Genetic mapping in sugarcane, a high polyploid, using bi-parental progeny: identification of a gene controlling stalk colour and a new rust resistance gene

Modern sugarcane cultivars (Saccharum spp) are highly polyploïd and aneuploid interspecific hybrids (2n=100–130). Two genetic maps were constructed using a population of 198 progeny from a cross between R570, a modern cultivar, and MQ76-53, an old Australian clone derived from a cross between Trojan (a modern cultivar) and SES528 (a wild Saccharum spontaneum clone). A total of 1,666 polymorphic markers were produced using 37 AFLP primer combinations, 46 SSRs and 9 RFLP probes. Linkage analysis led to the construction of 86 cosegregation groups for R570 and 105 cosegregation groups for MQ76-53 encompassing 424 and 536 single dose markers, respectively. The cumulative length of the R570 map was 3,144 cM, while that of the MQ76-53 map was 4,329 cM. Here, we integrated mapping information obtained on R570 in this study with that derived from a previous map based on a selfed R570 population. Two new genes controlling Mendelian traits were localized on the MQ76-53 map: a gene controlling the red stalk colour was linked at 6.5 cM to an AFLP marker and a new brown rust resistance gene was linked at 23 cM to an AFLP marker. Besides another previously identified brown rust resistance gene (Bru1), these two genes are the only other major genes to be identified in sugarcane so far.     

Haplotype structure around Bru1 reveals a narrow genetic basis for brown rust resistance in modern sugarcane cultivars

Modern sugarcane cultivars (Saccharum spp., 2n?=?100-130) are high polyploid, aneuploid and of interspecific origin. A major gene (Bru1) conferring resistance to brown rust, caused by the fungus Puccinia melanocephala, has been identified in cultivar R570. We analyzed 380 modern cultivars and breeding materials covering the worldwide diversity with 22 molecular markers genetically linked to Bru1 in R570 within a 8.2?cM segment. Our results revealed a strong LD in the Bru1 region and strong associations between most of the markers and rust resistance. Two PCR markers, that flank the Bru1-bearing segment, were found completely associated with one another and only in resistant clones representing efficient molecular diagnostic for Bru1. On this basis, Bru1 was inferred in 86?% of the 194 resistant sugarcane accessions, revealing that it constitutes the main source of brown rust resistance in modern cultivars. Bru1 PCR diagnostic markers should be particularly useful to identify cultivars with potentially alternative sources of resistance to diversify the basis of brown rust resistance in breeding programs.     

Development of leucine-rich repeat polymorphism, amplified fragment length polymorphism, and sequence characterized amplified region markers to the Cronartium ribicola resistance gene Cr2 in western white pine ( Pinus monticola )

White pine blister rust (WPBR), caused by Cronartium ribicola, is a devastating disease in Pinus monticola and other five-needle pines. Pyramiding a major resistance gene (Cr2) with other resistance genes is an important component of integrated strategies to control WPBR in P. monticola. To facilitate this strategy, the objective of the present study was to identify leucine-rich repeat (LRR) polymorphisms, amplified fragment length polymorphisms (AFLPs), and sequence characterized amplified region (SCAR) markers linked to the western white pine Cr2 (BSA) gene for precise gene mapping. Bulked segregant analysis and haploid segregation analysis allowed the identification of 11 LRR polymorphisms and five AFLP markers in the Cr2 linkage. The closest LRR markers were 0.53 Kosambi cM from Cr2 at either end. After marker cloning and sequencing, AFLP marker EacccMccgat-365 and random polymorphic DNA marker U570–843 were converted successfully into SCAR markers. For a potential application in marker-assisted selection (MAS), these two SCAR markers were verified in two western white pine families. This study represents the first report of LRR-related DNA markers linked to C. ribicola resistance in five-needle pines. These findings may help further candidate gene identification for disease resistance in a conifer species.     

