The study of the autocompatibilidad of the almendro has been the main aim of this thesis, awarded with the Young Prize Researchers of the SECH
The self-compatibility in almond tree: genetic structure of the Sf allele and modifications of its expression
El almendro, ' Prunus amygdalus' Batsch, is a diploid species belonging to the genus Prunus. Although the self-incompatibility (AI) is a major character in the plant Kingdom from the evolutionary point of view, the self-compatibility (AC) is a very critical agronomic character to ensure production. In this way, the AC has become one of the main objectives of the almond programs around the world. Although the AC has been identified in some varieties of almond tree, is very rare (Socias i Company et al., 2010a). The methods used to determine the AC in the almond tree have been observation of pollen tubes and fruit set fruit after controlled Pollinations.
The new molecular technicians have gone applying increasingly during these two last decades. In this way, it has achieved determine the genotype of the varieties and screens of a very fast way and effective. The AC has related with the presence of the allele 'Sf', which belongs to the series alélica 'S', and is dominant on the others alleles of AI. Thus always it had considered that the AI/AC was a qualitative character and no quantitative.
Between the pertinent individuals of the cruzamiento of the varieties of almendro 'Vivot' x 'Blanquerna', only 25% have been AC, whereas the proportion expected was a lot of elder, 50%. A genotipado previous revealed that 'Vivot' possessed the haplotipos 'Sf
' and 'S23
' and 'Blanquerna' 'Sf
' and 'S8
', by what the two varieties would have to be AC, although his phenotypes showed to be different. This difference fenotípica carried to investigate the possible distinct mechanisms of incompatibility that could take part in these plants. The two different versions of the haplotipo 'Sf', an active and AI ('Sfa
') and the another inactiva and AC ('Sfi
'), have showed a mutual recognition, by what the Sfa-RNasa of the style of ‘Vivot' has prevented the growth of the tube polínico Sfi of ‘Blanquerna' in a population of 80 individuals. These results suggest the presence of a locus modificador no tied to the locus 'S', which would be responsible of the control of the AI/AC in this population. For this proposed a model of recognition whereby the locus modificador acts so that it agrees with the phenotypes obtained in our family (Fernández i Martí et al., 2009to).
Although lately it has deepened a lot in the study of the locus 'S' to molecular level (Hanada et al., 2009b), his mechanism of action follows without being of the all established, by what suggests that other external genes are necessary in the AI. Once proposed that a locus modificador could take part in the mechanism of the AC in this family, during this thesis has pretended locate it in the genome of the almendro. For this has realised a ‘genome scan' using a hundred of scoreboards microsatélites (SSR) distributed by the 8 chromosomes of the genome. After building the maps of ligamiento, llevar analysis of QTLs associated to the phenotypes obtained and surprisingly located 2 QTL in our screens. These results propose the location for the first time in the family of the rosáceas of two new loci situated out of the locus 'S', and that find in the groups 6 and 8 (Fig. 1). This allows to suggest that the AI is a quantitative character and no qualitative, as it considered to date (Fernández i Martí et al., 2011).
It appears 1: Map of ligamiento of Vivot' x ‘Blanquerna', including the position of the locus 'S' and of the 2 new loci identified in G6 and G8.
On the other hand, the construction of a genomic bookshop (fósmido) in ‘Vivot' and ‘Blanquerna' has allowed to check that so much the sequences of the Sf
-RNasas as of the SFBf
were entirely identical, in spite of producing two expressesions fenotípicas so distinct (Fernández i Martí et al., 2010b).
As it has not been possible to find any mutation between both Sf, in spite of a big quantity of technicians realised (NepHGE, Analysis of transcription, Southern Blot, etc.) has suggested the implication of the metilación of the DNA in this population (Fernandez i Marti et al., 2012, submitted). The metilación can produce changes in the phenotypes or in the expressesion of the genes without altering the sequences of the DNA. The essays llevar in the transcurso of this thesis using the treatment of bisulfito for the conversion of the cytosines suggest that the DNA metilado would be the manager of the activation and/or inactivación of the haplotipo 'Sf', since it has found a mutation of timina to cytosine in the region ‘upstream' of the RNasa.
In fact, it has been able to check that when the sequence of the Sf
-RNasa is metilada, this inactiva the expressesion, by what this inhibition would translate in an expressesion AC, as it is the case of ‘Blanquerna'. In the contrary case, if the sequence of DNA is not metilada, as it is the case of ‘Vivot', the RNasa remains active and to consequence the plant will be AI. This discovery puts in evidence that the epigenética has influenced of positive way in the evolution of the species and that possibly, in his origin all the varieties of the species of 'Prunus' would be in his incompatible form, but that with different mutations, some of them silenciadas, ones has turned into compatible and others no.
Also have wanted to tackle appearances of modelling of proteins along this work. For this has built the structure in 3D of the RNasas of the alleles 'Sf', 'S23' and 'S8' by means of tools bioinformáticas. The main difference found was that in the structure of the 'Sf' there was a longer bow that in the others two RNasas AI. In the bibliography has described that the big bows are susceptible to the degradation proteolítica, by what this same phenomenon could take place in these RNasas and have a repercussion to structural and functional level between compatible and incompatible alleles in almendro (Fernandez i Marti et al., 2012).
