Cytological analyses of hybrids and derivatives of hybrids between durum wheat and Thinopyrum bessarabicum,using multicolour fluorescent GISH*

The wheat progenitors and other wild relatives continue to be important sources of genes for agronomically desirable traits, which can be transferred into durum wheat (Triticum turgidum; 2n = 4x = 28; AABB genomes) cultivars via hybridization. Chromosome pairing in durum × alien species hybrids provides an understanding of genomic relationships, which is useful in planning alien gene introgression strategies. Two durum cultivars, ‘Lloyd’ and ‘Langdon’, were crossed with diploid wheatgrass, Thinopyrum bessarabicum (2n = 2x = 14; JJ), to synthesize F₁ hybrids (2n = 3x = 21; ABJ) with Ph1. ‘Langdon’ disomic substitution 5D(5B) was used as a female parent to produce F₁ hybrids without Ph1, which resulted in elevation of pairing between durum and grass chromosomes – an important feature from the breeding standpoint. The F₁ hybrids were backcrossed to respective parental cultivars and BC₁ progenies were raised. ‘Langdon’ 5D(5B) substitution × Th. bessarabicum F₁ hybrids were crossed with normal ‘Langdon’ to obtain BC₁ progeny. Chromosome pairing relationships were studied in F₁ hybrids and BC₁ progenies using both conventional staining and fluorescent genomic in situ hybridization (fl‐GISH) techniques. Multicolour fl‐GISH was standardized for characterizing the nature and specificity of chromosome pairing: A–B, A–J and B–J pairing. The A–J and B–J pairing will facilitate gene introgression in durum wheat. Multicolour fl‐GISH will help in characterizing alien chromosome segments captured in the durum complement and in their location in the A and/or B genome, thereby accelerating chromosome engineering research.     

Changes in the meiotic pairing behaviour of a winter wheat-winter barley hybrid maintained for a long term in tissue culture, and tracing the barley chromatin in the progeny using GISH and SSR markers

The aim of the present study was to produce backcross progenies in a new winter wheat (‘Asakaze komugi’) × winter barley (‘Manas’) hybrid produced in Martonvasar. As no backcross seeds were obtained from the initial hybrids, young inflorescences of the hybrids were used for in vitro multiplication in three consecutive cycles until a backcross progeny was developed. The chromosome constitution of the regenerated hybrids was analysed using genomic in situ hybridization (GISH) after each in vitro multiplication cycle. The seven barley chromosomes were present even after the third in vitro multiplication cycle but abnormalities were observed. Sixteen BC; plants containing, according to GfSH analysis, one to three complete barley chromosomes, two deletion barley chromosomes and a dicentric wheat‐barley translocation were grown to maturity from the single backcross progeny. The barley chromatin was identified using 20 chromosome‐specific barley SSR markers. All seven barley chromosomes were represented in the BC: plants. A deletion breakpoint at FL ±0,3 on the 5HL chromosome arm facilitated the physical localization of microsatellite markers.     

Detection of wheat-barley translocations by genomic in situ hybridization in derivatives of hybrids multiplied in vitro

Wheat-barley translocations were identified by genomicin situ hybridization (GISH) in backcross progenies originating from in vitro regenerated wheat (Triticum aestivum L. cv. Chinese Spring) × barley (Hordeum vulgare L. cv. Betzes) hybrids. The regenerated hybrids were pollinated with the wheat line Martonvásári 9 kr1. Five translocated wheat-barley chromosomes were recovered among 51 BC₂F₂ progeny from the in vitro regenerated wheat × barley hybrids. All were single breakpoint translocations with the relative positions of the breakpoints ranging from the centromere to about 0.8 of the relative arm length. Of the four translocations with intercalary breakpoints, three were transfers of terminal barley segments to wheat chromosomes; one was a transfer of a terminal wheat segment to a barley chromosome. Because of the absence of diagnostic N-bands, the identity of three barley segments could not be determined; in one translocation the barley chromosome involved had a NOR so it must have been 5H or 6H, and the centric translocation was 4HS.2BL. Following selfing, homozygotes of four translocations were selected. The experiment suggests that in vitro culture conditions are conducive for major genome rearrangements in wheat-barley hybrids. This revised version was published online in July 2006 with corrections to the Cover Date.     

