Use of molecular markers to accelerate the breeding of common bean lines resistant to rust and anthracnose

The main goal of this work was to introduce resistance genes for rust, caused by Uromyces appendiculatus, and anthracnose, caused by Colletotrichum lindemuthianum, in an adapted common bean cultivar through marker-assisted backcrossing. DNA fingerprinting was used to select plants genetically closer to the recurrent parent which were also resistant to rust and to race 89 of C. lindemuthianum. DNA samples extracted from the resistant parent (cv. Ouro Negro), the recurrent parent (cv. Rudá), and from BC₁, BC₂ and BC₃ resistant plants were amplified by the RAPD technique. The relative genetic distances in relation to the recurrent parent varied between 9 and 59% for BC₁, 7 and 33% for BC₂, and 0 and 7% for BC₃ resistant plants. After only three backcrosses, five lines resistant to rust and anthracnose with, approximately, 0% genetic distance in relation to the recurrent parent were obtained. These lines underwent field yield tests in two consecutive growing seasons and three of them presented a good yield performance, surpassing in that sense their parents and most of the reference cultivars tested.     

The Gene Pools of the Grasspea (Lathyrus sativus L.)

Interspecific hybridization between the grasspea, Lathyrus sativus, and 15 wild species in Section Lathyrus is reported. Only two species, L. amphicarpos and L. cicera produced viable F¹ hybrids with low fertility when crossed with L. sativus as male parent. Crosses with six other species produced pods following pollination when L. sativus was the male, but seedlings were inviable, seeds did not germinate or pods were empty or had totally shrivelled seeds. When L. sativus was the female parent, only one cross with L. gorgoni produced an F¹, but the seedling was inviable. The germplasm resources of the grasspea are identified, with L. amphicarpos and L. cicera placed in the secondary gene pool and the other species in the tertiary gene pool. The definition of these germplasm resources is discussed in terms of grasspea improvement through plant breeding.     

A single dominant gene for downy mildew resistance in broccoli

Downy mildew, incited by Peronospora parasitica (Pers.: Fr.) Fr., is a destructive disease of broccoli (Brassica oleraceaL., Italica Group). Resistant cultivars represent a desirable control method to provide a practical, environmentally benign, and long-term means of limiting damage from this disease. Doubled-haploid (DH) lines developed by us exhibit a high level of downy mildew resistance at the cotyledon stage. To determine the mode of inheritance for this resistance, a resistant DH line was crossed to a susceptible DH line to make an F₁, from which F₂ and backcross (BC) populations were developed. All populations were evaluated for response to artificial inoculation with P. parasitica at the cotyledon stage. All F₁ plants (including reciprocals) were as resistant as the resistant parent, indicating no maternal effect for this trait. F₂ populations segregated approximately 3resistant to 1 susceptible, BC populations using the resistant parent as the recurrent parent contained all resistant plants, and the BC to the susceptible parent segregated 1 resistant to 1 susceptible. These results indicate that resistance is controlled by a single dominant gene. This gene should be easily incorporated into F₁ hybrids and used commercially to prevent downy mildew at the cotyledon stage. This revised version was published online in August 2006 with corrections to the Cover Date.     

Transfer of disease resistance within the genus Brassica through asymmetric somatic hybridization

Summary Asymmetric somatic hybrid plants between Brassica napus L. (oilseed rape genome AACC) and a transgenic line of Brassica nigra L. Koch (black mustard genome BB) were tested for their resistance against rapeseed pathogens Phoma lingam (black leg disease) and Plasmodiophora brassicae (club root disease). The transgenic B. nigra line used (hygromycin-resistant, donor) is highly resistant to both fungi, whereas B. napus (recipient) is highly susceptible. The asymmetric somatic hybrids were produced using the donor-recipient fusion method (with X-irradiation of donor protoplasts) reported by Zelcer et al. (1978) for the production of cybrids. Using hygromycin-B for selection, a total of 332 hybrid calli were obtained. Regenerants, resistant or susceptible to both diseases, were selected. Many hybrids expressed resistance to only one pathogen. Dot blot experiments showed that the asymmetric hybrid plants contained varying amounts of the donor genomic DNA. Furthermore, a correlation was detected between the radiation dose and the degree of donor DNA elimination.     

