A new source of resistance against coffee leaf rust from New-Caledonian natural interspecific hybrids between Coffea arabica and Coffea canephora

The development of cultivars resistant to coffee leaf rust caused by the fungal pathogen Hemileia vastatrix is a priority in coffee breeding. However, only very few descendants of interspecific hybrids between Coffea arabica and diploid relative species have been used as resistance source. Identification of new sources of resistance appeared therefore particularly worthwhile. Hybrid plants derived from interspecific hybridization between C. arabica and Coffea canephora and found in neo‐natural coffee tree populations of New Caledonia were therefore investigated. Amplified Fragment Length Polymorphism and microsatellites amplification were used to evaluate the genetic diversity of 14 hybrid plants, and rust resistance was evaluated by inoculation with a panel of rust races representing a large variability in virulence. An important genetic diversity was characterized in hybrid plants originating from introgressions into C. arabica from various C. canephora progenitors. On the 14 plants tested for leaf rust resistance, eight appeared resistant to all races investigated. Such plant material should represent a highly valuable resource for C. arabica breeding against coffee leaf rust.     

Detection by simple sequence repeat markers of introgression from Coffea canephora in Coffea arabica cultivars

Microsatellites or simple sequence repeats (SSR) were used to assess polymorphism among 16 Coffea arabica and four Coffea canephora accessions, and to identify DNA introgression fragments from C. canephora in four C. arabica lines. Thirty‐one primer pairs allowed for the identification of 92 polymorphic alleles distributed over 37 loci. The C. arabica accessions derived from the genetic bases ‘Typica’ and ‘Bourbon’ were grouped separately according to their genetic origin. Two genotypes derived from a spontaneous hybrid (C arabica×C. canephora) were classified with the C. canephora accessions from Central Africa. Coffea canephora from West Africa were separated from the other accessions studied. Four alleles related to introgression (i.e. present in C. canephora and introgressed lines, and absent in C. arabica) were identified. The SSR markers were used successfully for characterization of a particular cultivar (‘Veranero’) from Costa Rica, which is known for its late maturity.     

Genetic analysis of partial resistance to coffee leaf rust ( Hemileia vastatrix Berk & Br.) introgressed into the cultivated Coffea arabica L. from the diploid C. canephora species

The objective of this study was to identify polymorphic molecular markers associated with partial resistance to coffee leaf rust, Hemileia vastarix. A segregating F ₂ population derived from a cross between the susceptible Coffea arabica cv. Caturra and a C. canephora-introgressed Arabica line exhibiting high partial resistance was analyzed. Rust resistance measured as rust incidence (RI) and defoliation (DEF) was evaluated in field conditions in three consecutive years (2003–2005). During the 2003 season, which was characterized by favorable conditions for a rust epidemic, the F ₂ plants exhibited different levels of resistance ranging from very susceptible (50.1% for DEF and 49.5% for RI) to highly partial resistance (9.1% for DEF and 3.7% for RI). Molecular analysis enabled identification of seven polymorphic markers (5 AFLP and 2 SSR) exhibiting significant association with partial resistance. Coexistence of resistance homozygous alleles (RR) at codominant SSR loci was correlated with high resistance. This study is the first attempt to develop PCR-based sequence specific markers linked to partial rust resistance in coffee.     

Inheritance of coffee leaf rust resistance and identification of AFLP markers linked to the resistance gene

The most important disease of Coffea arabica is coffee leaf rust caused by the fungus Hemileia vastatrix. The purpose of this study was to characterize the inheritance of coffee resistance gene(s) to race II of this pathogen and to identify and map molecular markers linked to this trait. Different populations were used: F₂ (160 plants), BCr (20), and BCs (135), derived from a cross between the resistant genotype Híbrido de Timor UFV 427-15 and the susceptible cultivar Catuaí Amarelo UFV 2143-236 (IAC 30). The segregation analysis showed that the resistance of Híbrido de Timor to race II of the H. vastatrix is conferred by a single dominant gene. The amplification of 176 AFLP (Amplified fragment length polymorphism) primer combinations using bulked segregant analysis (BSA) allowed the identification of three molecular markers linked to the resistance gene. Genetic mapping of these three markers in the F₂ population indicated that they are distributed on both sides, flanking the resistance gene. The markers E.CTC/M.TTT405 and E.CGT/M.TGT300 were found linked to the resistance gene at 8.69 cM (LOD 18.91) and 25.10 cM (LOD 5.37), respectively, while E.CCT/M.TTC230 was localized on the other side of the gene, at 20.50 cM (LOD 6.15). These markers are the first rust resistance markers identified in Híbrido de Timor and can be useful for marker assisted selection in coffee breeding programs.     