Production and characterization of intergeneric hybrids between Brassica oleracea and a wild relative Moricandia arvensis

Intergeneric crosses were made between Brassica oleracea and Moricandia arvensis utilizing embryo rescue. Six F₁ hybrid plants were generated in the cross‐combination of B. oleracea × M. arvensis from 64 pods by the placenta‐embryo culture technique, whereas three plants were produced in the reciprocal cross from 40 pods by the ovary culture technique. The hybrid plants were ascertained to be amphihaploid with 2n = 23 chromosomes in mitosis and a meiotic chromosome association of (0–3)II + (17–23)I at metaphase I (M I). In the backcross with B. oleracea, some of these hybrids developed sesquidiploid BC₁ plants with 2n = 32 chromosomes that predominantly exhibited a meiotic configuration of (9II + 14I) in pollen mother cells. The following backcross of BC₂ plants to B. oleracea generated 48 BC₃ progeny with somatic chromosomes from 2n = 19 to 2n = 41. The 2n = 19 plants showed a chromosomal association type of (9II + 1I) and a chromosomal distribution type of (9¹/₂ + 9¹/₂) or (9 + 10) at M I and M II, respectively. These facts might suggest that they were monosomic addition lines (MALs) of B. oleracea carrying a single chromosome of M. arvensis that could offer potential for future genetic and breeding research, together with other novel hybrid progeny developed in this intergeneric hybridization.     

Alteration of the genomic composition of Solanum nigrum (+) potato backcross derivatives by somatic hybridization: selection of fusion hybrids by DNA measurements and GISH

Fusion experiments were performed with a first (BC₁‐6738) and a second (BC₂‐9017) generation backcross hybrid of 6x Solarium nigrum (+) 2x potato somatic hybrids with potato cultivars. Because no progeny was obtained from the BC₂ genotypes, alternative approaches were sought to overcome the sexual crossing barrier. Five potato genotypes, one of which contains the hygromycin resistance gene, were used in the fusion experiments. All vigorous regenerants were used for the estimation of nuclear DNA content using flow cytometry. Plants with a DNA content higher than that of the BC₁‐6738 or BC₂ genotypes were considered potential somatic hybrids. Forty‐nine potential somatic hybrids resulted from fusion experiments with BC₁‐6738, from which 20 grew vigorously in the greenhouse and flowered. After pollination with several 4x potato cultivars, eight genotypes produced seeded berries and five genotypes gave seedless berries. In addition, 11 of these 13 somatic hybrids were selected for genomic in situ hybridization (GISH) analysis to determine their genomic composition. Nine had exactly or approximately the expected number of 36 S. nigrum and 60 potato chromosomes. In one genotype, only 22 instead of 36 S. nigrum chromosomes were found and one potato chromosome was possibly missing. Only five potential somatic hybrids were detected among the 79 regenerants from BC₂‐9017 (+) 2x potato fusion experiments that were analysed by flow cytometry. Two of these hybrids were rather vigorous and did flower, but pollinations with potato have not yet set any berries.     

The genus Dasypyrum—part 2. Dasypyrum villosum—a wild species used in wheat improvement

Dasypyrum villosum (L.) P. Candargy is discussed as a species commonly used in wheat improvement. Chromosomal localization of the potentially useful traits and chromosomal position of some morphological and isozyme markes are shown. The investigations using molecular RAPD, AFLP, SSR, RFLP markers and in situ (GISH, FISH) hybridization experiments on D. villosum itself and in wide hybrids with Triticum are summarized. The article also presents the information about designation of D. villosum chromosomes and the current knowledge on the phenomenon of two-coloured D. villosum caryopses.     

偏凸-柱穗山羊草双二倍体与普通小麦不同杂种世代的染色体及性状分离特点

为探讨偏凸山羊草－柱穗山羊草双二倍体SDAU18在小麦遗传改良中的利用价值,以SDAU18和普通小麦品种烟农15及其9个杂种世代为材料,分析不同自交和回交世代染色体和性状分离的特点。结果表明,随自交和以烟农15为轮回亲本回交世代的增加,染色体数目逐渐减少,回交比自交能使后代的染色体数目更快趋近普通小麦的42条,至F₅和BC₃F₁代,染色体数目为42的植株已分别达93.9%和92.0%。与自交世代相比,回交后代减数第一分裂中期的花粉母细胞的染色体构型较为简单,回交次数过多不利于外源染色体与普通小麦染色体发生重组,一般应以回交2~3次为宜;随自交和回交世代的增进,杂种的育性提高,至F₃和BC₂F₁代育性基本稳定。在不同杂种世代可分离出具有矮秆、大穗、大粒、对白粉病、条锈病免疫或高抗及外观品质优良的变异类型,以F₃和BC₁F₁代的变异类型最丰富。     

