Biodiversità genetica nelle specie Juglans Regia L, Juglas Nigra L e nei relativi ibridi interspecifici
Author(s)
Pollegioni, Paola
Date Issued
February 10, 2009
Type
Doctoral Thesis
Abstract
Juglans nigra (Eastern black walnut) and Juglans regia (common or Persian walnut) are highly economically important species. The Persian walnut, an indigenous species in Eurasia from the Balkans to southwest China, is cultivated throughout the temperate regions of the world for its high quality wood and edible nuts. Black walnut, native to the Eastern part of North America, is a fast growing species with a dark-colored wood used in the manufacture of furniture and other wood products. Both species are monoecious and heterodichogamous, with the same number of chromosomes. Although phylogenetic analysis has demonstrated that black walnut and Persian walnut belong to different sections of genus Juglans, a hybrid between them, Juglans x intermedia Carr. (J. nigra x J. regia), can occur naturally. However the hybridization between black and Persian walnut is not common in nature, because of temporal separation between flower maturity of both parent species and some genetic incompatibility mechanisms as failure of fertilization (pre-zygotic factors) and embryo abortion (post-zygotic factors). The identification and selection of “hybridogenic” parents is the first step to obtain hybrid progeny. Although some hybridogenic trees were detected, controlled crosses remained yet unsuccessfully. Nevertheless, seed orchards were established in France generating the so-called NG23 x RA walnut hybrid. These open-pollinated seed orchards involved the deployment of one J. nigra plus tree as female parent and four J. regia plus trees as male parents to ensure enough pollen pressure. The identification of parental plants by phenological observations and their clonal propagation by grafting started in the 1980s and required more than ten years. Compared to the parental species, most J. x intermedia hybrids show increased vegetative vigour, soil adaptability, good wood quality, greater winter-hardiness and less susceptibility towards to pathogenic infection than J. regia. Common walnut is susceptible to the damage caused by pests, flooding, drought and resulted susceptible to some walnut disease as anthracnose, bacteriosis and bark cancer. Particularly the anthracnose caused by Gnomonia leptostyla (Fr.) Ces. is one of the most important diseases of walnut for timber production; symptoms develop on leaves, stem and fruit as irregular necrotic areas that are often surrounded by small chlorotic halos. In severe cases, these lesions may cause premature defoliation , fruit drop or poorly filled nuts. The selection of resistant genotypes toward the anthracnose disease could represent a valid alternative to the cultural and chemical (fungicides) management. In black walnut complete resistance to the anthracnose fungi has not been found although a wide range in susceptibility exists; studies indicated that the natural resistance to G. leptostyla is highly heritable, encouraging genetic breeding programs in walnut. Most resistance to pathogens in plants is based on gene-for-gene model: an R-gene product recognises a pathogen gene product in the host, conferring full or partial resistance. Plant disease (R) genes fall into four mainly families, but the vast majority of them are members of the cytoplasmic nucleotide binding site -leucine rich repeat (NBS-LRR)-containing R-gene family. The NBS region of the characterized R genes contain several highly conserved motifs including P-loop, kinase -2 and GLPL motif. The NBS-LRR proteins seem to be involved in recognizing pathogens and activating signal transduction pathways to induce defences responses. Specific pathogen recognition governed by receptor-like R gene products usually lead to a hypersensitive response, associated with the coordinated induction an integrated set of defence responses as cell wall rigidification, the extracellular generation of reactive oxygen species (ROS), accumulation of phenolic compounds, synthesis of phytoalexins, deposition of papillae and accumulation of pathogenesis-related proteins. The effect of juglone (5-hydroxy-1,4-naphoquinone) and his precursor hydrojuglone glucoside on anthracnose in black walnut were correlated with a partial resistance typical of juvenile leaves. Although the mechanisms underlying such results are still poorly understood, we supposed that the resistance to G. leptostyla has been classified as “race-specific resistant” mediated by an interaction between R-gene product and avirulant pathogen ligand. Recently a new strategy was proposed to amplify a large collection of R-gene and R-gene Analogues (RGA) fragments. The NBS-profiling approach is based on PCR amplification using an adapter primer for an adapter matching a restriction enzyme site and a degenerate primer targeting the conserved domains present in the NBS region. In this way it is possible to obtain DNA profiles with markers mainly present in R-genes and RGAs. The aims of this work consisted of (1) Application of ten microsatellite nuclear markers for the genetic characterization of J. nigra, J. regia, and new J. x intermedia plants in a mixed Italian population (Villa Mezzalira’s Park). In particular, for the first time this research was also designed to confirm the transferability of ten black walnut SSR loci to Persian walnut. (2) Retrospective selection of hybridogenic walnut trees by microsatellite parentage analysis of half-sib families collected in the Villa Mezzalira’s Park. Verify if this short term marker-assisted breeding tactic could represented an alternative way to
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detect new hybridogenic genotypes for walnut hybrid breeding programs. (3) Preliminary genetic analysis of Italian walnut germplasm (Azienda Ovile, CRA-ISPIO, Unità di Ricerca Forestale, Rome) using functional molecular markers tightly linked to R-gene and RGAs to detect genotypes resistant to anthracnose: application for the first time of NBS-profiling approach on forest species.