Interspecific hybridization between Coffea arabica L. and tetraploid C. canephora P. Ex Fr. II. Meiosis in F1 hybrids and back crosses to C. Arabica

Summary Melosis was studied in Coffea arabica, in induced tetraploid C. canephora, in their F₁ hybrid (arabusta hybrid) and in backcross generations of the hybrid with C. arabica as recurrent parent. Irregularities were observed, consisting of univalents (especially in the arabusta hybrid), multivalents (especially in tetraploid C. canephora) and uneven distribution of chromosomes at first anaphase. Chromosome distribution was improved by backcrossing. Meiotic irregularities wer negatively correlated with pollen fertility.     

Interspecific hybridization between Coffea arabica L. and tetraploid C. canephora P. ex Fr. I. Fertility in F1 hybrids and backcrosses to C. Arabica

Summary The interspecific (C. arabica × tetraploid C. canephora) F₁ hybrid showed a low but highly variable fertility. An almost complete restoration to normal fertility was observed in individual trees of BC₁ and BC₂ generations with C. arabica as recurrent parent. From a comparison of various tests of fertility, it was found that pollen viability is a satisfactory measure of fertility.     

Production of a new low-caffeine hybrid coffee and the biochemical mechanism of low caffeine accumulation

The GCAs are new tetraploid interspecific hybrids developed in Madagascar from Coffea eugenioides, C. canephora and C. arabica. Selected GCA having genotype UF1023 contained 0.37% DW caffeine and no detectable theobromine in green beans. Low caffeine accumulation in GCA plants is due mainly to the low biosynthetic activity of purine alkaloids, possibly the extremely weak N-methyltransferase reactions in caffeine biosynthesis. No significant catabolic activity of caffeine was found in GCA-UF1023, in common with almost all coffee plants including C. arabica.     

Genetic diversity for RAPD markers between cultivated and wild accessions of Coffea arabica

Summary Random amplified polymorphic DNA (RAPD) markers have been successfully employed to analyse the genetic diversity among cultivated and subspontaneous accessions of Coffea arabica. The narrow genetic base of commercial cultivars was confirmed. On the other hand, a relatively large genetic diversity was observed within the germplasm collection demonstrating the importance of collecting missions. Results suggested an East-West differentiation in Ethiopia, the primary centre of diversification of C. arabica. The large heterosis effect reported in intergroup hybrids could be related to such genetic differentiation. RAPD method appeared to be effective in resolving genetic variations and in grouping germplasm in C. arabica.     

Stable resistance against the leaf miner <Emphasis Type="Italic">Leucoptera coffeella</Emphasis> expressed by genetically transformed <Emphasis Type="Italic">Coffea canephora</Emphasis> in a pluriannual field experiment in French Guiana

Summary A pluriannual field trial of transgenic clones of Coffea canephora (the Robusta coffee tree) transformed for resistance to the lepidopteran coffee leaf miner Leucoptera coffeella was installed in French Guiana. Fifty-eight transformed clones produced by transformation of the C. canephora clone 126 were planted. They were harbouring the pEF1α constitutive promoter of Arabidopsis thaliana controlling either the Bacillus thuringiensis native gene for the cry1Ac insecticidal protein (eight clones) or a synthetic cry1Ac gene (53 clones). The vectors for the transformation were a strain of the bacterium Agrobacterium tumefaciens and one of Agrobacterium rhizogenes. The transformed clones were generally independent, presenting different integration patterns of the genetic construct. Four randomly distributed groups of five plants per transformed clone were planted along with 60 untransformed control trees. Over a 4-year period after plantation six releases of L. coffeella were performed. Mines on the leaves are the marks of larvae development and were counted on plants. A majority of the independent transformed clones harbouring the synthetic gene and transformed by the strain of A. tumefaciens displayed constantly much less mines than the control, therefore expressing a stable resistance. The need for complementary research is presented.     