Screening for Fusarium resistance in seedling populations of Asiatic hybrid lily

A test to select Fusarium resistant seedlings of the Asiatic hybrid lily is described. Young seedlings of 28 populations, obtained from an incomplete diallel between eight parents with different levels of Fusarium resistance, were tested for resistance. Significant differences in Fusarium resistance between and within populations were detected. The average percentage of selected seedlings ranged from 34% in resistant × resistant crosses to 2% in susceptible × susceptible crosses. Although resistant descendants were obtained in susceptible × susceptible crosses, using at least one resistant parent produced higher percentages of resistant seedlings. The resistance level of the parents correlated highly with the general combining ability for Fusarium resistance based on the seedling test. For eight populations, seedlings selected for Fusarium resistance and non-tested (control) seedlings of the same cross were compared, after propagation, in a clonal test. Variation between and within populations, found at seedling level, was confirmed at clonal level. A positive selection response was found for all eight populations. In the seedling test, approximately 18% of the seedlings were selected as resistant of which 15% (2.7% of seedlings tested) appeared to be susceptible escapes. Comparison between selection at seedling level and at clonal level indicated that approximately 25% of the seedlings tested were missed (rejected resistant plants) in the seedling test. The practical use of a seedling test for Fusarium resistance in lily breeding programs is discussed.     

Synthesis and preliminary characterization of a new species (amphidiploid) in Cucumis

A successful interspecific hybridization between cucumber (Cucumis sativus L., 2n = 14) and a wild it Cucumis species, C. hystrix Chakr. (2n = 24) was made via embryo rescue. Hybrid plants (2n = 19; 7 from cucumber and 12 from C. hystrix) were sterile, but morphologically uniform. Self-pollination and backcrossing of F₁ hybrid plants to either parent confirmed presence of both male- and female-sterility that were likely caused by lack of homology and improper pairing during meiosis. While the multiple-branching habit, densely brown hairs (on corolla and pistil), orange-yellow corolla, and ovate fruit of F₁ hybrid plants were similar to that of the C. hystrix parent, the appearance of the first pistillate flower was more similar to that of C. sativus parent. The diameter and internode length of the stem, and the shape and size of leaves and flowers were intermediate when compared to the parents. The chromosome number in the hybrid was doubled through somaclonal variation during embryo culture and regeneration process to restore the fertility. Pollen grains were released and fruits with viable seeds matured on fertile, synthetic amphidiploid plants. The results from flow cytometry indicated that, on average, 7.3% of the morphologically unique regenerants had the 4C DNA content of 2.35 pg relative to the 2C DNA content of the original F₁ hybrid at 1.17 pg and, therefore, were likely chromosome-doubled F₁ hybrids (2n = 38). Nutrition alanalysis indicated that the synthetic species had higher protein (0.78%)and mineral (0.35%) content compared to the normal pickling cucumber(0.62% and 0.27%, respectively), and could be considered a new Cucumis crop having a special place in the future agriculture. Preliminarily evaluation indicates that C. hystrix possesses a high level of root-knot nematode resistance, and that this resistance is partially expressed in the interspecific F₁ and chromosome-doubled F₁. This and the fact that the fruit morphology of the fertile amphidiploid differs during the growing season (e.g., short and long fruit) suggest that it could be useful in broadening the germplasm base of cucumber. This revised version was published online in July 2006 with corrections to the Cover Date.     

Characterization of resistance to powdery mildew (Erysiphe pisi) in a germplasm collection of Lathyrus sativus

Lathyrus sativus has a considerable potential for dry land‐farming systems but can be severely damaged by powdery mildew infection. Little is known about the availability of resistance against powdery mildew and the underlying mechanisms in the Lathyrus genus. A range of resistance reactions was identified in a collection of Iberian‐cultivated L. sativus germplasm. In most cases a compatible reaction to powdery mildew with no macroscopically visible necrosis was observed. Nevertheless, accessions with reduced disease severity despite being of a high infection type have been identified. The genes responsible for this partial resistance will be potentially useful for the development of durable resistant cultivars of Lathyrus spp., and may also be of value for the improvement of other related cultivated leguminous plants.     

Inheritance of adult-plant resistance to Phytophthora capsici in pepper

Summary Inheritance studies were conducted to determine the genetic basis of adult-plant resistance in pepper (Capsicum annuum L.) to Phytophthora capsici. F₁, backcrosses and F₂ populations were developed using the resistant parent Criollo de Morellos 334 and susceptible parents Agronômico 10-G and Yolo Wonder. Pepper plants, at 36 days post-emergence, were inoculated near the base of the stem with an inoculum suspension of 5×10⁴ zoospores/ml. Segregation ratios in the F₂ generation of 13 resistant to 3 susceptible plants fit a 2-gene model for resistance with dominant and recessive epistasis.     

