"How ""phenotypes"" are controlled by ""genotypes"" is the essential question in biology. Understanding natural ""genotype"" or ""genes"" and the mechanism mediated by the ""genotype"" or ""gene"" behind a ""phenotype"" facilitates a more feasible application of science in practical purpose and very important for society and Agriculture. To complement the rapid development of sequencing technique, which has been successfully used to predict potential ""genotypes"" for some agricultural ""phenotypes"" from natural species, functional validation of these predicted ""genotypes"", by interrupting the genes with T-DNA insertion or RNA interference, was traditionally used. However, these methods involve foreign DNA from bacterial in the genome to cause or maintain the interrupting effect, which needs to be GMO plants (defined as containing the foreign DNAs in an organism). On the other hand, the insertion of foreign genes in the genome could cause unexpected consequences such as an opposite effect (enhancing instead of knocking out the function of gene for study depending on the position of T-DNA insertion). CRISPR/Cas9 system has been extensively used as a genome editing tool recently, in which the endonuclease activity of Cas9 digest the specific target DNA sequence under the leading of CRISPR guide RNA sequence. One important feature of this method is that the foreign transgene is able to be excluded from the organism once the mutations are generated, which is not GMOs. So, one of the foundation objectives of this project is to develop an efficient CRISPR/Cas system for knocking out genes from natural species of Arabidopsis, which is an extensively used model plant and has made great contributions to even crop science. Based on this aim in a starting stage, an easy-to-use CRISPR vector toolbox has been developed from this project, with a design convenient to remove the foreign transgenes (non GMO plants). And with this toolbox, both single site mutation and fragment deletion in a given gene can be made.
With this methodology, the second issue to be addressed can be done, which is to understand the evolutionary function of immune genes (NBLRR resistant genes) in plants. Immune response mediated by Resistance (R) protein, with nucleotide binding/leucine rich repeat (NB-LRR or NLR) domains, is one of the most important strategies developed by plants. It is well known that NLRs is the most variable gene family and compose of comparable ratio of plant genome. The feature of extensive sequence diversity and presence/absence polymorphism among populations (presence in some accessions but absence from other accessions) indicates balancing selection and/or rapid diversification happened to this gene family during evolution. The objective for this part of project is to understand whether the relatively conserved NLR genes among 1001 Arabidopsis accessions have the same fitness contributions or the balanced fitness contributions to the ones with presence/absence polymorphism. This study will be important for the society because, on one hand, it will help to have a better understanding on the further interaction between pathogen and plants in a much longer time window, and on the other hand, the discovery of important NLR genes that are retained after long-term evolution and contribute to plant fitness will help plant breeders to improve plant performance.
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