Studies on Pigmentation of Fruits in Derivatives of Crosses between Cucurbita maxima Duch. ×C. moschata Poir.

Precocious fruit pigmentation was found to be recessive in Cucurbita maxima. When precocious C. maxima cultivars were crossed with C. moschata and C. equadorensis, the trait was found to be dominant. Following the introduction of precocious pigmentation into C. moschatta cv. ‘Waltham Butternut’, the character was found to be recessive. In addition, foliage yellowing, spotting and leaf variegation were associated with the character. At fruit maturity two new fruit colors were obtained; orange and green, vs the typical cream color of the cv. ‘Waltham Butternut’. It is suggested that at least two complementary genes from both parental species control orange fruit color, and a’ single gene is responsible for mature green pigmentation. Improvement of C. moschata cultivars is aimed at by assembling the beneficial effects of the various characters.     

Doubled haploids of Coffea canephora: development,fertility and agronomic characteristics

Summary Doubled haploids (DH) of Coffea canephora Pierre were developed using haploid embryos which occur spontaneously in association with polyembryony. The frequencies of polyembryonic seeds and haploid embryos varied according to the parental genotypes. However, production of a large number of DH seemed possible from all genotypes. More than 750 DHs produced from various genotypes were grown under field conditions and evaluated for different characters of agronomic importance. Approximately half of DH genotypes did not survive, suggesting a strong, negative effect of homozygosity. Inbreeding depression is particularly severe on general vigor and reproductive aspects. For several characters studied such as leaf shape, leaf rust resistance and hundred bean weight, considerable genetic variations were observed within and between groups of DHs constituted by the DHs produced from the same clone. Despite their low vigor and reduced fertility, the DHs of C. canephora offer new possibilities in genetic research and coffee breeding.     

Additional evidence for oligogenic inheritance of durable host resistance to coffee berry disease (<Emphasis Type="Italic">Colletotrichum kahawae</Emphasis>) in arabica coffee (<Emphasis Type="Italic">Coffea arabica</Emphasis> L.)

Breeding for host resistance to coffee berry disease (CBD) in arabica coffee (Coffea arabica) was initiated some 35–40 years ago in Kenya, Ethiopia and Tanzania in response to severe CBD epidemics. The release of CBD resistant cultivars to the coffee growers has been in progress since 1985. The resistance of cultivars like Ruiru 11 (Kenya) and Ababuna (and other cvs in Ethiopia) appears to be of a durable nature, since confirmed cases of a breakdown of host resistance under field conditions have not been reported over the past 20 years. Host resistance to the hemibiotrophic fungus Colletotrichum kahawae is of a quantitative nature, but nevertheless can be practically complete in some genotypes of arabica coffee. There is still no consensus on the genetics of CBD resistance, some claiming convincing evidence for oligogenes (1–3 major genes) and others for polygenes determining CBD resistance. Results from genetic studies with germplasm from the centre of genetic diversity for C. arabica in Ethiopia are presented here. These together with the recent identification of molecular markers associated with and the mapping of one major gene, provides additional evidence for oligogenic inheritance of CBD resistance. The development of cultivars combining yield and quality with durable host resistance to CBD has contributed greatly to increased sustainability of arabica coffee production in Africa. It has also considerable relevance to arabica coffee in Latin America and Asia, where CBD is still a quarantine disease but with a risk of becoming endemic one day, just as has happened earlier with coffee leaf rust (Hemileia vastatrix).     