Monogenic resistance in tomato to Fusarium oxysporum f. sp. lycopersici race 3

Summary Resistance to fusarium wilt, incited by Fusarium oxysporum (Schlecht.) f. sp. lycopersici (Sacc.) Snyder & Hansen race 3 in tomato (Lycopersicon esculentum Mill.) was discovered in LA 716, a L. pennellii accession. A resistant BC₁F₃ breeding line, E427, was developed from LA 716. E427 was crossed with the susceptible cv. Suncoast and F₁, BCP₁, BCP₂ (to Fla 7155, a susceptible parent) F₂, F₃, and BCP₂S₁ seeds were obtained. Segregation for resistance following root dip inoculation over three experiments indicated a single dominant gene controlled resistance. Five of the 12 BCP₁S₁'s segregated more susceptible plants, whereas one of the 12 segregated more resistant plants than expected (P<0.05). Three of 23 F₃ lines segregated more susceptible plants than expected while 1 of the 23 had more resistant plants than expected (P<0.05). Segregation in all other lines fit expected ratios. Five of the 23 F₃'s were homozygous resistant which was an acceptable fit to expectations (P=0.1–0.5). The gene symbol I ₃ is proposed for resistance to race 3 of the wilt pathogen. Deviations from expected ratios in data reported here and for other breeding lines indicate an effect of modifier genes and/or incomplete penetrance. Plant age at inoculation and seed dormancy did not affect results.Florida Agricultural Experiment Station Journal Series No. 8101.     

Segregation distortion of Brassica carinata derived black rot resistance in Brassica oleracea

Three segregating F₂ populations were developed by self-pollinating 3 black rot resistant F₁ plants, derived from across between black rot resistant parent line 11B-1-12 and the susceptible cauliflower cultivar ‘Snow Ball’. Plants were wound inoculated using 4 isolates ofXanthomonas campestris pv. campestris (Xcc) race 4, and disease severity ratings of F₂ plants from the three populations were scored. A total of 860 arbitrary oligonucleotide primers were used to amplify DNA from black rot resistant and susceptible F₂ plants and bulks. Eight RAPD markers amplified fragments associated with completely disease free plants following black rot inoculation,which segregated in frequencies far lower than expected. Segregation of markers with black rot resistance indicates that a single, dominant major gene controls black rot resistance in these plants. Stability of this black rot resistance gene in populations derived from 11B-1-12 may complicate introgression into B. oleracea genotypes for hybrid production. This revised version was published online in July 2006 with corrections to the Cover Date.     

The transfer of triazine resistance from Brassica napus L. to B. oleracea L. II. Morphology,fertility and cytology of the F1 hybrid

Summary F₁ hybrids of triazine resistant Brassica napus and triazine susceptible B. oleracea were morphologically intermediate to the parent species. Of 49 hybrids examined, 44 had 28 chromosomes, two had 37, one had 38 and two had 56. The 38-chromosome plant was thought to be a matromorph, the others, A₁C₁C (28), A₁C₁CC (37) or A₁A₁C₁C₁CC (56) type hybrids. Pollen stainability averaged 9.0% in the sesquidiploid, 32.0% in the tetraploids and 89.5% in the hexaploids. All the interspecific hybrids were resistant to 1.0×10^-4 mol L^-1 atrazine. The sesquidiploid hybrids produced gametes with chromosome numbers ranging from 9 to 17 and the tetraploid hybrid gametes had chromosome numbers from 15 to 22. Most hybrids produced self-seed. The partial fertility of these hybrids may permit their backcrossing to one or both parents.     

Intergeneric crosses for the transfer of resistance to the beet cyst nematode from Raphanus sativus to Brassica napus

Summary The possibilities to transfer important traits and in particular resistance to the beet cyst nematode (Heterodera schachtii, abbrev. BCN) from Raphanus sativus to Brassica napus were investigated. For these studies B. napus, R. sativus, the bridging hybrid ×Brassicoraphanus (Raparadish) as well as offspring of the cross ×Brassicoraphanus (Raparadish) ×B. napus were used. Reciprocal crosses between B. napus and R. sativus were unsuccessful, also with the use of embryo rescue. Crosses between ×Brassicoraphanus as female parent and B. napus resulted in a large number of F₁ hybrids, whereas the reciprocal cross yielded mainly matromorphic plants. BC₁, BC₂ and BC₃ plants were obtained from backcrosses with B. napus, which was used as the male parent. F₁ hybrids and BC plants showed a large variation for morphology and male and female fertility. Cuttings of some F₁ and BC₁ plants, obtained from crosses involving resistant plants of ×Brassicoraphanus, were found to possess a level of resistance similar to that of the resistant parent. These results and indications for meiotic pairing between chromosomes of genome R with those of the genomes A and/or C suggest that introgression of the BCN-resistance of Raphanus into B. napus may be achieved.     

