Source: UNIVERSITY OF ILLINOIS submitted to
GENOME-WIDE SCREENING FOR CIS AND TRANS REGULATION OF ALLELE-SPECIFIC GENE EXPRESSION IN MAIZE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0221221
Grant No.
(N/A)
Project No.
ILLU-802-384
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2009
Project End Date
Sep 30, 2012
Grant Year
(N/A)
Project Director
Bohn, M. O.
Recipient Organization
UNIVERSITY OF ILLINOIS
2001 S. Lincoln Ave.
URBANA,IL 61801
Performing Department
Crop Sciences
Non Technical Summary
This project proposal is based on the hypothesis that artificial selection on regulatory sequence variants could aid plant breeding efforts to improve crop productivity. Screening for allele-specific gene expression would seem to be a logical first step. Recent technological advances in high-throughput DNA sequencing and the availability of the complete maize genome provide the means to deeply sequence transcriptomes and to digitally count the number of transcripts. New studies in mammalian model species demonstrated the power and reproducibility of this approach in contrast to microarrays. The overall objective of the proposed project is to identify DNA sequence variants in regulatory regions of the maize genome. The specific research objectives are to (1) evaluate elite maize inbreds and progeny derived from their crosses using a diallel mating scheme for plant life history characteristic and agronomic performance, (2) evaluate genome-wide gene expression of immature ears of these maize genotypes using the Illumina Genome Analyzer II platform, and (3) use a novel quantitative genetic approach to genome-wide screen for allele-specific mRNA expression variation in maize. The knowledge gained in this project will be used to develop allele-specific DNA markers located in regulatory regions of the maize genome. Together with the new insights into the molecular basis of dominance, epistasis, and heterosis, these DNA markers will enable designing more efficient and innovative marker-assisted breeding strategies. We also envision that isolation and modification of the found regulatory DNA sequences will open the door to pioneering biotechnology approaches for increasing crop productivity.
Animal Health Component
(N/A)
Research Effort Categories
Basic
60%
Applied
40%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20115101080100%
Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1510 - Corn;