Identification of a major QTL for adult plant resistance to coffee leaf rust (Hemileia vastatrix) in the natural Timor hybrid (Coffea arabica x C. canephora)

Most of the commercial varieties of coffee (Coffea arabica L.) derived from the Timor hybrid (TH) have been shown to contain major genes for coffee leaf rust (CLR) resistance. To identify markers tightly linked to such genes, an F₂ mapping population derived from a cross between ‘Caturra’ (susceptible variety) and the TH‐derived DI.200 line (highly resistant) was generated. Using expressed sequence information and a bioinformatics approach, both targeted region amplified polymorphism (TRAPs) markers and simple sequence repeat (SSR) markers were identified. Phenotypic evaluations in the field and under controlled conditions confirmed the existence of one quantitative trait locus for CLR resistance. Four candidate SSR markers were associated with high CLR resistance. They spanning a region of 2.5 cM designated Q_{CLR_4} located within chromosome 4 of the international C. canephora map. The presence of this region was confirmed in a set of elite lines and commercial varieties. The Q_{CLR_4} region corresponds to a new and genetically independent S_H locus that could potentially be useful in gene pyramiding with other genes to enhance rust resistance in TH derivatives.     

Expression of resistance to Atherigona soccata in F1 hybrids involving shoot fly-resistant and susceptible cytoplasmic male-sterile and restorer lines of sorghum

In recent years, cytoplasmic male‐sterility (CMS) has been recognized as a potential danger to the stability of crop production and resistance to insect pests in sorghum. Therefore, the influence of CMS on the expression of resistance to sorghum shoot fly was studied at the ICRISAT, Patancheru, India using the interlard fishmeal technique. The experimental material consisted of 12 restorer, 12 CMS and the maintainer lines, and their 144 F₁ hybrids. Shoot fly‐resistant CMS lines were preferred for oviposition and had more damage because of deadhearts than the corresponding maintainer lines. The hybrids based on shoot fly‐resistant CMS × resistant restorer lines were significantly less preferred for oviposition than the hybrids based on other cross combinations and exhibited the highest frequency (69.1%) of shoot fly‐resistant hybrids. The hybrids based on glossy and trichomed parents had the highest frequency (>90%) of hybrids with glossy and trichome traits, emphasizing the need to transfer these traits into both parents for better expression in the F₁ hybrids. The expression pattern of trichome density, leaf glossiness and leaf sheath pigmentation in the F₁ hybrids and their parents suggested that the interactions between cytoplasmic and nuclear genes possibly control the expression of traits associated with resistance to sorghum shoot fly in the F₁ hybrids.     

Combining Ability of Doubled Haploids in Coffea canephora P.

Fifty-five doubled haploids (DH) of Coffea canephora were crossed with either heterozygous genotypes or DH in order to study their combining ability. Three agronomic trials were established. Marked hybrid vigour was observed for all characters analyzed including yield. Large differences were evident among top-crosses involving different DH produced from the same parental clone reflecting the high level of heterozygosity of clones. Factorial mating design analysis indicated that all genetic variance was attributable to additive effects in estimates of yield as well as plant height and leaf characteristics. The general combining ability variance component was also predominant for stem girth and susceptibility to leaf rust, although effects due to interaction were detected. Some hybrid combinations had yield comparable to standard clonal varieties. The implications of such results for breeding of Coffea canephora are discussed. Particularly, the development of F₁ hybrid varieties is envisaged.     

Improvement of seed yield in Vicia faba L. By using experimental mutagenesis. II. Comparison of gamma-radiation and ethyl-methane-sulphonate (EMS) in production of morphological mutants

Summary Gamma-rays and EMS have been used in two dosages to test their effectiveness in inducing mutations in one major (cv. Aguadulce) and one minor (cv. Manfredini) Vicia faba cultivar. The plants of the M₁ and M₂ generations showed reduced emergence, survival, and fertility. EMS was more effective than gammarays in inducing mutations giving a mutation frequency in M₁ and a frequency of M₂ mutants from two to four times higher. The two mutagenic agents do not seem to differ in mutation spectrum. The minor cv. Manfredini was more sensitive to the lower dosage of gamma-rays than the major cv. Aguadulce. Both mutagens proved to be effective in enlarging the morphological variation of both cultivars.Research work supported by C.N.R. special grant I.P.R.A.-Sub-project 1. Paper N. 515.     

Medium, Explant and Genotype Factors Influencing Shoot Regeneration in Oilseed Brassica spp.