Pre and posthaustorial resistance to rusts in <Emphasis Type="Italic">Lathyrus cicera</Emphasis> L.

Lathyrus cicera has a high potential as fodder crop in dry areas, but can in particular environments be damaged by rust. Little is known on the availability of resistance against rust fungi and the underlying mechanisms in L. cicera germplasm. The present study assessed and characterised macro and microscopically the resistance to rust fungi Uromyces pisi and U. viciae-fabae, in a collection of L. cicera accessions. A wide range of disease reaction was found in the germplasm collection against the different rust species. L. cicera accessions were highly resistant to U. viciae-fabae being hypersensitive response the most frequent reaction. On the contrary, most accessions showed a compatible interaction with U. pisi, with varying levels of partial resistance, although cases of hypersensitivity were also identified. Differences on germination, orientated germ tube growth and appressoria differentiation were observed but were in general of marginal importance to explain the resistance to U. pisi among the L. cicera accessions. Resistance was due, to a combination of pre and post-haustorial mechanisms.     

Flatpea crossing methods and germplasm screening for reduced 2,4-diaminobutyric acid composition

Summary Flatpea (Lathyrus sylvestris L.) is a potentially valuable forage legume, but it contains high levels of 2,4-diaminobutyric acid (A₂bu), a compound that can have adverse effects on some animals, including rats and poultry. This study evaluated intercrossed pollination methodology needed for cultivar improvement and characterized intercrossed progeny for A₂bu concentration. Pollen germination on an artificial medium, using pollen collected during different stages of flower development, was used to identify appropriate stages for flower emasculation. Pollen dehiscence occurred at an early-bud stage when petal color was first evident. Slightly more mature buds contained pollen with substantially higher in vitro germination than pollen from early-bud stage flowers. Lines that produced high numbers of seeds per pollination were identified from crosses between flatpea accessions. Cross-pollination resulted in significantly higher seed set than selfing. Pollination using caged honeybees was the most efficient method of hybridization, although the degree of inbreeding in bee-pollinated plants could not be determined. Intercrossed progeny produced by honeybee pollination contained a lower mean concentration of foliar A₂bu than the mean for flatpea accessions.Abbreviations HPLC high performance liquid chromatography - OPA ortho-phthalaldehyde     

Chromosomal structural changes in Capsicum annuum L. and C. chinense Jacq.

Summary Wide hybridizations between M. arvensis and Brassica amphidiploid species (B. napus and B. juncea) were carried out in order to incorporate desirable traits of M. arvensis into Brassica crops. Crossing barriers between them were present without the use of in vitro techniques. F₁ hybrids have been produced through ovary culture, when M. arvensis were used as a female parent. Higher hybrid embryo productivity (3.07 embryos per pollination) was obtained in the cross of M. arvensis x B. napus than in that of M. arvensis x B. juncea (0.79 embryos). The hybridity was confirmed by morphology, cytology, isozyme and Southern analyses. The first backcrossing progenies and open pollinated ones were produced.     

Application of in vitro organ culture in wide-cross breeding of rapeseed

Oil radish (Raphanus sativus var. raphanistroides Makino) is resistant to drought and low temperature. In order to breed more resistant cultivars of rapeseed, the wide cross between rapeseed (Brassica napus L.) and oil radish was made. Rapeseed was not compatible with oil radish, and the frequency of hybrid plants (F₁) was very low. Moreover, the hybrid plants were sterile. In order to recover the intergeneric hybrids (F₁), the in vitro organ culture technique was applied in our experiments. The frequency of hybrid plants (F₁) was increased up to 25.55% by means of in vitro culture of pollinated ovaries. Some fertile amphidiploid hybrid plants were obtained by means of colchicine treatment of small buds obtained from cultured flower receptacle segments of hybrid plants (F₁). It is suggested that the technique of in vitro culture of pollinated ovaries and flower receptacle segments is useful in the wide-cross breeding of rapeseed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Selection for resistance to Plasmodiophora brassicae Wor. in oriental subspecies of Brassica rapa L.