A New Source of Resistance to Pseudocercosporella herpotrichoides, Cause of Eyespot Disease of Wheat, Located on Chromosome 4V of Dasypyrum villosum

Resistance to Pseudocercosporella herpotrichoides in five wheat cultivars, accession W6 7283 of Dasypyrum villosum, and ‘Chinese Spring’ disomic addition lines of the D. villosum chromosomes IV, 2V, 4V, 5V, 6V and 7V, was evaluated in seedlings by measuring disease progress 6 weeks after inoculation with a β—glucuronidase—transformed strain of the pathogen and by visual estimates of disease severity. D. villosum and the disomic addition line of chromosome 4V were as resistant as wheat cultivars ‘VPM—1’ and ‘Cappelle Desprez’, but less resistant than ‘Rendezvous’. Resistance of the chromosome 4V disomic addition line was equivalent to that of D. villosum.‘Chinese Spring’ and disomic addition lines of IV, 2V, 5V, 6V and 7V were all susceptible. These results confirm Sparaguee's (1936) report of resistance in D. villosum to P. herpotrichoides and establish the chromosomal location for the genes controlling resistance. The presence of chromosome 4V in the addition line and its homocology to chromosome 4 in wheat were confirmed by Southern analysis of genomic DNA using chromosome group 4-specific clones. This genetic locus is not homoeologous with other known genes for resistance to P. herpotrichoides located on chromosome group 7, and thus represents a new source of resistance to this pathogen.     

A Robertsonian translocation from Thinopyrum bessarabicum into bread wheat confers high iron and zinc contents

Development of wheat–alien translocation lines has facilitated practical utilization of alien species in wheat improvement. The production of a compensating Triticum aestivum–Thinopyrum bessarabicum whole‐arm Robertsonian translocation (RobT) involving chromosomes 6D of wheat and 6E^b of Th. bessarabicum (2n = 2x = 14, E^bE^b) through the mechanism of centric breakage–fusion is reported here. An F₂ population was derived from plants double‐monosomic for chromosome 6D and 6E^b from crosses between a DS6E^b(6D) substitution line and bread wheat cultivar ‘Roushan’ (2n = 6x = 42, AABBDD) as female parent. Eighty F₂ genotypes (L1–L80) were screened for chromosome composition. Three PCR‐based Landmark Unique Gene (PLUG) markers specific to chromosomes 6D and 6E^b were used for screening the F₂ plants. One plant with a T6E^bS.6DL centric fusion (RobT) was identified. A homozygous translocation line with full fertility was recovered among F₃ families and verified with genomic in situ hybridization (GISH). Grain micronutrient analysis showed that the DS6E^b(6D) substitution line and T6E^bS.6DL stock have higher Fe and Zn contents than the recipient wheat cultivar ‘Roushan’.     

Chromosomal Stability in the Backcross Progenies of Pentaploid Hybrids between Avena sativa L. and A. maroccana Gdgr.

In a backcrossing programme to transfer desirable characters from wild Avena maroccana Gdgr. to cultivated oats, A. sativa L., meiotically stable plants in BC₁F₃ and BC₂F₂ progenies were isolated. The recovery of stable genotypes with 2n = 6×= 42 chromosomes indicated that two backcrosses are enough for such a programme. The cytological observations in various backcross generations are presented and discussed.     

An evaluation of the potential of intergeneric gene transfer between Brassica napus and Sinapis arvensis

The possibility of gene transfer between Brassica napus and Sinapis arvensis was evaluated. Six spring-type cultivars of B. napus and four strains of S. arvensis were reciprocally crossed through controlled crosses. No hybrid was yielded from any cross. However, one hybrid with 28 chromosomes was obtained from B. napus×S. arvensis through ovule culture. The hybrid plant was highly sterile and set no seed on open pollination. Two F₂ plants, with 35 and 36 chromosomes respectively, were obtained through self-pollination by hand. Backcross of B. napus produced 23 plants carrying some characteristics of S. arvensis, but backcross to S. arvensis failed to produce a plant. The chromosome counts of the BC₁F₁ plants indicated that gametes with more than nine chromosomes were favoured during the meiosis. The data demonstrated that gene transfer from S. arvensis to B. napus was very difficult under controlled cross and backcross, while to transfer genes from B. napus to S. arvensis would be extremely remote even under the most favorable conditions.     

