oryza sativa grown across India for its high yield and quality. However, this cultivar is susceptible to several diseases and pests including rice blast. The analysis of near isogenic lines indicated the presence of a resistance gene, Pi-1(t) in the donor cultivar C101LAC which is highly resistant to the rice blast fungus Magnaporthe grisea (M. grisea). C101LAC was crossed with susceptible indica rice cultivar (BPT 5204) to generate the mapping population. A mendelian segregation ratio of 3:1 for resistant to susceptible F₂ plants using bulk segregation analysis confirmed the presence of a major gene pi-1(t) by simple sequence repeats marker RM224 to the highly virulent blast isolate DRR 001.     

湖南桃江病圃稻瘟病菌的无毒基因及水稻抗瘟单基因联合抗性分析

【目的】鉴定湖南省桃江病圃稻瘟病菌无毒基因型,为合理搭配种植湖南省水稻抗瘟品种和抗病育种提供依据。【方法】在湖南桃江病圃采集水稻品种"丽江新团黑谷"(LTH)稻瘟菌病样,用单孢分离法分离稻瘟病菌单孢并纯化获得单孢菌株,用针刺离体法将菌株接种到以"LTH"为轮回亲本培育而成的24个含单抗瘟基因的水稻5叶期第5叶片上,对供试菌株进行无毒基因鉴定,并应用联合致病性系数和联合抗病性系数分析抗瘟基因组合间的互作。【结果】供试92个稻瘟病单孢菌株含有全部的24个无毒基因,对24个已知含单抗瘟基因的水稻材料表现出不同程度的毒力水平,含水稻抗瘟基因Pi-20对供试菌株抗菌频率最高,达54.35%;通过联合致病性系数和联合抗病性系数分析抗瘟基因组合间的互作,结果表明最佳搭配组合为Pi-20×Pi-k~s(RAC=0.28,PAC=0.23)。【结论】湖南省桃江病圃稻瘟病菌致病力较强,24个抗瘟基因多已感病化,含抗性基因Pi-20与Pi-k、Pi-k~s、Pi-3组合的水稻品种目前可在湖南省推广利用,但需研究引进新的抗瘟基因。     

Linkage analysis of a fertility restoring mutant generated from CMS rice

 DNA polymorphism between a cytoplasmic male-sterile rice line II-32A, the male-fertile maintainer counterpart II-32B, a fertile revertant (T24), as well as two commercial indica restorers, was analyzed with randomly amplified polymorphic DNA (RAPD). A very low degree of polymorphism was found between the revertant T24 and II-32A compared with that of indica rice varieties. This result, together with agronomic and genetic evidence, suggests the revertant to be a product of a nuclear mutation. An analysis of polymorphism between II-32A and the revertant T24 with 510 RAPD decamer primers identified the co-segregating markers OPB07₆₄₀ and OPB18₁₀₀₀ to be linked to a sterile allele of the restoring locus in the revertant T24, at a distance of 5.3 cM. RAPD analysis of a mapping population of Tesanai2/CB with primer OPB07 revealed linkage of OPB07₆₄₀ with RG374 (10.8 cM) and RG394 (8.8 cM) on chromosome 1. Thus the restorer gene, designated Rf ₅, was tentatively localized between RG374 and RG394 on chromosome 1 and appears to be independent of other mapped restorer genes in rice. Received: 11 November 1997 / Accepted: 17 December 1997     

Sequence variations at a complex microsatellite locus in rice and its conservation in cereals

In an attempt to study changes associated with microsatellites in rice, the DNAs of cultivated rice, including indica and japonica varieties, and wild rice genotypes were amplified by the polymerase chain reaction with primers flanking the (GATA) _n and (AC) _n repeats at a microsatellite-containing locus OS1E6 (Genebank accession number AFO16647) previously reported from a PstI rice (var. Malkolam) genomic library in pUC18. Eight alleles of varying sizes were obtained which were cloned and sequenced. Sequencing data indicated that the size variations of the different alleles were due to differences in the repeat number as well as to sequence variations in the region flanking the microsatellite motifs. In order to study the presence of this complex microsatellite-containing locus of rice in different cereals, their DNAs were amplified using primers flanking the OS1E6 locus. It was found that this locus was present in the various cereal genotypes analyzed, indicating its conservation across different cereal members. Received: 10 March 2000 / Accepted: 14 April 2000     

Identification of flanking SSR markers for a major rice gall midge resistance gene <Emphasis Type="Italic">Gm1</Emphasis> and their validation