Characterization and fine mapping of <Emphasis Type="Italic">RppQ</Emphasis>, a resistance gene to southern corn rust in maize

Southern corn rust (SCR) is a fungal disease caused by Puccinia polysora Underw, which can infect maize and may result in substantial yield losses in maize production. The maize inbred line Qi319 carries the SCR resistance gene RppQ. In order to identify molecular markers linked to the RppQ gene, several techniques were utilized including random amplified polymorphic DNA (RAPD), simple sequence repeat (SSR), and amplified fragment length polymorphism (AFLP). In addition, sequence characterized amplified region (SCAR) techniques combined with bulked segregant analysis (BSA) were used. Seven RAPD markers, eight SSR markers, and sixty-three AFLP primer combinations amplified polymorphisms between two parents and two bulk populations. A large F₂ population was used for genetic analysis and for fine mapping of the RppQ gene region. One AFLP polymorphic band, M-CAA/E-AGC₃₂₄, was converted to a SCAR marker, MA7, which was mapped to a position 0.46 cM from RppQ. Finally, the RppQ gene was mapped between the SCAR marker MA7 and the AFLP marker M-CCG/E-AGA₁₅₇ with distances of 0.46 and 1.71 cM, respectively.     

Application of synteny across Poaceae to determine the map location of a sugarcane rust resistance gene

A major rust resistance gene has been identified in a self-progeny of the sugarcane cultivar R570. Until now, this gene was known to be linked to a marker revealed by the sugarcane probe CDSR29 but unassigned to any linkage group of the current genetic map. We used synteny relationships between sugarcane and three other grasses in an attempt to saturate the region around this rust resistance gene. Comparison of sugarcane, sorghum, maize and rice genetic maps led to the identification of homoeologous chromosome segments at the extremity of sorghum linkage group D, rice linkage group 2, maize linkage group 4 and in the centromeric region of maize linkage group 5. One hundred and eighty-four heterologous probes were selected and tested for cross-hybridization with sugarcane DNA; 106 produced a good hybridization signal and were hybridized on 88 individuals of the R570 selfed progeny. Two hundred and seventeen single-dose markers were added to the R570 genetic map, of which 66% mapped to linkage group VII, together with the rust resistance gene. This gene has now been mapped to the end of a co-segregating group consisting of 19 RFLP markers. None of the mapped loci were located closer to the gene than CDSR29. The gene thus appears to reside at the edge of a ’’synteny cluster’’ used to describe the different grass genomes. Received: 12 January 2000 / Accepted: 21 March 2000     

A high-resolution map of the H1 locus harbouring resistance to the potato cyst nematode Globodera rostochiensis

The resistance gene H1 confers resistance to the potato cyst nematode Globodera rostochiensis and is located at the distal end of the long arm of chromosome V of potato. For marker enrichment of the H1 locus, a bulked segregant analysis (BSA) was carried out using 704 AFLP primer combinations. A second source of markers tightly linked to H1 is the ultra-high-density (UHD) genetic map of the potato cross SH × RH. This map has been produced with 387 AFLP primer combinations and consists of 10,365 AFLP markers in 1,118 bins (). Comparing these two methods revealed that BSA resulted in one marker/cM and the UHD map in four markers/cM in the H1 interval. Subsequently, a high-resolution genetic map of the H1 locus has been developed using a segregating F₁ SH × RH population consisting of 1,209 genotypes. Two PCR-based markers were designed at either side of the H1 gene to screen the 1,209 genotypes for recombination events. In the high-resolution genetic map, two of the four co-segregating AFLP markers could be separated from the H1 gene. Marker EM1 is located at a distance of 0.2 cM, and marker EM14 is located at a distance of 0.8 cM. The other two co-segregating markers CM1 (in coupling) and EM15 (in repulsion) could not be separated from the H1 gene.Communicated by J.G. Wenzel     

SSR and SCAR mapping of a multiple-allele male-sterile gene in Chinese cabbage (Brassica rapa L.)

The genic multiple-allele inherited male-sterile gene Ms in Chinese cabbage (Brassica rapa L.) was identified as a spontaneous mutation. Applying this gene to hybrid seed production, several B. rapa cultivars have been successfully bred in China. A BC₁ population (244 plants) was constructed for mapping the Ms gene. Screening 268 simple sequence repeat (SSR) markers which cover the entire genome of Chinese cabbage was performed with bulked segregant analysis (BSA). On the basis of linkage analysis, the Ms gene was located on linkage group R07. In addition, through the amplified fragment length polymorphism (AFLP) and the sequence-characterized amplified region (SCAR) techniques combining BSA, two SCAR markers which were converted from corresponding AFLP markers flanked the Ms gene. Finally, a genetic map of the Ms gene was constructed covering a total interval of 9.0 cM. Two SCAR markers, syau_scr01 and syau_scr04, flanked the Ms gene at distances of 0.8 and 2.5 cM, respectively. All the SSR markers (cnu_m273, cnu_m030, cnu_m295, and syau_m13) were mapped on the same side of the gene as syau_scr04, the nearest one of which, syau_m13, was mapped at a distance of 3.3 cM. These SSR and SCAR markers may be useful in marker-assisted selection and map-based cloning. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Identification of a major quantitative trait locus (QTL) for yellow spot (<Emphasis Type="Italic">Mycovellosiella koepkei</Emphasis>) disease resistance in sugarcane

