Final Report Summary - BREED4FUTURE (Breeding for a sustainable agriculture: quality and resistance in the frame of H2020)
The project titled “Breed4Future” aims by using the state of the art of genome sequencing technologies, to identify the casual genes associated with disease resistance in a sustainable agriculture and understanding how plants defend themselves from pathogens. The specific research objectives proposed were: (i) to identify molecular mechanisms determining disease resistance across the Compositae and (ii) to establish syntenic relationships within the family as well as with other plant species (Rosaceae) and (iii) to gain expertise in excellent research to be applied in commercial crops to reduce the gap between research and market in the landscape of H2020.
The period of the Outgoing phase at the Genome Center UCDavis was mainly focused on research training activities of Dr. Fernandez as mentioned in Annex 1, part B of the Breed4Future project. The first part was oriented in learning molecular and bioinformatics tools. The second part consisted in the literature review on resistance genes and plant genome assembly methods. The fellow gained experience by using available database from 25 plant genome sequences, such as Arabidopsis thaliana, Zea mays, Populus trichocarpa, Lactuca sativa, Theobroma cacao, Medicago truncatula, Vitis vinifera, Prunus persica, Fragaria vesca, Carica papaya, Solanum lycopersicum, Citrus clementina, and many others. These genomes were used for molecular evolution of resistance genes, comparative genomics and DNA variation (SNP, SSR and CNVs). Dr. Fernandez also generated REN-SEQ libraries for 10 lettuce cultivars. The aim of this task was dual. First of all, he wanted to set up a new protocol called REN-SEQ (Sequence Enrichment for Targeted Sequencing), by using the two most powerful NGS platforms “Illumina” and “PacBio”. The second aim was the re-annotation of the NB-LRR gene family from sequenced plant genomes and rapid mapping of resistance loci in segregation populations. This method enables discovery and annotate of new pathogen resistance gene family members in plant genome sequences.
On the other hand, we were able to identify and classify for the first time in the compositae family the LRR-RLK genes, which are associated to many important roles in plants, such as growth and cell development and defense response. Although RLK subfamilies have been previously studied in some plant species, no comprehensive study has been performed on this gene family in lettuce, which has a high economic importance and is frequent targets for emerging pathogens. Within Breed4futre, we performed an in silico analysis to identify and classify LRR-RLK homologues in the predicted proteomes of many plant species, such as arabidopsis, citrus, tomato, rice, peach, almond, tobacco, papaya or grape. In addition we used large-scale phylogenetic approaches to elucidate the evolutionary relationships of the LRR-RLKs and their cell surface immune receptors.
In accordance with the goals of the second part of the project in Spain, Dr. Fernandez, taking part of an international consortium, has sequenced the novo genome of almond, and re-sequence 48 varieties at 16X using Illumina, PAcbio and Oxford Nanopore platforms. On the other hand, through bioinformatics tools, we have identified the major genes that are supposed to confer resistance to Xanthomonas in almond. I carried out an in silico analysis in order to establish orthology with genes from this model species and that may be associated with Xap in the almond ‘Forastero’ cultivar. The sequence similarity of Xa25, Xa26 and Xa29, which are the major genes conferring resistance to Xanthomonas oryzae pv. oryzae in rice, and encoding NB-LRR proteins, were blasted (p) against the genome of the almond ‘Forastero’. Three relevant hits corresponding to the genes pdulcis7A006414P1, pdulcis7A010546P1 and pdulcis7A007653P1 were retrieved. Additionally, I investigated and blasted those almond genes against the peach reference genome, which is another significant Prunus species. In fact the three hits, identified in peach to ppa018004m, ppa019022m and ppa026334m genes, correspond to proteins with an NB-LRR domain as showed in the Rosaceae peptide database (http://pathways.rosaceae.org).
Dr. Fernandez has also been investigating the gene family associated to drought tolerance in Prunus. A previous root transcriptomic analysis by Illumina in an almond x peach hybrid rootstock served us to identify a number of drought-responsive gene families. The goal was to study the evolution of these gene families among different woody plant species. This approach included 15 genes belonging to the Homeodomain superfamily, 12 genes encoding different Heat Shock Proteins (HSPs), 12 genes encoding several Late Embryogenesis Abundant (LEA) proteins and 7 genes encoding different Dehydration-Responsive Element-Binding (DREB) transcription factors (TFs). The drought stress genes were extracted from genome assemblies of nine plant species (Prunus dulcis, P. persica, Malus domestica Festuca vesca, Camellia sinensis, Arabadopsis. thaliana, Quercus rubra, Pinus taeda and P. lambertiana) representing higher plant diversity and used for phylogenetic analysis by using the maximum-likelihood approach (RAxML). The resultant phylogenetic trees exhibited different patterns of evolution. There has been expansion of specific gene families. In some cases, expansion happened prior to speciation, whereas in other cases, it happened afterwards. Further approaches are being undertaken in order to characterize the evolutionary pathway of drought stress genes in the Rosaceas and the other plant species.