Host-plant resistance is the preferred strategy for management of Asian rice gall midge (Orseolia oryzae), a serious pest in many rice-growing countries. The deployment of molecular markers linked to gall midge resistance genes in breeding programmes can accelerate the development of resistant cultivars. In the present study, we have tagged and mapped a dominant gall midge resistance gene, Gm1, from the Oryza sativa cv. W1263 on chromosome 9, using SSR markers. A progeny-tested F₂ mapping population derived from the cross W1263/TN1 was used for analysis. To map the gene locus, initially a subset of the F₂ mapping population consisting of 20 homozygous resistant and susceptible lines each was screened with 63 parental polymorphic SSR markers. The SSR markers RM316, RM444 and RM219, located on chromosome 9, are linked to Gm1 at genetic distances of 8.0, 4.9 and 5.9 cM, respectively, and flank the gene locus. Further, gene/marker order was also determined. The utility of the co-segregating SSR markers was tested in a backcross population derived from the cross Swarna/W1263//Swarna, and allelic profiles of these markers were analysed in a set of donor rice genotypes possessing Gm1 and in a few gall midge-susceptible, elite rice varieties.     

Fine-mapping and molecular marker development for <Emphasis Type="Italic">Pi56(t),</Emphasis> a NBS-LRR gene conferring broad-spectrum resistance to <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis> in rice

The major quantitative trait locus qBR9.1 confers broad-spectrum resistance to rice blast, and was mapped to a ~69.1 kb region on chromosome 9 that was inherited from resistant variety Sanhuangzhan No 2 (SHZ-2). Within this region, only one predicted disease resistance gene with nucleotide binding site and leucine-rich repeat (NBS-LRR) domains was found. Specific markers corresponding to this gene cosegregated with blast resistance in F₂ and F₃ populations derived from crosses of susceptible variety Texianzhan 13 (TXZ-13) to SHZ-2 and the resistant backcross line BC-10. We tentatively designate the gene as Pi56(t). Sequence analysis revealed that Pi56(t) encodes an NBS-LRR protein composed of 743 amino acids. Pi56(t) was highly induced by blast infection in resistant lines SHZ-2 and BC-10. The corresponding allele of Pi56(t) in the susceptible line TXZ-13 encodes a protein with an NBS domain but without LRR domain, and it was not induced by Magnaporthe oryzae infection. Three new cosegregating gene-specific markers, CRG4-1, CRG4-2 and CRG4-3, were developed. In addition, we evaluated polymorphism of the gene-based markers among popular varieties from national breeding programs in Asia and Africa. The presence of the CRG4-2 SHZ-2 allele cosegregated with a blast-resistant phenotype in two BC₂F₁ families of SHZ-2 crossed to recurrent parents IR64-Sub1 and Swarna-Sub1. CRG4-1 and CRG4-3 showed clear polymorphism among 19 varieties, suggesting that they can be used in marker-assisted breeding to combine Pi56(t) with other target genes in breeding lines.     

The three important traits for cooking and eating quality of rice grains are controlled by a single locus in an elite rice hybrid, Shanyou 63

The cooking and eating quality of the rice grain is one of the most serious problems in many rice-producing areas of the world. In this study, we conducted a molecular marker-based genetic analysis of three traits, amylose content (AC), gel consistency (GC) and gelatinization temperature (GT), that are the most important constituents of the cooking and eating quality of rice grains. The materials used in the analysis included F₂ seeds, an F_2:3 population, and an F₉ recombinant inbred-line population from a cross between the parents of ’Shanyou 63’, the most widely grown hybrid in rice production in China. Segregation analyses of these three generations showed that each of the three traits was controlled by a single Mendelian locus. Molecular marker-based QTL (quantitative trait locus) analyses, both by one-way analysis of variance using single marker genotypes and by whole-genome scanning with MAPMAKER/QTL, revealed a single locus that controls the expression of all three traits. This locus coincided with the Wx region on the short arm of chromosome 6, indicating that all three traits were either controlled by the Wx locus or by a genomic region tightly linked to this locus. This finding has provided clues to resolving the molecular bases of GC and GT in future studies. The results also have direct implications for the quality improvement of rice varieties. Received: 5 January 1999 / Accepted 30 January 1999     

Development of EST-derived CAPS and AFLP markers linked to a gene for resistance to ryegrass blast (Pyricularia sp.) in Italian ryegrass (Lolium multiflorum Lam.)

Ryegrass blast, also called gray leaf spot, is caused by the fungus Pyricularia sp. It is one of the most serious diseases of Italian ryegrass (Lolium multiflorum Lam.) in Japan. We analyzed segregation of resistance in an F₁ population from a cross between a resistant and a susceptible cultivar. The disease severity distribution in the F₁ population suggested that resistance was controlled by a major gene (LmPi1). Analysis of amplified fragment length polymorphisms with bulked segregant analysis identified several markers tightly linked to LmPi1. To identify other markers linked to LmPi1, we used expressed sequence tag-cleaved amplified polymorphic sequence (EST-CAPS) markers mapped in a reference population of Italian ryegrass. Of the 30 EST-CAPS markers screened, one marker, p56, flanking the LmPi1 locus was found. The restriction pattern of p56 amplification showed a unique fragment corresponding to the resistant allele at the LmPi1 locus. A linkage map constructed from the reference population showed that the LmPi1 locus was located in linkage group 5 of Italian ryegrass. Genotype results obtained from resistant and susceptible cultivars indicate that the p56 marker is useful for introduction of the LmPi1 gene into susceptible germplasm in order to develop ryegrass cultivars with enhanced resistance to ryegrass blast.     