In this study we used amplified fragment length polymorphism (AFLP) and microsatellite (short sequence repeat or SSR) markers to identify a major quantitative trail locus (QTL) for yellow spot (Mycovellosiella koepkei) disease resistance in sugarcane. A bi-parental cross between a resistant variety, M 134/75, and a susceptible parent, R 570, generated a segregating population of 227 individuals. These clones were evaluated for yellow spot infection in replicated field trials in two locations across two consecutive years. A χ²-test (χ² at 98% confidence level) of the observed segregation pattern for yellow spot infection indicated a putative monogenic dominant inheritance for the trait with a 3 (resistant):1(susceptible) ratio. The AFLP and SSR markers identified 666 polymorphisms as being present in the resistant parent and absent in the susceptible one. A genetic map of M 134/75 was constructed using 557 single-dose polymorphisms, resulting in 95 linkage groups containing at least two markers based on linkages in coupling. QTL analysis using QTLCartographer v1.17d and MAPMAKER/QTL v1.1 identified a single major QTL located on LG87, flanked by an AFLP marker, actctc10, and an SSR marker, CIR12284. This major QTL, which was found to be linked at 14 cM to an AFLP marker, was detected with LOD 8.7, had an additive effect of −10.05% and explained 23.8% of the phenotypic variation of yellow spot resistance.     

Development and validation of CAPS and AFLP markers for white rust resistance gene in<Emphasis Type="Italic"> Brassica juncea</Emphasis>

White rust, caused by Albugo candida, is a very serious disease in crucifers. In Indian mustard (Brassica juncea), it can cause a yield loss to the extent of 89.9%. The locus Ac2(t) controlling resistance to white rust in BEC-144, an exotic accession of mustard, was mapped using RAPD markers. In the present study, we developed: (1) a more tightly linked marker for the white rust resistance gene, using AFLP in conjunction with bulk segregant analysis, and (2) a PCR-based cleaved amplified polymorphic sequence (CAPS) marker for the closely linked RAPD marker, OPB06₁₀₀₀. The data obtained on 94 RILs revealed that the CAPS marker for OPB06₁₀₀₀ and the AFLP marker E-ACC/M-CAA₃₅₀ flank the Ac2(t) gene at 3.8 cM and 6.7 cM, respectively. Validation of the CAPS marker in two different F₂ populations of crosses Varuna × BEC-144 and Varuna × BEC-286 was also undertaken, which established its utility in marker-assisted selection (MAS) for white rust resistance. The use of both flanking markers in MAS would allow only 0.25% misclassification and thus provide greater efficiency to selection.Communicated by C. Möllers     

A putative major gene for rust resistance linked with a RFLP marker in sugarcane cultivar ‘R570’

Inheritance of resistance to rust was investigated in the self progeny of the sugarcane cultivar R570 also used to build a RFLP genetic map. Resistance was evaluated through both field and controlled greenhouse trials. A clear-cut 3 (resistant) 1 (susceptible) segregation indicative of a probable dominant resistant gene was observed. This is the first documented report of a monogenic inheritance for disease resistance in sugarcane. This gene was found linked at 10 cM with an RFLP marker revealed by probe CDSR29. Other minor factors involved in the resistance were also detected.     

