Effetti a livello fenotipico, citogenetico e genomico conseguenti all'introgressione in frumento duro di segmenti cromosomici della specie affine Thinopyrum ponticum
Author(s)
Virili, Maria Elena
Date Issued
June 29, 2016
Type
Doctoral Thesis
Abstract
To meet the numerous challenges that wheat breeding is facing today, such as climate changes and rise in global food demand, a promising approach consists of widening the crop genetic basis by resorting to alien genes from wild wheat relatives. In fact, recent progress in chromosome engineering, through which alien chromosome segments can be transferred to wheat chromosomes, enables targeting of even complex traits, such as those underlying root system architecture, that are of great importance for yield performance of crop species. Previous studies on three durum wheat-Thinopyrum ponticum near-isogenic recombinant lines (NIRLs), containing 23%, 28% and 40% of their distal 7AL arm replaced by homoeologous portions of the alien 7AgL arm of the Thinopyrum ponticum (R5, R112 and R23, respectively), had indicated the presence of yield-contributing QTL. In the present study, root system architecture (RSA) and root and shoot biomass of the same NIRLs were evaluated. The study was conducted by using two different micro-methods, i.e. paper no-roll (PNR) and rhizotrons (RHZ). The results obtained from both methods showed that presence of 7AgL portions significantly enhanced several root traits involved in yield performance and nutrient uptake in the three NIRLs compared to their controls lacking any 7AgL segment. In particular, presence of the 5% 7AgL segment differentiating the NIRL with 28% 7AgL from that with 23% 7AgL, determined a significant increase in most of the analysed traits, such as spread of root angle (+17% and +37% with PNR and RHZ, respectively), average root diameter (+14% and +34%), root biomass (+31% and +38%) and radical apparatus length. The results obtained allow to functionally dissect the 40% 7AgL spanning the alien content of the three NIRLs, and hence to associate the genetic control of several root traits to defined 7AgL portions. The same comparative analysis has been conducted on bread wheat-Thinopyrum ponticum lines, such as the T4 translocation line (7DS.7DL/7AgL; the 7AgL segment of this lines include the segments present in the 3 durum wheat NIRLs) and the KS24 line, characterized by another 7AgL accession of the Thinopyrum ponticum (called 7el2L). Proves conduced in bread wheat confirmed 7AgL alleles specificity to determinate the positive contribution for analysed RSA traits. Were conducted further investigations, with paper roll (PR) method, on the same recombination/translocation lines of durum and bread wheat relating to architecture and root biomass in water stress conditions. It was confirmed the positive contribution of 7AgL segments to RSA traits even under limited water conditions (particularly for biomass and root length related traits).
A further aspect that the same materials above described have allowed to analyse concerned epigenetic alterations (particularly DNA methylation) potentially resulting from the introduction of alien genetic material in durum wheat genome. Little is known about epigenetic effects resulting from the transfer of small amounts of genetic material from wild relatives species, especially after several generations from the creation of such "partial hybrids". For the evaluation
of these changes, have been used an immunoassay vs 5-mC and the MSAP (Methylation Sensitive Amplification Polymorphism) method in different tissues (leaves, anthers) and physiological stages (Z1.3, Z2. 2-Z2.3, I and II mitotic division of micro-gametogenesis) of durum wheat-Th. ponticum recombinant lines. In particular the MSAP method allowed, through the use of three different enzymes with different sensitivity to methylated cytosines (EcoRI; MspI, HpaII) and 8 pairs of selective primers, to carry out both a quantitative analysis and a sequence-specific analysis of methylation changes in the four development phases and also between recombinant (HOM +) and control lines (not carriers of 7AgL segments, HOM-). Both methods have shown in the HOM+ and HOM- NIRLs a conservation of the ~ 90% for methylation profiles, even though there was hyper-methylation associated with the presence of wild segments. Through the MSAP system have been analysed 574 "GGCC" sites, which showed a different methylation both in relation to tissue and development phase but also in relation to the 3 7AgL segments; associated to the presence / absence of the 7AgL were identified 173 methylation polymorphisms, 123 of which were attributable to the recombinant R23. This recombinant showed, qualitatively and quantitatively, the main methyl alterations compared to R5 and R112 NIRLs. The results showed that the effects in genome-wide DNA methylation levels in genotypes derived from interspecific transfer of limited size chromosomal segments are mainly limited, at least to the BC4-5F7 generation; It was however highlighted a proportionality between size of the transferred 7AgL segments and extent of changes in the methylation pattern and furthermore has been suggested the possible involvement of the gene Sd (Segregation distortion), present in the 12% 7AgL exclusive of R23, in the most marked epigenetic modifications observed in the NIRL R23 HOM+.
Additional information
Dottorato di ricerca in Biotecnologie vegetali
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