Field Of Science
1080 - Genetics;
Goals / Objectives
The complete sequence of the maize genome promises to facilitate basic maize genetics research and translation of results through breeding into products for growers and consumers. The need now is to establish new, contemporary conduits to better enable the translation. The paradigm is that of the pharmaceutical industry, characterized by companies with a continuous pipeline stretching from basic research through evaluating suitability for product development, actual development, evaluation of final product and a tight, effective integration of companies with universities and regulatory authorities. For the hybrid seed corn industry, corn breeding in the public sector often serves the valuable role of evaluating whether or not novel genes or alleles with newly discovered, promising functions are suitable for transferring into commercial hybrids. An example of this sort of evaluation is the study of genes involved in heterosis, one of the most prized goals in maize improvement. An increasing number of laboratories have started evaluating the genetic diversity in worldwide maize germplasm for new alleles that may prove beneficial to maize improvement. Through this project, we want to extend these evaluations using populations derived from a core set of highly elite maize inbreds recently released from PVP protection by the seed industry. Seed companies have very successfully exploited heterosis through selective breeding for decades, with little evidence of exhausting the variability necessary for further gain. We propose that it is important to determine (1) the subset of allelic diversity within this small but representative set of elite inbreds as it relates to much larger, more extensive maize diversity collections, (2) how this allelic diversity is organized within and among elite inbreds, and (3) how it promotes the production of superior performing hybrids. The overall objective of the proposed project is to identify DNA sequence variants in regulatory regions of the maize genome. The specific research objectives are: Objective 1 - Evaluate elite maize inbreds and progeny derived from their crosses using a diallel mating scheme for a comprehensive set of plant life history characteristics and agronomially important traits, with specific focus on the ear shoot. Objective 2 - Evaluate genome-wide gene expression of developing ears of elite maize inbreds and their progeny. Objective 3 - Conduct a quantitative genetic analysis of gene expression to identify regulatory regions in the maize genome. In this project we will take advantage of very recent developments in the area of short-read sequencing technologies for transcriptional analysis. The possibility of using new generation sequencing platforms for determining mRNA expression levels was first successfully investigated using cDNA libraries obtained from mammalian (human and mouse) tissues. However, in this project, we will combine the power of high-throughput transcriptome sequencing with plant-specific crossing designs and a novel quantitative genetic approach in order to investigate the regulation of allele-specific mRNA expression in maize.
Project Methods
Inferred changes in cis- and trans-regulatory sequences are associated with evolutionary divergence of gene expression. Divergence in phenotypic expression of many quantitative traits accompanied the divergence in gene expression and the divergence in gene expression was driven in large part by natural selection acting upon variation in regulatory DNA sequence. We hypothesize that artificial selection on regulatory sequence variants could aid plant breeding efforts to improve crop productivity. Screening for allele-specific gene expression would seem to be a logical first step. Recently a quantitative genetic approach for a genome-wide screen of allele-specific mRNA expression (GASE) variation was presented. The approach was based on a diallel mating design commonly used in maize breeding to estimate general combining ability (GCA) and specific combining ability (SCA) variance among homozygous inbreds and their F1 hybrid progeny. In the diallel mating scheme, for a set of m homozygous inbreds, all possible matings are made to create a set of m(m-1)/2 F1 hybrids. The parental phenotypic marginal means provide GCA estimates of each parental inbred while deviations of observed phenotypic values from pair-wise GCA estimates yield SCA estimates. Quantitative genetics theory ascribes variability in GCA to cumulative single-locus, and intra-gametic epistatic variance while attributing SCA variance to cumulative single-locus, and inter-gametic epistatic variation. In the GASE analysis mRNA concentration from an expressed gene is regarded as a phenotypic trait for which GCA variance is attributed to variability in cis-acting regulatory elements, whereas SCA variability is ascribed to variation in trans-acting, or a combination of cis- and trans-acting, elements. Experimental evidence of both types of gene expression control has been obtained and GASED was successfully applied to a sample of Arabidopsis lines and F1 hybrids. We propose implementing the procedure to maize using a more comprehensive and superior statistical model. This model is enhanced by supplementing the parental inbreds and F1 hybrids in the experimental design with F2 and backcross entries to obtain added degrees of freedom for estimation of additional, more meaningful, parameters. All plant materials were developed using elite maize inbreds LH1, LH123HT, PHG39, and PHG84 and comprise six F1 hybrids derived from their crosses using a half diallel design, the six F2 populations, and the twelve backcross (BC1) populations derived by crossing each F1 hybrid with its two parental inbreds. All 28 genotypes were evaluated for a comprehensive set of traits and ear tissue from all genoyptes was sampled four days after anthesis in 2009. Total RNA will be extracted and copied into cDNA fragments. These will be sequenced using second generation high throughput sequencing technology. Plot means of phenotypic traits and plot transcript levels will be used in a trait-by-trait analysis of variance (ANOVA) using our augmented GASE model. Multivariate analysis approaches will be used to relate gene expression profiles to agronomic performance. Seed of maize inbreds was obtained from the National Plant Germplasm System.