The role of key-flowering genes in Prunus dwarfing rootstocks has also been studied during this third year of the Marie Curie project and led us to identify genes that will be applied shortly in our almond and peach breeding programs. Although Spain is placed as the third almond producer`s country after USA and Australia, the crop has suffered a drastic change with a high density planting system and a low vigour rootstocks. This will represent an important challenge in order to understand how the trees develop and how different varieties and rootstock adapt to these moderns planting systems. In this Breed4future project, we have studied the vigour of the trees at molecular level of two different rootstocks, “Rootpac-40” and “Garnem”, representing different vigour with and without the scion. Additionally we have carried out a phenotypic study by using several combinations rootstock/scion in a commercial orchard. For studying the molecular bases we have approached the expression of a gene family FT/TFL1 responsible for inducing and repressing the flowering time with complementary functions, which aimed to reduce the juvenile period of the tree. For phenotyping we have monitored four traits in order to increase our knowledge of the tree development: shoot length, TCSA (Trunk Cross-Sectional Area), number of flower buds and number of vegetative buds. Expression profiles through quantitative PCR of the four genes studied (FT1, BFT, TFL1 and MFT), which are associated to flowering time, revealed that the genes TFL1 (ppa012369m; delay flowering) and BFT (ppa021829m; delay flowering) presented different expressions among rootstocks without the scion grafted in “Rootpac-40”, whereas in “Garnem” no differences at expression level were observed. On the other hand, FT1 (ppa012320m; advance flowering) and MBF (ppa012388m; delay flowering) showed the same expression profile in rootstocks with and without the scion “Rootpac- 40” and “Garnem”. Further studies are undertaken in order to elucidate the physiological function of those genes in Prunus.
The understanding and identification of genes conferring disease resistance in the Rosaceae, as well as resistance to drought tolerance will help in designing new breeding strategies in these crop families. Hence, these important findings along with the use of a more sustainable approach, such as CRISPR/Cas9, will be one of the solutions to reduce the global warming impact and improve human nutrition and health. Consequently, developing plants that confer disease resistance to Xap and other pathogens will reduce considerably the use of chemical products, which are widely known for their negative impact on health and environment.
The period of the Outgoing phase at the Genome Center UCDavis was mainly focused on research training activities of Dr. Fernandez as mentioned in Annex 1, part B of the Breed4Future project. The first part was oriented in learning molecular and bioinformatics tools. The second part consisted in the literature review on resistance genes and plant genome assembly methods. The fellow gained experience by using available database from 25 plant genome sequences, such as Arabidopsis thaliana, Zea mays, Populus trichocarpa, Lactuca sativa, Theobroma cacao, Medicago truncatula, Vitis vinifera, Prunus persica, Fragaria vesca, Carica papaya, Solanum lycopersicum, Citrus clementina, and many others. These genomes were used for molecular evolution of resistance genes, comparative genomics and DNA variation (SNP, SSR and CNVs). Dr. Fernandez also generated REN-SEQ libraries for 10 lettuce cultivars. The aim of this task was dual. First of all, he wanted to set up a new protocol called REN-SEQ (Sequence Enrichment for Targeted Sequencing), by using the two most powerful NGS platforms “Illumina” and “PacBio”. The second aim was the re-annotation of the NB-LRR gene family from sequenced plant genomes and rapid mapping of resistance loci in segregation populations. This method enables discovery and annotate of new pathogen resistance gene family members in plant genome sequences.
On the other hand, we were able to identify and classify for the first time in the compositae family the LRR-RLK genes, which are associated to many important roles in plants, such as growth and cell development and defense response. Although RLK subfamilies have been previously studied in some plant species, no comprehensive study has been performed on this gene family in lettuce, which has a high economic importance and is frequent targets for emerging pathogens. Within Breed4futre, we performed an in silico analysis to identify and classify LRR-RLK homologues in the predicted proteomes of many plant species, such as arabidopsis, citrus, tomato, rice, peach, almond, tobacco, papaya or grape. In addition we used large-scale phylogenetic approaches to elucidate the evolutionary relationships of the LRR-RLKs and their cell surface immune receptors.