It appears 2: Diagram esteroscópico in 3D of the structure modelizada of the RNasas 'S8', 'S23' and 'S8' of the almendro, showing the elements of the secondary structure (to). Superficial representation of the structure modelizada of the same RNasas.
The construction of a genomic bookshop (fósmido) in 'Vivot' and 'Blanquerna' has allowed to check that so much the sequences of the Sf-RNasas as of the SFBf were entirely identical
On the other hand, in the transcurso of this thesis doctoral and of complementary way to the works realised to determine the car-compatibility, have wanted to evaluate other characters that begin to have a big importance so much for the program of improvement of the APPOINTMENT as from the nutritional punto, and obviously of big importance for the consumer. These characters are the related with the quality of the almond. During two years have evaluated in the same population of 'Vivot' x 'Blanquerna' diverse chemical components, like the acids grasos (oleico, linoleico, palmítico, esteárico and palmitoleico), tocoferoles (α and β), proteins or total content in oil. On the other hand they have evaluated of parallel way other physical characters of the seed and of the shell, like weight, width, geometrical diameter, size, etc. For all they, has determined for the first time in almendro his genetic control, finding around 25 QTLs with some percentages of upper explanation to 75% in the majority of the cases and covering practically all the genome of the almendro (Fernandez i Marti et al., 2012; Font i Forcada et al., 2012).
It appears 3: Map of ligamiento of 'Vivot' x 'Blanquerna', including the position of the QTL associated to the quality of the fruit.
Finally, it examined molecularmente the vegetal material of the bank of germoplasma of the almendro situated in the APPOINTMENT of Aragon, which is the one of reference of the FAO and the INIA, being one of the most important to world-wide level (Fernandez i Marti et al., 2009c).
This thesis has been multidisciplinary, in which they have worked with characters so important from the punto agronómico like nutritional (compatibility, sour grasos, tocoferoles, etc). All these results obtained are directly related with the aims of the project of Genetic Improvement of the Almendro that comes developing in the APPOINTMENT of Aragon and of the that have derived the varieties of almendro of greater success at present in the crop of the almendro in Spain and go to be serve to increase the efficiency of this line of investigation.
- Fernández i Martí To, Hanada, T., Alonso, J.M., Yamane, H., Tao, R., Partners i Company R., 2009to. To modifier locus affecting the expressesion of the S-RNase gene could be the cause of breakdown of self-incompatibility in almond. Sex. Plant Reprod. 22: 179-186.
- Fernández i Martí, To., Alonso, J.M., Espiau, M.T., Blond-Cabetas, M.J., Partners i Company, R. 2009b. Genetic diversity in Spanish and foreign almond germplasm assessed by Molecular characterization with SSRs. J. Amer. Soc. Hortic. Sci. 134: 535-542.
- Hanada, T., Fukuta, K., Yamane, H., Esumi, T., Gradziel, T.M., Dandekar, A.m., Fernández i Martí, To., Alonso, J.M., Partners i Company, R., Tao, R., 2009. Cloning and characterization of self-Compatible Sf haplotype in almond (Prunus dulcis (Mill.) D.To. Webb.). HortScience 44, 609-613.
- Fernández i Martí To, Hanada, T., Alonso, J.M., Yamane, H., Tao, R., Partners i Company R., 2010. The almond Sf haplotype shows To double expressesion despite its comprehensive genetic identity. Scientia Hortic. 125: 685-691.
- Partners i Company, R., Fernández i Martí, À., Kodad, Or., Alonso, J.M., 2010. Self-compatibility evaluation in almond: strategies, achievements and failures. HortScience. 45, 1-5.
- Fernandez i Marti To., Howad, W., Tao, R., Alonso, J.M., Arús, P and Partners i Company, R. 2011. Identification of QTL associated with self-compatibility in Prunus. Tree Genet. Genomes 7: 629-639.
- Fernández i Martí To, Wirthensohn M, Alonso JM, Partners i Company R, Hrmova M. 2012to. Molecular modeling of S-RNases involved in almond self-incompatibility. Frontiers in Plant Science (doi: 10.3389/fpls.2012.00139).
- Fernández i Martí To, Font i Forcada C, Partners i Company R. 2012b. Genetic analysis for physical nut traits in almond (2012). Tree Genet. genomes (doi: 10.1007/s11295-012-0566-8).
- Fernández i Marti To, Akagi T, Hanada T, Tao R and Partners i Company R. 2012c. Self-compatibility in Prunus species could be linked to DNA methylation. (submitted).
- Font i Forcada C, Fernández i Martí To, Partners i Company R. 2012. Mapping QTLs for kernel composition in almond. BMC Genetics 13:47.
- Partners i Company R, Kodad Or, Fernández i Martí To, Alonso JM. 2012. Pollen tube growth and self-compatibility in almond. Plants (doi:10.3390/plants10x000x).