The effects of culture media, explants and genotypes on shoot regeneration in oilseed Brassica species were examined in this study. The maximum shoot regeneration frequency was obtained in Murashige and Skoog medium supplemented with 3 mg l^?1 6‐benzylaminopurine and 0.15 mg l^?1 1‐naphthaleneacetic acid. The addition of 2.5 mg l^?1 AgNO₃ was very beneficial to shoot regeneration in B. napus and Ag₂S₂O₃ (10 mg l^?1) was even superior to AgNO₃ (2.5 mg l^?1). Explant age, explant type and carbon source also significantly affected shoot regeneration. Four‐day‐old seedlings of cotyledonary explants showed the maximum shoot regeneration frequency and number of shoots per explant. Of the four explants – peduncles, hypocotyls, cotyledons and leaf petioles – cotyledons produced the highest shoot regeneration frequency (56.67 %). Four carbon sources – glucose, maltose, starch and sucrose – were compared for their respective effects on shoot regeneration from cotyledonary explants. Sucrose appeared to be the best carbon source for shoot regeneration with the highest shoot regeneration frequency (76.00 %). Considerable variation in shoot regeneration from cotyledonary explants was observed both between and within Brassica species. The shoot regeneration frequency ranged from 10.00 % for cv. R5 (B. rapa) to 83.61 % for cv. N1 (B. napus). Two B. napus, one B. carinata and one B. juncea cultivars exhibited shoot regeneration frequency higher than 70 %. In terms of the number of shoots produced per explant, B. rapa showed the highest variation, ranging from 5.64 for cv. R3 to 1.33 for cv. R5. Normal plantlets were regenerated from all induced shoots and developed normally. The regenerated plants were fertile and identical with the source plants.     

Genetics of disease resistance in urdbean (Vigna mungo (L.) Hepper) to the leaf spots caused by Colletotrichum truncatum (Schw.) Andrus & Moore

Summary The resistance sources among various test cultivars of urdbean to Colletotrichum truncatum, a leaf spotting pathogen, were identified and genetics of resistance was worked out by studying F₁, F₂ and F₃ generations of crosses between resistant cultivars and the susceptible cv. Kulu 4 and of those among the resistant parents. The resistance was found to be controlled by single dominant genes and the resistance genes were non-allelic.     

Genome organization in coffee as revealed by EST PCRRFLP,SNPs and SSR analysis

An EST-based PCR-RFLP method was employed to gain insight into genome organization in eight allopolyploid Coffea arabica cultivars and seven diploid coffee species. The PCR-amplified products at 19 EST loci were digested with 46 different restriction enzymes and size fractioned in agarose gels. Most often, the sum of the fragments length was double or more than the PCR product. In arabica, this condition could be explained by assuming the presence of duplicated loci in paralogous chromosomes and this was supported by considerable evidence of multiple loci SSR amplification. Based on the RFLP analysis, 12 EST loci were polymorphic. The level of polymorphism was higher in different species compared to the arabica varieties. Sequencing of the amplified products revealed a SNP frequency of 0.021 among diploid species and of 0.007 among arabica varieties. We propose that the involvement of two genomes in C. arabica maintains a residual level of heterozygosity in the form of paralogous chromosomes, while the self-fertilization in this species tends to drive of homozygosity. The heterozygosity of paralogous chromosomes in arabica creates valuable polymorphism essential for species diversity and survival in various ecological niches, while self-fertility tends to preserve in homozygosity many genes of functional significance.     

Assessment of quantitative resistance of rice cultivars to Xanthomonas campestris pv. oryzae: A comparison of assessment methods

Summary Lesion length, leaf length, and leaf width were measured on infected leaves two weeks after clip inoculation of 64 rice cultivars with two virulent isolates of Xanthomonas campestris pv. oryzae (X.c. pv. oryzae). No significant correlation was found between the lesion length and the leaf dimensions, indicating that physical leaf size does not affect the spread of the bacteria once these have entered the leaf. Lesion length is therefore an acceptable parameter for assessing resistance to (X.c. pv. oryzae), and is to be preferred above the parameter percentage diseased leaf area (% DLA), especially when small differences between genotypes are to be assessed. The confounding influence of differences in leaf length can cause large changes in the ranking order of cultivars when assessed by the % DLA. For this reason lesion length is a better assessor of the value of a quantitative resistance for breeding and research purposes than % DLA.