In accordance with the goals of the second part of the project in Spain, Dr. Fernandez, taking part of an international consortium, has sequenced the novo genome of almond, and re-sequence 48 varieties at 16X using Illumina, PAcbio and Oxford Nanopore platforms. On the other hand, through bioinformatics tools, we have identified the major genes that are supposed to confer resistance to Xanthomonas in almond. I carried out an in silico analysis in order to establish orthology with genes from this model species and that may be associated with Xap in the almond ‘Forastero’ cultivar. The sequence similarity of Xa25, Xa26 and Xa29, which are the major genes conferring resistance to Xanthomonas oryzae pv. oryzae in rice, and encoding NB-LRR proteins, were blasted (p) against the genome of the almond ‘Forastero’. Three relevant hits corresponding to the genes pdulcis7A006414P1, pdulcis7A010546P1 and pdulcis7A007653P1 were retrieved. Additionally, I investigated and blasted those almond genes against the peach reference genome, which is another significant Prunus species. In fact the three hits, identified in peach to ppa018004m, ppa019022m and ppa026334m genes, correspond to proteins with an NB-LRR domain as showed in the Rosaceae peptide database (http://pathways.rosaceae.org).
Dr. Fernandez has also been investigating the gene family associated to drought tolerance in Prunus. A previous root transcriptomic analysis by Illumina in an almond x peach hybrid rootstock served us to identify a number of drought-responsive gene families. The goal was to study the evolution of these gene families among different woody plant species. This approach included 15 genes belonging to the Homeodomain superfamily, 12 genes encoding different Heat Shock Proteins (HSPs), 12 genes encoding several Late Embryogenesis Abundant (LEA) proteins and 7 genes encoding different Dehydration-Responsive Element-Binding (DREB) transcription factors (TFs). The drought stress genes were extracted from genome assemblies of nine plant species (Prunus dulcis, P. persica, Malus domestica Festuca vesca, Camellia sinensis, Arabadopsis. thaliana, Quercus rubra, Pinus taeda and P. lambertiana) representing higher plant diversity and used for phylogenetic analysis by using the maximum-likelihood approach (RAxML). The resultant phylogenetic trees exhibited different patterns of evolution. There has been expansion of specific gene families. In some cases, expansion happened prior to speciation, whereas in other cases, it happened afterwards. Further approaches are being undertaken in order to characterize the evolutionary pathway of drought stress genes in the Rosaceas and the other plant species.
The role of key-flowering genes in Prunus dwarfing rootstocks has also been studied during this third year of the Marie Curie project and led us to identify genes that will be applied shortly in our almond and peach breeding programs. Although Spain is placed as the third almond producer`s country after USA and Australia, the crop has suffered a drastic change with a high density planting system and a low vigour rootstocks. This will represent an important challenge in order to understand how the trees develop and how different varieties and rootstock adapt to these moderns planting systems. In this Breed4future project, we have studied the vigour of the trees at molecular level of two different rootstocks, “Rootpac-40” and “Garnem”, representing different vigour with and without the scion. Additionally we have carried out a phenotypic study by using several combinations rootstock/scion in a commercial orchard. For studying the molecular bases we have approached the expression of a gene family FT/TFL1 responsible for inducing and repressing the flowering time with complementary functions, which aimed to reduce the juvenile period of the tree. For phenotyping we have monitored four traits in order to increase our knowledge of the tree development: shoot length, TCSA (Trunk Cross-Sectional Area), number of flower buds and number of vegetative buds. Expression profiles through quantitative PCR of the four genes studied (FT1, BFT, TFL1 and MFT), which are associated to flowering time, revealed that the genes TFL1 (ppa012369m; delay flowering) and BFT (ppa021829m; delay flowering) presented different expressions among rootstocks without the scion grafted in “Rootpac-40”, whereas in “Garnem” no differences at expression level were observed. On the other hand, FT1 (ppa012320m; advance flowering) and MBF (ppa012388m; delay flowering) showed the same expression profile in rootstocks with and without the scion “Rootpac- 40” and “Garnem”. Further studies are undertaken in order to elucidate the physiological function of those genes in Prunus.
The understanding and identification of genes conferring disease resistance in the Rosaceae, as well as resistance to drought tolerance will help in designing new breeding strategies in these crop families. Hence, these important findings along with the use of a more sustainable approach, such as CRISPR/Cas9, will be one of the solutions to reduce the global warming impact and improve human nutrition and health. Consequently, developing plants that confer disease resistance to Xap and other pathogens will reduce considerably the use of chemical products, which are widely known for their negative impact on health and environment.