Production of intergeneric hybrids between Brassica juncea and Diplotaxis virgata through ovary culture, and the cytology and crossability of their progenies

The cytogenetic study was investigated in the intergeneric F₁ hybrid, F₂and backcross progenies (BC₁). The plants used were Brassica juncea(2n=36) and Diplotaxis virgata(2n=18). Three intergeneric F₁ hybrids between two species were produced through ovary culture. They showed 36 chromosomes. It might consist one genome of B. juncea and two genomes of D. virgata. The morphology of the leaves resembled that of B. juncea. The color of the petals was yellow that was like in D. virgata. The size of the petal was similar to that of B. juncea. The mean pollen fertility was15.3% and the chromosome associations in the first meiotic division were(0–1)_IV+(0–2)_III+(8–12)_II+(12–20)_I. Many F₂ and BC₁seeds were harvested after open pollination and backcross of the F₁ hybrids withB. juncea, respectively. The F₂seedlings showed different chromosome constitutions and the range was from 28 to54 chromosomes. Most seedlings had 38chromosomes followed by 36, 40 and 54. The BC₁ seedlings also showed different chromosome constitutions and the range was from 29 to 62. Most seedlings had both 40and 54 chromosomes followed by 36, 46 and52. In the first meiotic division of F₂ and BC₁ plants, a high frequency of bivalent associations was observed in all the various kinds of somatic chromosomes. Many F₃ and BC₂ seeds were obtained by self-pollination and open pollination of both F₂ and BC₁ plants, and by backcrossing both F₂ and BC₁plants with B. juncea, respectively,especially, three type progeny with 36, 40or 54 chromosomes. The somatic chromosomes of the F₃ and BC₂ plants were further investigated. The bridge plants between B. juncea and D. virgata with 36 chromosomes may be utilized for breeding of other Brassica crops as well as B. juncea. This revised version was published online in July 2006 with corrections to the Cover Date.     

Detection of chromosomes carrying genes for seed dormancy of wheat using the backcross reciprocal monosomic method

Preharvest sprouting (PHS) in wheat represents a major constraint to the production of high‐quality grain. Genetic variation for tolerance to PHS is associated with seed dormancy. The present study was initiated to detect homologous chromosome variation associated with seed dormancy genes in a PHS tolerant cultivar ‘Zenkojikomugi’ (Zen) and ‘Chinese Spring’ (CS) using the backcross reciprocal monosomic method. The most striking effect on variation in seed dormancy was associated with chromosome 3A, and followed by group 4 chromosomes. These chromosomes of Zen increased dormancy compared with the respective CS homologues. Chromosomes 2D and 7D of Zen, and 6A of CS seemed to contribute smaller positive effects on dormancy. Chromosomes 2A of CS and Zen, as well as 3B, were equally effective in enhancing dormancy. The chromosome 3 A factors were independent of the grain‐colour gene R‐A1 because Zen was found to carry the white‐grained R‐A1a allele, which was identical to that of CS, and there was no difference in dormancy between the reciprocal F₁ seeds.     

Rapid Incorporation of D-genome Chromosomes into A and/or B Genomes of Hexaploid Triticale

