New Paper in PLosONE

Anthony Becker was an undergraduate summer intern in the lab as part of the  Danforth Center NSF-REU program. He did a great job learning how to code in R and the basics of mapping genetics.  Tony worked to apply methods developed to map genes using  older microarrays to newer, high density single nucleotide polymorphism (SNP) arrays. His efforts resulted in a new paper in PLoS ONE: Bulk Segregant Analysis Using Single Nucleotide Polymorphism Microarrays. Tony has stayed on in the lab to help out with various and sundry R projects.

Here is the Abstract of the paper:

Bulk segregant analysis (BSA) using microarrays, and extreme array mapping (XAM) have recently been used to rapidly identify genomic regions associated with phenotypes in multiple species. These experiments, however, require the identification of single feature polymorphisms (SFP) between the cross parents for each new combination of genotypes, which raises the cost of experiments. The availability of the genomic polymorphism data in Arabidopsis thaliana, coupled with the efficient designs of Single Nucleotide Polymorphism (SNP) genotyping arrays removes the requirement for SFP detection and lowers the per array cost, thereby lowering the overall cost per experiment. To demonstrate that these approaches would be functional on SNP arrays and determine confidence intervals, we analyzed hybridizations of natural accessions to the Arabidopsis ATSNPTILE array and simulated BSA or XAM given a variety of gene models, populations, and bulk selection parameters. Our results show a striking degree of correlation between the genotyping output of both methods, which suggests that the benefit of SFP genotyping in context of BSA can be had with the cheaper, more efficient SNP arrays. As a final proof of concept, we hybridized the DNA from bulks of an F2 mapping population of a Sulfur and Selenium ionomics mutant to both the Arabidopsis ATTILE1R and ATSNPTILE arrays, which produced almost identical results. We have produced R scripts that prompt the user for the required parameters and perform the BSA analysis using the ATSNPTILE1 array and have provided them as supplemental data files.

 

and here is a non-technical summary.

In order to understand all of life, it is necessary to identify the genes underlying all facets of an organism. The process of mapping to a gene has historically been a time and resource intensive endevour. One of the limiting steps was the identification of DNA differences that can be used for mapping (markers) between two lines that have differences in a given trait. Significant advances in sequencing and microarray technologies have enabled the creation of silicon arrays with hundreds of thousands of features for assaying single DNA base changes (single nucleotide polymorphisms, SNP). For any given pair of crop lines, the arrays will have tens of thousands of features that  can be used as markers. In this paper, we show that a SNP array designed for the model plant Arabidopsis can be used  for several established mapping techniques with improved speed and cost.  Large sequencing resources are available for many crop plants which would allow these approaches to be used in economically important crops, such as Maize, Soybean and Cotton. These resources will enable plant breeders and producers to make rapid strides in crop improvement

 

New paper in Plant Cell

Our paper "Arabidopsis NPCC6/NaKR1 Is a Phloem Mobile Metal Binding Protein Necessary for Phloem Function and Root Meristem Maintenance" was just published in Plant Cell.  This paper was mainly the result of the hard work of newly minted Ph.D Hui Tian from John Ward's Lab at UMN.  She worked with me to clone the gene, finally finding it when the causal deletion of only 7 bp disrupted a single oligo on the Arabidopsis tiling array. It's a fascinating gene, encoding a protein that moves through the phloem, the part of the plants vasculature responsible for moving solutes away from leaves.

Here is the abstract:

SODIUM POTASSIUM ROOT DEFECTIVE1 (NaKR1; previously called NPCC6)encodes a soluble metal binding protein that is specificallyexpressed in companion cells of the phloem. The nakr1-1 mutantphenotype includes high Na⁺, K⁺, Rb⁺, and starch accumulationin leaves, short roots, late flowering, and decreased long-distancetransport of sucrose. Using traditional and DNA microarray-baseddeletion mapping, a 7-bp deletion was found in an exon of NaKR1that introduced a premature stop codon. The mutant phenotypeswere complemented by transformation with the native gene orNaKR1-GFP (green fluorescent protein) and NaKR1-β-glucuronidasefusions driven by the native promoter. NAKR1-GFP was mobilein the phloem; it moved from companion cells into sieve elementsand into a previously undiscovered symplasmic domain in theroot meristem. Grafting experiments revealed that the high Na⁺accumulation was due mainly to loss of NaKR1 function in theleaves. This supports a role for the phloem in recirculatingNa⁺ to the roots to limit Na⁺ accumulation in leaves. The onsetof root phenotypes coincided with NaKR1 expression after germination.The nakr1-1 short root phenotype was due primarily to a decreasedcell division rate in the root meristem, indicating a role inroot meristem maintenance for NaKR1 expression in the phloem.

And here is a non-technical summary:

A major problem for world agriculture is the growing decrease in avaialable arable land. More and more we are working in solils that impart a stress on the plants that make up the crops we depend on. In order for plants to survive without being able to move out of unfavorable soil environments, they adjust the biochemical composition of their tissues through a wide variety of mechanisms.  One of these mechanisms is to move  elements such as sodium (Na) and potassium (K) from tissue to tissue, including from the root to the shoot and back again.  Understanding the molecular basis of these mechanisms will enable the production of crops that are better able to respond to the changing environment  and increase yields with fewer inputs.  In this study, we identified and characterized a gene which is important for loading Na  into the phloem, the 'veins' of the plant responsible for moving molecules out of the leaves to the seeds and roots. The protein also moves into the  phloem. Plants without a functional form of this gene, called NAKR1, have altered levels of Na, K and starch in the leaves, have shorter roots and flower later than plants with a functional copy of NAKR1.  These results will lead to a better understanding of how plants distribute elements between tissues and ultimately will allow for crop improvement strategies that deal with poor soil quality.