Ten microsatellites tested in the mixed walnut population collected in Villa Mezzalira’s Park amplified in both species, producing fragments of variable size; eight (7.14 %) were common, 68 (60.7 %) amplified in J. nigra and 36 (32.1 %) in J. regia only (private alleles). Indices of genetic diversity revealed high level of variability. The Principal Coordinate Analysis on the basis of total 112 alleles divided the total sample set (138 plants) into three main groups: J. nigra (82), J. regia (49) and J. x intermedia hybrids (7). Performing the microsatellite fingerprinting, a triploid hybrid plant with two genome parts of J. nigra and one part of J. regia was identified (N21). The cytological analysis proved this triploid state showing 48 somatic chromosomes. The mother testing analysis of the 7 diploid hybrids by exclusion method indicated one putative hybridogenic mother plant (N17). The sequence analysis of amplified fragments confirmed the cross-species amplification of SSR. Inter-specific differences between alleles were due not only to simple changes in the number of repeats but also to mutations in the flanking regions.
The same battery of 10 SSR primer pairs was used to perform the DNA fingerprinting and parentage tests of 461 progeny individuals (eight half-sib families) collected in the Villa Mezzalira’s Park. The high levels of polymorphism detected positively influenced the exclusion and identity probabilities. The assignment analysis revealed the presence of 198 diploid J. x intermedia hybrids among the total of the progeny seedlings (42.9%). Maternity checks were carried on all individuals and few errors of sampling (0.06 %) were found. Four distinct hybridogenic mother trees were identified: as expected N17 but also N23, N24 J. nigra genotypes and the triploid hybrid plant N21. These three black walnut plants showed a different reproductive success rates. Paternity of 198 diploid hybrids detected in four open-pollinated families were inferred by using the most-likelihood approach based on nine microsatellite loci. Differential male reproductive success was observed among pollen donors within the research site. In the production of hybrid progeny the male reproductive success was unevenly distributed both in amount and in space. In particular 49 (47.5 %) of the total diploid hybrids detected in four half-sib families were sired by only three J. regia genotypes. Distance and orientation between trees may influence pollination, but it is probably not a major determinant of pollen success. The timing of pollen release and genetic incompatibilities could be a plausible explanations for the observed fertilization pattern. This retrospective selection may represent an alternative measure to the traditional approach offering a method to identify new parental combinations at a reasonable cost, with no phonological and morphological data available of the trees.
A total of 132 Juglans plants, conserved in Azienda Ovile, were sampled and analyzed using neutral (SSRs) and functional markers (NBS). This germplasm was compared to 597 plants located in the Villa Mezzalira’s Park and already genotyped. Microsatellite fingerprinting analysis of 132 samples confirmed the former classification based on morphological observations; the assignment analysis classified 63 genotypes as common walnut (J. regiaRA) and 69 samples as black walnut (J. nigraJN). In addition according to the percentage of leaf necrosis area these genotypes were further divided in three main group: (68) J. nigraJN resistant, (1) J. nigraJN susceptible, and (63) J. regiaRA susceptible to anthracnose. Phenological observations indicated that the different leaf emergence rates noted in this study (J. nigra and J. regia was late and early emerging species respectively) could have some association with resistance to anthracnose. Nevertheless this hypothesis didn’t justified the presence of a susceptible genotype J. nigra. A total of 4 primer NBS-enzyme combinations (RsaI-NBS1, RsaI-NBS5A6, MseI-NBS1 and MseI-NBS5A6) amplified in Juglans spp. These primers were designed from a part of the conserved P-loop motif for NBS1 and of kinase-2 for NBS5A6. Out of the total (320 bands), 48 fragments were common, 144 fragments amplifying in J. nigra and 128 in J. regia only. A deeply analysis of NBS marker distributions revealed the presence of a putative candidate R-gene linked marker correlated to resistance/susceptibility to anthracnose in walnut. In the NBS1-RsaI combination, all susceptible plants, 63 J.regiaRA and one J. nigraJN, showed a common band of 110 bp in length which was not present on the 68 resistant J.nigraJN plants.
Additional information
Dottorato di ricerca in Ecologia forestale
Subjects
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