Recipient Organization
MARQUETTE UNIVERSITY
915 W WISCONSIN AVE RM 175
MILWAUKEE,WI 53233
Performing Department
Biological Sciences
Non Technical Summary
The sabbatical project "Establishment of an Efficient Transformation System for Functional Analysis of Cold Tolerance Genes in Rice" is performed by PD Michael Schläppi from Marquette University, and fits the A1152 Physiology of Agricultural Plants program area "mechanisms of plant response to abiotic stresses", but also touches on "plant growth and developmental processes". Rice (Oryza sativa L.) yield would be enhanced if growers could plant two weeks earlier in the season to better utilize the spring rain and avoid the high nighttime temperatures of mid-summer, which decrease grain quality and yield. The objectives of the project are to receive training at an outstanding rice transformation facility of the Chinese Academy of Sciences, and to establish an efficient transformation pipeline for functional analysis of putative cold tolerance genes from rice. A minimum of 10 cold tolerance candidate genes will then be selected from our ongoing QTL and gene-fine mapping efforts and functionally analyzed by overexpressing them in a cold sensitive INDICA-type rice accession, and, conversely, by knocking them out via CRISPR-Cas9 technology in a cold tolerant JAPONICA-type accession. This will allow us to identify novel cold tolerance genes and provide loci for marker assisted breeding to improve the chilling tolerance of U.S. rice cultivars. By planting chilling tolerant rice varieties earlier, growers will use less irrigation water from aquifers, thus decreasing the environmental impact.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
We intend to establish a high-efficiency transformation system of standard and recalcitrant rice cultivars to test probable rice cold tolerance genes as candidates to ultimately enhance chilling tolerance of commercial rice grown in the United States. Rice as a crop originates from subtropical regions and is prone to chilling injury leading to poor germination, necrosis of plant organs, slow chlorophyll synthesis, delayed development, and reduced seed set. Cold tolerant cultivars will allow rice producers to plant early while reducing risks in seedling establishment caused by cold weather, and reduce costs through the use of spring rains. During this sabbatical project, we aim to use functional genomics approaches to test cold tolerance candidate genes to advance our understanding of the molecular and cellular mechanisms governing the quantitative traits of chilling tolerance in rice.We have recently identified 48 chilling tolerance quantitative trait loci (QTL) and generated a list of associated candidate genes that could be used to improve different cold tolerance traits in U.S. rice cultivars. The 48 QTL were identified using a genome-wide association study (GWAS) approach by screening the USDA Rice Mini-Core (RMC) collection for five cold tolerance indices at the germination and young seedling stages, while our collaborators at the Dale Bumpers National Rice Research Center in Arkansas identified an additional 71 QTL by screening the Rice Diversity Panel 1 (RDP1), for cold tolerance during germination (42 QTL) and at the reproductive stage (29 QTL). Although some QTL overlapped in the two studies, there are genes associated with over 50 cold tolerance QTL that need to be subjected to functional analysis by overexpressing (OX) alleles from cold tolerant varieties in cold sensitive cultivars and vice versa, by knocking out (KO) those same alleles in cold tolerant cultivars. Because it would be prohibitive to pay a rice transformation facility to generate more than 100 different OX and KO lines, we came to the conclusion that it will be beneficial for our current NIFA-AFRI #2016-67013-24587 grant funded project to elucidate the "mechanisms of cold stress tolerance responses in rice" to perform transformation experiments in our own lab. Therefore, the main objective of this sabbatical project is to establish an efficient rice transformation system in our own lab for high-throughput screening of transgenic lines with enhanced (OX) or reduced (KO) chilling tolerance phenotypes as a functional test to identify strong-effect cold tolerance genes.The main rational for this project is that, despite a large amount of mapped cold tolerance QTL, very few bona fide cold tolerance genes are known. A major bottleneck is that bioinformatics based genomics analyses need to be paired with functional genomics approaches that manipulate cold tolerance candidate genes in planta. To this end, an efficient transformation assay for cold sensitive, recalcitrant rice accessions to overexpress alleles of probable cold tolerance genes needs to be established. The lab of Dr. Chengcai Chu, a collaborator of this sabbatical project, has already developed such a system and will provide the necessary training. This sabbatical fellowship not only will allow us to establish the system in our lab, but also to use an uninterrupted research year to generate a critical number of transgenic lines to identify novel cold tolerance genes via functional genomics.For this sabbatical project, we have the following specific aims:Aim 1: To receive 6 weeks of training in rice transformation of standard and recalcitrant rice cultivars from our collaborator at the Chinese Academy of Sciences in Beijing, and to establish an efficient rice transformation system at Marquette University.Aim 2: To select 10 of the most probably cold tolerance candidate genes based on QTL analysis and fine-mapping, and to clone their cDNAs into rice-specific OX and KO vectors conducive for Agrobacterium tumefaciens mediated transformation.Aim 3: To introduce OX constructs into the recalcitrant, cold sensitive INDICA-type Kasalath variety, and to introduce KO constructs into the standard, cold tolerant JAPONICA-type Zhong Hua 11 variety.