Two series of progenies were developed from hybrids between octoploid (AABBDDRR) and tetraploid triticale ((AB)(AB)RR). One arose from the successive selfing of the F₁s, while the second was established after one backcross of the F₁ hybrids with the respective 8 × triticale parent. Altogether, 250 F₃ and BC₁F₂ lines were developed, of which 112 were karyotyped in the F₄/F₅ or in BC₁F₃/BC₁F₄ generations using C-banding and SDS-PAGE. The 112 lines represented 61 different karyotypes, of which 39 appeared to be stabilized, having pairs of homologous wheat chromosomes only, while 22 karyotypes exhibited 1—3 heterologous pairs. The frequency of karyotypically stabilized lines originating from the series with one backcrossing was much higher (79.5 %) than those derived from the successive selfing of the F₁ hybrids (51.7%). Six lines had the pure hexaploid triticale chromosome constitution. The frequency of disomic substitutions of D genome chromosomes for their homoeologous A and/or B genome chromosomes ranged from one to six per line with an average of 1.7. Except for 3B and 6B all possible D(A/B) substitutions were obtained. Chromosomes ID and 3D substituted for their homoeologues with the highest frequency, while the substitution of chromosome 4D for 4A or for 4B was the least frequent. D(R) substitutions were found in eight lines only. A complete set of 6x triticale lines was established in which chromosome ID was present in all possible combinations, i.e. single 1D(1A/1B/1R) disomic substitutions as well as disomic ID addition.     

Cytogenetic Study of a Trigeneric (Triticale × Trileymus) Hybrid

Summary In this study a new trigeneric hybrid involving species from the Triticum, Secale and Leymus was produced by crossing octoploid triticale (Jinsong49) with octoploid tritileymus (950059). The chromosome constitution of the parental amphiploid, trigeneric hybrid and its progenies were studied. Genomic in situ hybridization (GISH) analysis showed that Jinsong49 and 950059 had 44 wheat chromosomes, and 12 rye chromosomes, 12 L. mollis chromosomes respectively. The mean meiotic configuration of trigeneric hybrid F₁ was 13.17 I + 20.82 II + 0.37 III + 0.02 IV. GISH results indicated the trigeneric hybrid F₁ had 6 rye chromosomes and 6 Leymus chromosomes. In the selfed derivatives of the trigeneric hybrids, while the number of selfed generation increased, the mean number of chromosomes tends to decrease gradually and slowly. GISH results revealed that most plant tested in the progeny population had 8–12 rye chromosomes, and no Leymus chromosomes were detected. The results indicated that rye chromosomes can be preferentially transmitted in the progenies of trigeneric hybrid than Leymus chromosomes.     

Identification of four genes for stable hybrid sterility and an epistatic QTL from a cross between Oryza sativa and Oryza glaberrima

To further understand the nature of hybrid sterility between Oryza sativa and Oryza glaberrima, quantitative trait loci (QTL) controlling hybrid sterility between the two cultivated rice species were detected in BC₁F₁ and advanced backcross populations. A genetic map was constructed using the BC₁F₁ population derived from a cross between WAB450-16, an O. sativa cultivar, and CG14, an O. glaberrima cultivar. Seven main-effect QTLs for pollen and spikelet sterility were detected in the BC₁F₁. Forty-four sterility NILs (BC₆F₁) were developed via successive backcrosses using pollen sterility plants as female and WAB450-16 as the recurrent parent. Seven NILs, in which the target QTL regions were heterozygous while the other QTL regions as well as most of the reminder of the genome were homozygous for the WAB450-16 allele, were selected as the QTL identification materials. BC₇F₁ for the seven NILs showed a continuous variation in pollen and spikelet fertility. The four identified pollen sterility QTLs were located one each on chromosomes 1, 3, 7 and 7. Pollen sterility loci qSS-3 and qSS-7a were on chromosomes 3 and 7, respectively, which coincides with the previously identified S19, and S20, while loci qSS-1 and qSS-7b on chromosomes 1 and 7L appear distinct from all previously reported loci. An epistatic interaction controlling the hybrid sterility was detected between qSS-1 and qSS-7a.     

Interspecific cross of Brassica oleracea var. alboglabra and B. napus : effects of growth condition and silique age on the efficiency of hybrid production, and inheritance of erucic acid in the self-pollinated backcross generation

Interspecific hybrids were produced from reciprocal crosses between Brassica napus (2n = 38, AACC) and B. oleracea var. alboglabra (2n = 18, CC) to introgress the zero-erucic acid alleles from B. napus into B. oleracea. The ovule culture embryo rescue technique was applied for production of F₁ plants. The effects of silique age, as measured by days after pollination (DAP), and growth condition (temperature) on the efficiency of this technique was investigated. The greatest numbers of hybrids per pollination were produced under 20°/15°C (day/night) at 16 DAP for B. oleracea (♀) × B. napus crosses, while under 15°/10°C at 14 DAP for B. napus (♀) × B. oleracea crosses. Application of the ovule culture technique also increased the efficiency of BC₁ (F₁ × B. oleracea) hybrid production by 10-fold over in vivo seed set. The segregation of erucic acid alleles in the self-pollinated backcross generation, i.e. in BC₁S₁ seeds, revealed that the gametes of the F₁ and BC₁ plants carrying a greater number of A-genome chromosomes were more viable. This resulted in a significantly greater number of intermediate and a smaller number of high-erucic acid BC₁S₁ seeds.     