Progress 10/01/09 to 09/30/12

Outputs
OUTPUTS: This project is based on the hypothesis that artificial selection on regulatory sequence variants could aid plant breeding efforts to improve crop productivity. Screening for allele-specific gene expression would seem to be a logical first step. The overall objective of this project is to identify DNA sequence variants in regulatory regions of the maize genome. For this experiment [Experiment 1] the inbreds LH1, LH123HT, PHG39, and PHG84 were used. LH1 and PHG39 belong to the Stiff Stalk heterotic group, whereas inbreds LH123 and PHG84 belong to the non-Stiff Stalk heterotic group. All were formerly parents of commercially distributed maize hybrids, and plant variety protection and/or utility patents of each have recently expired. The four parental inbreds, the six F1 hybrids derived from their crosses using a half diallel design, the six F2 populations obtained by selfing each F1 hybrid, and the twelve backcross populations derived by crossing each F1 hybrid with its two parental inbreds were developed in the 2007 and 2008 summer nurseries at the University of Illinois, Urbana, Illinois. All genotypes were grown in an experiment consisting of 28 entries in Urbana in 2009 and 2010. Ear tissue from all 28 genoyptes grown in three replications was sampled four days after anthesis. This experiment was embedded in a larger set of germplasm developed from 66 bi-parental crosses between 12 elite maize inbreds of which LH1, LH123HT, PHG39, and PHG84 are a subset. We developed for each of the 66 crosses populations of 25 recombinant inbred lines (RILs). Together with Experiment 1, the 12 parental inbreds, the F1 hybrids, and the segregating F1 derived F2 populations were evaluated for a comprehensive set of 57 traits (agronomic traits, processing traits, architectural traits) in 2009-2012 [Experiment 2], and all 1,500 RILs were evaluated in 2011 for plant and ear height, male and female flowering, as well as leaf length, leaf width, and leaf angle [Experiment 3]. Ears of RILs were harvested and their phenotyping is in progress. Applying the Eberhart and Gardner general model we found for Exp1 that out of 18 ear traits only six (kernels per row, cob length, cob weight, grain fill, ear weight, and grain weight) varied significantly for any genetic component of variation: only non-additive effects were significant. The F1 hybrid genotypic effect estimates were positively and highly correlated. Of the ear branching traits, only kernels per row varied significantly among genotypes. A principal component analysis was conducted and the first traits principal component accounted for nearly ninety percent of the variation with nearly equal loadings on each trait. This component, a manifestation of ear length and lateral branching during ear development, characterized by kernels per row, and highly correlated with grain yield, was named the kernels per row phenotype. The loadings on the corresponding first principal component of the F1 hybrids reflected a known pattern of yield heterosis in crosses among the parents. In the larger germplasm set evaluated in Exp2, we confirmed that substantial additive, dominance, and epistatic variation was present. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Corn breeding research in the public sector involves identifying novel genes, alleles, and breeding procedures that have promising commercial applications, but this role is stymied due to limited access to commercial quality lines and hybrids. Utilization of lines recently released from Plant Variety Protection (PVP) may be a solution. We characterized inbreds representative of North American proprietary germplasm accessed following expiration of their U.S. PVP certificates. Experimental material derived from ex-PVP lines will be an important component of corn breeding research on increased productivity. In this research, hybrid mapping populations as developed in this project will be invaluable for detection of non-additive quantitative trait loci and association effects in yield related traits. The knowledge gained in this project will be used to develop allele-specific DNA markers located in regulatory regions of the maize genome. Together with the new insights into the molecular basis of dominance, epistasis, and heterosis, these DNA markers will enable the designing of more efficient and innovative marker-assisted breeding strategies. We also envision that isolation and modification of the found regulatory DNA sequences will open the door to pioneering biotechnology approaches for increasing crop productivity.

Publications

  • No publications reported this period


Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: This project is based on the hypothesis that artificial selection on regulatory sequence variants could aid plant breeding efforts to improve crop productivity. Screening for allele-specific gene expression would seem to be a logical first step. Recent technological advances in high-throughput DNA sequencing and the availability of the complete maize genome provide the means to deeply sequence transcriptomes and to digitally count the number of transcripts. The overall objective of this project is to identify DNA sequence variants in regulatory regions of the maize genome. The specific research objectives are to (1) evaluate elite maize inbreds and progeny derived from their crosses using a diallel mating scheme for plant life history characteristic and agronomic performance, (2) evaluate genome-wide gene expression of immature ears of these maize genotypes using the Illumina Genome Analyzer II platform, and (3) use a novel quantitative genetic approach to genome-wide screen for allele-specific mRNA expression variation in maize. For this experiment the homozygous inbreds LH1, LH123HT, PHG39, and PHG84 were used. Inbreds LH1 and PHG39 belong to the Stiff Stalk heterotic group, whereas inbreds LH123 and PHG84 belong to different lineages in the non-Stiff Stalk heterotic group. All were formerly parents of commercially distributed maize hybrids, and plant variety protection and/or utility patents of each have recently expired. The four parental inbreds, the six F1 hybrids derived out of their crosses using a half diallel design, the six F2 populations obtained by selfing each F1 hybrid, and the twelve backcross (BC1) populations derived by crossing each F1 hybrid with its two parental inbreds were developed in the 2007 and 2008 summer nurseries at the University of Illinois Research and Education Center in Urbana, Illinois, and increased in our 2008/2009 winter nursery. All genotypes were grown in a designed experiment consisting of 28 entries in Urbana, Illinois, in the summer seasons 2009 and 2010 and evaluated for a comprehensive set of plant life history characteristics and agronomically important traits with specific focus on the ear shoot. Ear tissue from all 28 genoyptes grown in three replications (total number of samples N = 84) was sampled four days after anthesis. The extraction of total RNA from pooled ear shoot base samples obtained from a single plot was completed in 2011. The statistical analysis showed that genotypic variation in kernels per row was highly correlated with cob length, cob weight, grain fill, ear weight, and grain weight per ear. Only non-additive sources of variation were significant genetic components of these traits. Inter-correlations of genotypic variation among the traits were quite high, with the first principal component of the correlation matrix accounting for 90% of the variation among traits. Trait weights on the first principal component vector were nearly equal. The combined statistical analysis of the field experiments and the quantitative-genetic interpretation of the results is in progress. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The knowledge gained in this project will be used to develop allele-specific DNA markers located in regulatory regions of the maize genome. Together with the new insights into the molecular basis of dominance, epistasis, and heterosis, these DNA markers will enable designing more efficient and innovative marker-assisted breeding strategies. We also envision that isolation and modification of the found regulatory DNA sequences will open the door to pioneering biotechnology approaches for increasing crop productivity.