Project Methods
1. Training in establishing a high-throughput rice transformation pipelines: To establish a rice transformation system at Marquette University, the PD will receive six weeks of training at the rice transformation facility of Dr. Chengcai Chu at the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, who is a collaborator of the sabbatical grant project. His transformation facility has an impressive track record not only for chilling tolerant JAPONICA accessions such as Nipponbare and Zhong Hua 11, but also for transformation of the chilling sensitive aus accession Kasalath, an INDICA-type cultivar. The Kasalath accession will be a suitable background for introducing JAPONICA alleles of putative chilling tolerance genes, while Zhong Hua 11 will be used to knock out one or several candidate genes at once using the recently developed CRISPR/Cas9 system. Zhong Hua 11 will be used, because it has a faster life cycle than Nipponbare, thus reducing the time to get T1 seeds from T0 plants. The general time frame to get T1 seeds from T0 plants is usually 4-6 months. Transgenic T1 seeds and seedlings will be evaluated in growth chamber based assays for changes in cold tolerance traits. During the six weeks of training in the summer of 2018, the PD will examine examples of all stages of rice transformation, from callus induction, callus infection with Agrobacterium tumefaciens, selection against Agrobacterium overgrowth and for T-DNA integration, regeneration of seedlings, root induction, and growth chamber-based production of T1 seeds from individual T0 events. The PD will learn "tricks" from the transformation facility operators, such as when to use glass vials instead of plastic ones, how much to dilute the Agrobacterium suspension, what transformation vectors and selection work best, how long to select, what signs of distress to look for, etc. After the training, the PD will establish an efficient rice transformation pipeline at Marquette University, based on previous experience in generating rice callus from several JAPONICA accessions and in regenerating non-transformed plants from them.2. Identify and clone putative cold tolerance genes based on QTL mapping: We will select at least 10 probable cold tolerance genes using our previous and current QTL mapping efforts and using several functional genomics criteria. First, we will use the Rice Expression Profile Database (http://ricexpro.dna.affrc.go.jp) to select cold and other abiotic stress tolerance annotated genes that are expressed in QTL appropriate tissues and correct developmental stages. For instance, Low Temperature Germinability candidate genes should be expressed during germination and Low Temperature Seedling Survivability candidate genes in young seedlings or roots. Second, we will mine previous gene expression profiling studies to further narrow down genes that are differentially expressed between the JAPONICA-type and INDICA-type parents before or during cold stress application. Third, to select the 10 most probable candidate genes, we will mine various databases to identify significant single nucleotide polymorphisms (SNPs) producing non-synonymous amino acid changes or affecting regulatory regions between the cold tolerant and sensitive parents. Some databases are publically available, such as SNP-Seek and RiceVarMap, while recent resequencing data of the Rice Mini-Core by our collaborator Dr. Chu is accessible to us as privileged information. And forth, we will validate expression and SNP data of those 10 genes by qPCR and sequencing analysis of genomic DNA and cDNA isolated from cold tolerant and sensitive accessions.For this specific aim, the 10 most probable rice cold tolerance genes will be cloned before and during the six-week training session into rice specific OX binary vectors obtained from the Chu rice transformation facility. In parallel, we will generate CRISPR-Cas9 based KO constructs for the 10 genes. Upon return from the transformation training session, the OX and KO vectors will be used to establish the rice transformation pipeline at Marquette University.3. Overexpression and knockout of selected, putative chilling tolerance genes in respective rice varieties: To functionally analyze putative chilling tolerance genes associated with previously mapped QTL, JAPONICA alleles of approx. 10 genes that, based on functional genomics, fall into the categories outlined above will be introduced into rice callus made from Kasalath, a cold sensitive INDICA-type accession. Kasalath is expected to have INDICA-type alleles of the 10 selected genes that most likely associate with the cold sensitive phenotype of Kasalath. The rationale for the OX analysis is that if a candidate is a bona fide cold tolerance gene, OX in Kasalath will have a positive effect on at least one of the five cold tolerance indices we previously developed for QTL mapping. In parallel, CRISPR-Cas9 constructs of the same genes will introduced into Zhong Hua 11 callus to KO the JAPONICA alleles. The rationale for this is that KO of a bona fide cold tolerance gene in a JAPONCIA accession will reduce cold tolerance scores of the transgenic T1 plants compared to wild type plants for at least one of the five cold tolerance indices we previously developed for QTL mapping. Transgenic T1 seeds are expected to be available at the end of the proposed sabbatical period, and various cold tolerance assays will be performed during that time, but might continue beyond the sabbatical grant period.