Identification of Two Blast Resistance Genes in a Rice Variety, Digu

Blast, caused by Magnaporthe grisea is one of most serious diseases of rice worldwide. A Chinese local rice variety, Digu, with durable blast resistance, is one of the important resources for rice breeding for resistance to blast (M. grisea) in China. The objectives of the current study were to assess the identity of the resistance genes in Digu and to determine the chromosomal location by molecular marker tagging. Two susceptible varieties to blast, Lijiangxintuanheigu (LTH) and Jiangnanxiangnuo (JNXN), a number of different varieties, each containing one blast resistance gene, Pik^s, Pia, Pik, Pi‐b, Pi‐k^p, Pi‐ta², Pi‐ta, Pi‐z, Pi‐i, Pi‐k^m, Pi‐z^t, Pi‐t and Pi‐11, and the progeny populations from the crosses between Digu and each of these varieties were analysed with Chinese blast isolates. We found that the resistance of Digu to each of the two Chinese blast isolates, ZB13 and ZB15, were controlled by two single dominant genes, separately. The two genes are different from the known blast resistance genes and, therefore, designated as Pi‐d(t)1 and Pi‐d(t)2. By using bulked segregation method and molecular marker analysis in corresponding F₂ populations, Pi‐d(t)1 was located on chromosome 2 with a distance of 1.2 and 10.6 cM to restriction fragment length polymorphism (RFLP) markers G1314A and G45, respectively. And Pi‐d(t)2 was located on chromosome 6 with a distance of 3.2 and 3.4 cM to simple sequence repeat markers RM527 and RM3, respectively. We also developed a novel strategy of resistance gene analogue (RGA) assay with uneven polymerase chain reaction (PCR) to further tag the two genes and successfully identified two RGA markers, SPO01 and SPO03, which were co‐segregated toPi‐d(t)1 and Pi‐d(t)2, respectively, in their corresponding F₂ populations. These results provide essential information for further utilization of the Digu's blast resistance genes in rice disease resistance breeding and positional cloning of these genes.     

SSRs for Marker-Assisted Selection for Blast Resistance in Rice (Oryza sativa L.)

Rice blast caused by the fungus Magnaporthe oryzae is one of the most devastating diseases of rice in nearly all rice growing areas of the world including Malaysia. To develop cultivars with resistance against different races of M. oryzae, availability of molecular markers along with marker-assisted selection strategies are essential. In this study, 11 polymorphic simple sequence repeat (SSR) markers with good fit of 1:2:1 ratio for single gene model in F₂ population derived from the cross of Pongsu seribu 2 (Resistant) and Mahsuri (Susceptible) rice cultivars were analysed in 296 F₃ families derived from individual F₂ plants to investigate association with Pi gene conferring resistance to M. oryzae pathotype. Parents and progeny were grouped into two phenotypic classes based on their blast reactions. Chi-square test for the segregation of resistance and susceptibility in F₃ generation fitted a ratio of approximately 3:1. Association of SSR markers with phenotypic trait in F₃ families was identified by statistical analysis. Four SSR markers (RM413, RM5961, RM1233 and RM8225) were significantly associated with blast resistance to pathotype 7.2 of M. oryzae in rice (p ≤ 0.01). These four markers accounted for about 20% of total phenotypic variation. So, these markers were confirmed as suitable markers for use in marker-assisted selection and confirmation of blast resistance genes to develop rice cultivars with durable blast resistance in Malaysian rice breeding programmes.     

Developments of functional markers for <Emphasis Type="Italic">Fom</Emphasis>-<Emphasis Type="Italic">2</Emphasis>-mediated fusarium wilt resistance based on single nucleotide polymorphism in melon (<Emphasis Type="Italic">Cucumis melo</Emphasis> L.)