Validation and identification of molecular markers linked to the leaf rust resistance gene <Emphasis Type="Italic">Lr</Emphasis>28 in wheat

Leaf rust resistance gene Lr28 controls one of the important resistances in the Indian subcontinent against the most prevalent Puccinia triticina pathotype 77-5. Pyramiding Lr28 with other resistance genes would therefore, provide durable resistance against rust, a process that can be facilitated by DNA markers. A microsatellite marker wmc313 linked to Lr28 at a distance of 5.0 cM was identified in the population HD2329 × HW2037. The marker was validated in another population developed from WL711× CS + Lr28: 2D/2M 3/8 (acc. 2956) as well as in a few near- isogenic lines (NILs) carrying gene Lr28. Compared to the previously reported marker TPSCAR SCS421₅₇₀, wmc313 is more closely linked to Lr28. Both these markers flanking the gene should be useful in the deployment of Lr28 into the breeding program using marker-assisted selection allowing pyramiding with other effective genes to confer durable resistance.     

An anchored linkage map for sugar beet based on AFLP,SNP and RAPD markers and QTL mapping of a new source of resistance to <Emphasis Type="Italic">Beet necrotic yellow vein virus</Emphasis>

Rhizomania, caused by Beet necrotic yellow vein virus (BNYVV), is an important sugar-beet disease worldwide and can result in severe losses of root yield and sugar content. We have identified a major QTL for BNYVV resistance from a new source in a segregating population of 158 individuals. The QTL explained an estimated 78% of the observed phenotypic variation and the gene conferring the partial resistance is referred to as Rz4. AFLP was used in combination with bulked segregant analysis (BSA) to develop markers linked to the resistance phenotype. AFLP marker analysis was extended to produce a linkage map that was resolved into nine linkage groups. These were anchored to the nine sugar-beet chromosomes using previously published SNP markers. This represents the first anchored sugar-beet linkage map to be published with non-anonymous markers. The final linkage map comprised 233 markers covering 497.2 cM, with an average interval between markers of 2.1 cM. The Rz4 QTL and an Rz1 RAPD marker were mapped to chromosome III, the known location of the previously identified BNYVV resistance genes Rz1, Rz2 and Rz3. The availability to breeders of new resistance sources such as Rz4 increases the potential for breeding durable disease resistance.     

AFLP markers linked to resistance against Striga gesnerioides race 1 in cowpea (Vigna unguiculata).

Amplified fragment length polymorphism (AFLP) analysis was used in combination with bulked segregant analysis (BSA) to identify molecular markers linked to two cowpea (Vigna unguiculata (L.) Walp.) genes conferring resistance to Striga gesnerioides race 1. After AFLP analysis of an F2 population derived from a cross between the resistant cultivar Gorom and the susceptible cultivar Tvx 3236, seven AFLP markers were identified that are linked to Rsg3, the gene conferring race I resistance in 'Gorom'. The distances between these markers and Rsg3 ranged from 9.9 to 2.5 cM, with two markers, E-AGA/M-CTA460 and E-AGA/M-CAG300, flanking Rsg3 at 2.5 and 2.6 cM, respectively. Analysis of a second F2 population derived from the cross between 'Tvx 3236' and the resistant cultivar IT81D-994 identified five AFLP markers linked to the race 1 resistance gene 994-Rsg present in 'IT81D-994'. The two markers showing the tightest linkage to the 994-Rsg locus were E-AAG/M-AAC450 and E-AAG/M-AAC150 at 2.1 and 2.0 cM, respectively. Two of the markers linked to 994-Rsg, E-AGA/M-CAG300 and E-AGA/M-CAG450, were also linked to Rsg3. The identification of molecular markers in common between the two sources of race 1 resistance suggests that either Striga resistance genes are clustered in these plants or that these loci are allelic. Mapping of the resistance loci within the cowpea genome revealed that three markers linked to Rsg3 and (or) 994-Rsg are located on linkage group 6.     

Genomic distribution and characterization of EST-derived resistance gene analogs (RGAs) in sugarcane

A large sugarcane EST (expressed sequence tag) project recently gave us access to 261,609 EST sequences from sugarcane, assembled into 81,223 clusters. Among these, we identified 88 resistance gene analogs (RGAs) based on their homology to typical pathogen resistance genes, using a stringent BLAST search with a threshold e-value of e(-50). They included representatives of the three major groups of resistance genes with NBS/LRR, LRR or S/T KINASE domains. Fifty RGAs showed a total of 148 single-dose polymorphic RFLP markers, which could be located on the sugarcane reference genetic map (constructed in cultivar R570, 2n=approximately 115). Fifty-five SSR loci corresponding to 134 markers in R570 were also mapped to enable the classification of the various haplotypes into homology groups. Several RGA clusters were found. One cluster of two LRR-like loci mapped close to the only disease resistance gene known so far in sugarcane, which confers resistance to common rust. Detailed sequence comparison between two NBS/LRR RGA clusters in relation to their orthologs in rice and maize suggests their polyphyletic origins, and indicates that the degree of divergence between paralogous RGAs in sugarcane can be larger than that from an ortholog in a distant species.     