Publications

  • No publications reported this period


Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: This project is based on the hypothesis that artificial selection on regulatory sequence variants could aid plant breeding efforts to improve crop productivity. Screening for allele-specific gene expression would seem to be a logical first step. Recent technological advances in high-throughput DNA sequencing and the availability of the complete maize genome provide the means to deeply sequence transcriptomes and to digitally count the number of transcripts. New studies in mammalian model species demonstrated the power and reproducibility of this approach in contrast to microarrays. The overall objective of this project is to identify DNA sequence variants in regulatory regions of the maize genome. The specific research objectives are to (1) evaluate elite maize inbreds and progeny derived from their crosses using a diallel mating scheme for plant life history characteristic and agronomic performance, (2) evaluate genome-wide gene expression of immature ears of these maize genotypes using the Illumina Genome Analyzer II platform, and (3) use a novel quantitative genetic approach to genome-wide screen for allele-specific mRNA expression variation in maize. For this experiment the homozygous inbreds LH1, LH123HT, PHG39, and PHG84 were used. Inbreds LH1 and PHG39 belong to the Stiff Stalk heterotic group, whereas inbreds LH123 and PHG84 belong to different lineages in the non-Stiff Stalk heterotic group. All were formerly parents of commercially distributed maize hybrids, and plant variety protection and/or utility patents of each have recently expired. The four parental inbreds, the six F1 hybrids derived out of their crosses using a half diallel design, the six F2 populations obtained by selfing each F1 hybrid, and the twelve backcross (BC1) populations derived by crossing each F1 hybrid with its two parental inbreds were developed in the 2007 and 2008 summer nurseries at the University of Illinois Research and Education Center in Urbana, Illinois, and increased in our 2008/2009 winter nursery. All genotypes were grown in a designed experiment consisting of 28 entries in Urbana, Illinois, in the summer seasons 2009 and 2010 and evaluated for a comprehensive set of plant life history characteristics and agronomically important traits with specific focus on the ear shoot. Ear tissue from all 28 genoyptes grown in three replications (total number of samples N = 84) was sampled four days after anthesis. The extraction of total RNA from pooled ear shoot base samples obtained from a single plot is underway. The combined statistical analysis of the field experiments and the quantitative-genetic interpretation of the results is in progress. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The knowledge gained in this project will be used to develop allele-specific DNA markers located in regulatory regions of the maize genome. Together with the new insights into the molecular basis of dominance, epistasis, and heterosis, these DNA markers will enable designing more efficient and innovative marker-assisted breeding strategies. We also envision that isolation and modification of the found regulatory DNA sequences will open the door to pioneering biotechnology approaches for increasing crop productivity.

Publications

  • No publications reported this period