Three single nucleotide polymorphism (SNP) sites in which amino acids had changed were detected by sequence analysis within the leucine-rich repeat (LRR) region of the Fom-2 gene. Cleaved amplified polymorphic sequence (CAPS) and allele-specific PCR (AS-PCR) methods were employed to explore the SNP validation linked to fusarium wilt resistance in the F₁ and F₂ generations simultaneously. Homozygous- and heterozygous-resistant genotypes and homozygous-susceptible genotype could be clearly distinguished using the CAPS method, and three detected SNP sites were observed to be linked to fusarium wilt resistance, with a segregation ratio of 1:2:1 in the F₂ generation. In addition, heterozygous-resistant and homozygous-susceptible genotypes could be clearly distinguished in the F₁ generation using the AS-PCR method, showing a 3:1 segregation in terms of resistant and susceptible genotypes in the F₂ generation. We therefore developed SNP-based functional markers (FMs) and identified some melon germplasm resistant to fusarium wilt by FM analysis within melon species. In conclusion, the SNP-based FMs originating from the SNP site of the Fom-2 LRR region were determined to be linked to fusarium wilt resistance and showed promise in the enhancement of breeding in melon.     

Deletion of a large genomic segment in tobacco varieties that are resistant to potato virus Y (PVY)

Recessive alleles (va, va ¹ , va ² , etc) of the tobacco Va locus confer resistance to potato virus Y (PVY). To elucidate the mechanism underlying this resistance, we attempted to identify randomly amplified polymorphic (RAPD) markers that reveal polymorphism between two nearly isogenic lines (NILs) that differ in their susceptibility to PVY. Using each of 500 primers and 800 pairs of primers, we identified over 100 RAPD fragments that differed between the NILs. We applied these RAPD primers or primer combinations to an F₂ population obtained from a cross between the susceptible line BY4 and the resistant va ² -bearing NIL, F55. It was found that only 10 RAPD markers were polymorphic between resistant and susceptible plants. Unexpectedly, these markers were all linked to Va. All 10 RAPD markers were present in all 8 susceptible varieties tested. At least one RAPD marker was not detected in 8 out of 10 resistant varieties. Southern analysis revealed that the sequences of markers were not present in the genomes of resistant varieties, and the markers were found in individually distinct positions on the chromosomes of susceptible tobacco varieties. These results strongly suggest that the resistance conferred by va is due to deletions at the Va locus governing susceptibility to PVY. Received: 20 May 1999 / Accepted: 17 August 1999     

Population Structure of Magnaporthe grisea from North-western Himalayas and its Implications for Blast Resistance Breeding of Rice

Neutral and pathogenicity markers were used to analyse the population structure of Magnaporthe grisea rice isolates from the north‐western Himalayan region of India. Random amplified polymorphic DNA (RAPD)‐based DNA fingerprinting of 48 rice isolates of M. grisea with five primers (OPA‐04, OPA‐10, OPA‐13, OPJ‐06 and OPJ‐19) showed a total of 65 RAPD bands, of which 54 were polymorphic. Cluster analysis of 48 rice isolates of M. grisea on the basis of these 65 RAPD bands revealed the presence of high genotypic diversity and continuous DNA fingerprint variation in the pathogen population. No correlation was observed between RAPD patterns and virulence characteristics of the pathogen. The observed population structure contrasted with presumed clonal reproductive behaviour of the pathogen and indicated the possibility of ongoing genetic recombination in the pathogen population. Analysis of the virulence organization of five RAPD groups (RG1–RG5) using 20 rice genotypes comprising at least 15 resistance genes revealed that no combination of resistance genes would confer resistance against all RAPD fingerprint groups present in the M. grisea rice population. The possible implications of the observed population structure of M. grisea for blast resistance breeding have been discussed.     

Spread of herbicide‐resistant weedy rice (red rice,Oryza sativa L.) after 5 years of Clearfield rice cultivation in Italy

The weedy relative of cultivated rice, red rice, can invade and severely infest rice fields, as reported by rice farmers throughout the world. Because of its close genetic relationship to commercial rice, red rice has proven difficult to control. Clearfield (Cl) varieties, which are resistant to the inhibiting herbicides in the chemical group AHAS (acetohydroxyacid synthase), provide a highly efficient opportunity to control red rice infestations. In order to reduce the risk of herbicide resistance spreading from cultivated rice to red rice, stewardship guidelines are regularly released. In Italy, the cultivation of Cl cultivars started in 2006. In 2010, surveillance of the possible escape of herbicide resistance was carried out; 168 red rice plants were sampled in 16 fields from six locations containing Cl and traditional cultivars. A first subsample of 119 plants was analysed after herbicide treatment and the resistance was found in 62 plants. Of these 119 plants, 78 plants were randomly selected and analysed at the level of the AHAS gene to search for the Cl mutation determining the resistant genotype: the Cl mutation was present in all the resistant plants. Nuclear and chloroplast microsatellite markers revealed a high correlation between genetic similarity and herbicide resistance. The results clearly show that Cl herbicide‐resistant red rice plants are present in the field, having genetic relationships with the Cl variety. Finding plants homozygous for the mutation suggests that the crossing event occurred relatively recently and that these plants are in the F₂ or later generations. These observations raise the possibility that Cl red rice is already within the cultivated rice seed supply.