Identification of quantitative trait loci contributing to Fusarium wilt resistance on an AFLP linkage map of flax (Linum usitatissimum)

 An AFLP genetic linkage map of flax (Linum usitatissimum) was used to identify two quantitative trait loci (QTLs) on independent linkage groups with a major effect on resistance to Fusarium wilt, a serious disease caused by the soil pathogen Fusarium oxysporum (lini). The linkage map was constructed using a mapping population from doubled-haploid (DH) lines. The DH lines were derived from the haploid component of F₂ haploid-diploid twin seed originating from a cross between a polyembryonic, low-linolenic-acid genotype (CRZY8/RA91) and the Australian cultivar ‘Glenelg’. The AFLP technique was employed to generate 213 marker loci covering approximately 1400 cM of the flax genome (n=15) with an average spacing of 10 cM and comprising 18 linkage groups. Sixty AFLP markers (28%) deviated significantly (P<0.05) from the expected segregation ratio. The map incorporated RFLP markers tightly linked to flax rust (Melamspora lini) resistance genes and markers detected by disease resistance gene-like sequences. The study illustrates the potential of the AFLP technique as a robust and rapid method to generate moderately saturated linkage maps, thereby allowing the molecular analysis of traits, such as resistance to Fusarium wilt, that show oligogenic patterns of inheritance. Received: 8 December 1997 / Accepted: 7 April 1998     

Identification and Validation of Molecular Markers Associated with Pachymetra Root Rot and Brown Rust Resistance in Sugarcane Using Map- and Association-based Approaches

Marker-assisted selection for traits that are difficult to screen for, such as resistance to many sugarcane diseases, has the potential to facilitate the development of improved cultivars in sugarcane. Pachymetra root rot (PRR) and brown rust resistance ratings were obtained over two years for 192 I1 progeny (progeny produced by two heterozygous, non-inbred parental lines) of a sugarcane (Saccharum spp. hybrid) cross between two elite sugarcane clones, Q117 and 74C42. Approximately 1000 single-dose markers, including microsatellite (SSR), amplified fragment length polymorphism (AFLP) and restriction fragment length polymorphism (RFLP) markers, were scored across the population and maps containing approximately 400 markers were constructed for each parent. At p ≤ 0.01, two genomic regions, one from the female Q117 map and a different region from the 74C42 male map, plus an unlinked bi-parental simplex marker (single-dose marker present in both parents) were identified as associated with PRR over both years of data collection. These regions explained between 6 and 16% of the phenotypic variation. An additional region was identified in the female map as associated with PRR at p ≤ 0.01 in one year and p ≤ 0.05 in the second year. This region explained between 4 and 8% of the phenotypic variation. For brown rust, two genomic regions, one from the female map and one from the male map, plus an unlinked marker from both maps, were identified as associated with brown rust resistance at p ≤ 0.01 over two years of phenotypic data. Each region explained between 7 and 18% of the phenotypic variation. Several additional regions were identified in both maps as associated with brown rust at p ≤ 0.01 in one year and p ≤ 0.05 in the second year. These regions also explained between 5 and 11% of the phenotypic variation. To validate these markers and determine whether they would be useful in alternative germplasm, markers from each genomic region associated with PRR or brown rust were screened across a set of 154 elite sugarcane clones; PRR and brown rust ratings were available for 131 and 72 of the clones, respectively. For PRR, three of the 6 markers tested remained significantly associated (p ≤ 0.01) with resistance ratings in the elite clone set. For brown rust, only one of the seven markers tested remained significantly associated (p ≤ 0.01) with resistance in the elite clone set, with one other marker associated at p ≤ 0.05. These results suggest that these markers could be broadly effective in selecting for PRR and/or brown rust resistance in sugarcane breeding programs.     