Genetics of spike length in durum wheat

Gene effects were analyzed using mean spike length of 12 populations, viz., both parents, F₁, F₂, first back cross generation, BC₁ and BC₂, second backcross generations, BC₁₁,BC₁₂, BC₂₁ and BC₂₂ along with BC₁ self and BC₂ self derived by selfing BC₁ and BC₂populations of three crosses involving six diverse cultivars of Triticum durumto determine the nature of gene actions governing spike length through generation mean analysis under normal and late sown environments. The six-parameter model was adequate in most of the cases to explain genetic variation among the generation means under both the sowing environments. Additive (d) gene effect was significant in all the cases, whereas dominance (h) gene effect was not so frequently observed significant. Epistatic effects, particularly digenic types were predominant over additive and dominance effects in most of the cases under both normal and late sown environments except in the cross Cocorit 71 × A-9-30-1 (normal sown).Additive × dominance × dominance (y), trigenic interaction played significant role in controlling the inheritance of this trait in the cross HI 8062 × JNK-4W-128under late sown condition. Duplicate epistasis was observed in the cross HI 8062× JNK-4W-128 (normal sown). Non-fixable gene effects were of higher magnitude than fixable gene effects in almost all cases, confirmed the major role of non-additive gene effects to control the inheritance of spike length in durum wheat. Significant heterosis over better parent was not observed. Similarly, inbreeding depression was not commonly observed. Favourable and suitable environment must be considered before finalizing breeding programme for its simple inheritance to get desirable improvement for high grain yield. Hybridization systems, such as biparental mating and / or diallel selective mating, which exploit both additive and non-additive gene effects, simultaneously, could be useful in the improvement of spike length in durum wheat. This revised version was published online in July 2006 with corrections to the Cover Date.     

Morphological and cytogenetic studies on the hybrid between bread wheat and <Emphasis Type="Italic">Psathyrostachys huashanica</Emphasis> Keng ex Kuo

Psathyrostachys huashanica Keng ex Kuo (2n = 2x = 14, NsNs), a source of wheat stripe rust, take-all fungus, and powdery mildew resistance with tolerance to salinity and drought, has been successfully hybridized as the pollen parent to bread wheat without using immature embryo rescuing culture for the first time. All of the CSph2b × P. huashanica hybrid seeds germinate well. Backcross derivatives were successfully obtained. F₁ hybrids were verified as intergeneric hybrids on the basis of morphological observation, cytological and molecular analyses. The results obviously showed the phenotypes of the hybrid plants were intermediate between bread wheat and P. huashanica. Chromosome pairing at MI of PMCs in the F₁ hybrid plants was low, and the meiotic configuration was 26.80 I + 0.60 II (rod). Cytological analysis of the hybrid plants revealed the ineffectiveness of the ph2b gene on chromosome association between the parents. Eight RAPD-specific markers for Ns genome were selected for RAPD analysis, and the results indicated that F₁ hybrids contained the Ns genome of P. huashanica. Furthermore, the significance of the finding for bread wheat improvement was discussed.     

Transfer of powdery mildew resistance from Brassica carinata to Brassica oleracea through embryo rescue

Interspecific hybrid plants and backcross 1 (BC₁) progeny were produced through sexual crosses and embryo rescue between Brassica carinata accession PI 360883 and B. oleracea cvs Titleist’and‘Cecile’to transfer resistance to powdery mildew to B. oleracea. Four interspecific hybrids were obtained through application of embryo rescue from crosses with B. carinata as the maternal parent, and their interspecific nature confirmed through plant morphology and random amplified polymorphic DNA (RAPD) analysis. Twenty‐one BC₁ plants were obtained through sexual crosses and embryo rescue although embryo rescue was not necessary to produce first backcross generation plants between interspecific hybrids and B. oleracea. All interspecific hybrids and eight of the BC₁ plants were resistant to powdery mildew.