Summary Selection for resistance to Plasmodiophora brassicae Wor. in oriental groups of Brassica rapa L.Two hundred and sixty-five cultivars of leafy, oriental bassicas were tested for resistance to 18 collections of Plasmodiophora brassicae, the causal agent of clubroot. The tests were conducted in the greenhouse at low and high level inoculum concentrations. Eleven cultivars of B. rapa pe-tsai, five cultivars of B. rapa pak-choy and three cultivars of B. rapa choy-sum consistently segregated for resistance at the lower concentration of inoculum (1000 spores/ml). All 265 cultivars were susceptible at the higher concentration (1 000 000 spores/ml). Three cultivars were used in pedigree and recurrent selection schemes for increased resistance. After three cycles of selfing resistant individuals, significantly more resistant S₃ lines were derived from each cultivar. Lines derived from two cultivars. Chinese White and PI 257236, continued to improve with each cycle of selection and demonstrated increased resistance to higher levels of inoculum (up to 1 000 000 spores/ml) New cultivars based on intercrosses of S₂ resistant individuals also had significantly better resistance than the original cultivar. After two cycles of selection in the third cultivar, PI 419007, resistance did not increase and its S₂ mass did not differ significantly from the original cultivar. Evidence that indicates resistance is pathotype-non-differential and offers an alternative to major gene, pathotype-differential types of resistance currently being introduced to the leafy oriental brassicas from other Brassica rapa groups.     

Production of interspecific hybrids between Glycine max and G. tomentella through embryo culture

Summary Interspecific hybrids have been obtained in an incompatible cross between Glycine max and G. tomentella through the in vitro culture of hybrid embryos. The percentage of successful pod setting in the crosses averaged 12.8% but there were marked differences depending on the soybean cultivar used as the female parent. Hybrid embryos at globular to heart stages were extracted from the embryo sac 15–25 days after pollination and cultured in vitro. Hybrid plants were successfully obtained by culturing the embryos on B5 medium supplemented with 0.1 mg/l IBA followed by culture on B5 medium supplemented with 0.1 mg/l TBA plus 0.25 mg/l 2-iP. The F1 plants resembled the wild male parent in growth form, but had an intermediate leaf shape between that of the parents.     

The transfer of triazine resistance from Brassica napus L. to B. oleracea L. III. First backcross to parental species

Summary Atrazine resistant Brassica napus × B. oleracea F₁ hybrids were backcrossed to both parental species. The backcrosses to B. napus produced seeds in both directions but results were much better when the F₁ hybrid was the pollen parent. Backcrosses to B. oleracea failed completely but BC₁s were rescued by embryo culture both from a tetraploid hybrid (2n = 4x = 37; A₁C₁CC) and sesquidiploid hybrids (2n = 3x = 8; A₁C₁C). Progeny of crosses between the tetraploid hybrid and B. oleracea had between 25 and 28 chromosomes. That of crosses between the sesquidiploid hybrid and B. oleracea had between 21 and 27. A few plants that had chromosome counts outside the expected range may have originated from either diploid parthenogenesis, unreduced gametes or spontaneous chromosome doubling during in vitro culture. Pollen stainability of the BC₁s ranged from 0% to 91.5%. All the BC₁s to B. oleracea were resistant to atrazine.     

Chromosomal location of resistance to Septoria tritici in seedlings of a synthetic hexaploid wheat, Triticum spelta and two cultivars of Triticum aestivum

Chromosomal location of resistance to two virulent Argentinean isolatesof Septoria tritici was studied in two wheat (Triticum aestivumL.) cultivars (Cappelle-Desprez & Cheyenne), a synthetic hexaploid(Synthetic 6x) and Triticum spelta in seedlings. Substitution lines of these(resistant or moderately resistant) genotypes into (susceptible) ChineseSpring were selected from a previous screening. For Synthetic 6x,resistance was clearly located in chromosome 7D. Chinese Spring with the7D chromosome substituted by Synthetic 6x showed almost completeresistance, similar to the level of Synthetic 6x. For the substitutions withCappelle-Desprez, Cheyenne, and T.spelta there were no lines with abehaviour similar to the resistant parent. However, some substitutions weremore resistant than the susceptible parent suggesting that severalchromosomes could be involved in the resistance of these genotypes toSeptoria leaf blotch.