SCAR and CAPS mapping of CRb, a gene conferring resistance to Plasmodiophora brassicae in Chinese cabbage ( Brassica rapa ssp. pekinensis)

Clubroot disease, caused by Plasmodiophora brassicae Wor., is highly damaging for Chinese cabbage. The CR (clubroot resistant) Shinki DH (doubled haploid) line of Chinese cabbage carries a single dominant gene, CRb, which confers resistance to the P. brassicae races 2, 4, and 8. An F₂ population derived from a cross between the CR Shinki DH line and a susceptible line, 94SK, was used to map the CRb gene. Inoculation of F₃ families with SSI (single-spore isolate) resulted in a 1:2:1 segregation ratio. Use of the AFLP technique combined with bulked segregant analysis allowed five co-dominant AFLP markers, and four and seven dominant AFLP markers linked in coupling and repulsion, respectively, to be identified. Six of the 16 AFLP markers showing low frequencies of recombination with the CRb locus among 138 F₂ lines were cloned. A reliable conversion procedure allowed five AFLP markers to be successfully converted into CAPS and SCAR markers. An F₂ population (143 plants) was analyzed with these markers and a previously identified SCAR marker, and a genetic map around CRb covering a total distance of 6.75 cM was constructed. One dominant marker, TCR09, was located 0.78 cM from CRb. The remaining markers (TCR05, TCR01, TCR10, TCR08, and TCR03) were located on the other side of CRb, and the nearest of these was TCR05, at a distance of 1.92 cM.Communicated by R. Hagemann     

Identification of molecular markers linked to Rdr1, a gene conferring resistance to blackspot in roses

Blackspot resistance in the tetraploid rose genotype 91/100–5 had been characterised previously as a single dominant gene in duplex configuration. In the present study a tetraploid progeny (95/3) segregating for the presence of the blackspot resistance gene Rdr1 were screened with 868 RAPD and 114 AFLP primers/primer combinations. Seven AFLP markers were found to be linked to Rdr1 at distances between 1.1 and 7.6 cM. The most closely linked AFLP marker was cloned and converted into a SCAR marker that could be screened in a larger population than the original AFLP and was linked at a distance of 0.76 cM. The cloned fragment was used as an RFLP probe to locate the marker on a chromosome map of diploid roses. This is the first report of markers linked to a resistance gene in roses, and the possibilities of using them for a marker-assisted selection for blackspot resistance as well as for map-based cloning approaches are discussed. Received: 23 December 1999 / Accepted: 25 March 2000     

普通小麦Qz180中一个抗条锈病基因的分子作图(英文)

普通小麦(Triticum aestivum L.)材料Qz180具有良好的抗条锈病特性,经基因推导发现其含有一个优良的抗条锈病的基因,暂定名为YrQz。用Qz180与感病材料铭贤169和WL1分别杂交构建了两个F_2群体,用条中30号条锈菌小种对这两个群体进行的抗性测验表明,YrQz为显性单基因遗传。通过SSR和AFLP结合BSA的方法对这个基因进行了分子作图,结果鉴定出与YrQz连锁的2个SSR标记和2个AFLP标记。根据SSR标记的染色体位置,该基因被定位在2B染色体的长臂上,位于两个SSR位点Xgwm388和Xgwm526之间;两个AFLP标记P35M48(452)和P36M61(163)分别位于该基因的两侧,遗传距离分别为3.4cM和4.1cM。     

Characterization and high-resolution mapping of a late blight resistance locus similar to R2 in potato

Identification of resistance (R) genes to Phytophthora infestans is an essential step in molecular breeding of potato. We identified three specific R genes segregating in a diploid mapping population. One of the R genes is located on chromosome 4 and proved phenotypically indistinguishable from the Solanum demissum-derived R2, although S. demissum is not directly involved in the pedigree of the population. By bulked segregant analysis combined with a resistance assay, a genetic linkage map of the R2-like locus was constructed with 30 coupling and 23 repulsion phase AFLP markers. Two markers flanking the R2-like locus were applied to screen an extended population of 1,586 offspring. About 103 recombinants were selected, and an accurate high-resolution map was constructed. The R2-like resistance was localized in a 0.4 cM interval and was found co-segregating with four AFLP markers, which can be used to isolate the R2-like gene by map-based gene cloning. By analyzing race-specificity and R gene-specific molecular markers, we also found that an R1-like gene and an additional unknown R gene are segregating in the population.