Genetic Maize

Potentially promiscuous pollen from corn tassels by circulating via Flickr.

Many people, including me, are concerned about potential harm to crop biodiversity from gene flow. Most people’s concern focuses on transgenics. There is a certain probability, albeit small, that transgenes will end up in the progeny of non-transgenic plants, weedy relatives of the crop, or wild relatives that grow nearby due to pollen flow. Transgenes can also be moved from place to place by accidental or purposeful movement of seeds.

How much transgene flow is actually happening is a subject of some controversy, but what about gene flow between non-transgenic plants?

There is potential for problems whenever plants that aren’t supposed to cross stray from their intended mates. Some things to think about include how gene flow happens at the field and genetic levels and what characteristics of the genes themselves can affect permanence of contaminating genes once they get into a variety they shouldn’t be in.

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Volker Brendel, professor of bioinformatics at Iowa State, spoke at the Maize Genetics Conference about the need for a better system of community annotation of the maize genome. The genome of the popular maize inbred line B73 is sequenced, but we don’t actually know what a lot of the code stands for. It’s going to take a lot of collaborative effort to discover and annotate (explain) the function of each gene and to put all of that information in one place so it will be useful.
Volker reminds us that the Arabidopsis 2010 funding is running out, so we need to assess the plant genetics situation. How many genes do we know the function of? There is still much to learn.
Maize is uniquely positioned to replace Arabidopis as a focus for basic plant research due to the many resources that are already established, the most important of which is the extensive maize genetics community (he didn’t say it, but there is another reason why maize is a better choice than Arabidopsis right now – all of our major grains are very closely related, so work on maize applies to rice, wheat, sorghum, and more). The community needs to work together in the annotation process, assigning functions to the genes that have been sequenced, putting the data from a variety of sources together to make a bigger picture. Each researchers has a favorite gene (pathway, organelle, etc) – how can each of the researchers contribute to the annotation process?
PlantGDB is a comparative genomics site funded by NSF has information on 14 species, including maize, which is very useful. However, no matter how clever the computer programs are, the human touch is still needed. Filling in information on any of these species helps us to better understand all of them. On the site, community members can flag genes for which the models don’t seem to fit, and can contribute alternative explanations. The final goal is to have every gene model approved by the relevant community member(s). When a person annotates a gene, the PlantGDB committee reviews it, approves it, and the information is shortly available on the site. Annotating the genes you are working on is your civil duty, something you owe due to public funding you receive.
After Volker’s talk, the attendees discussed what is the public’s role in the attenuation process should be. There are a lot of cases where the the gene model can be checked without any lab work, simply by looking at the sequences. Some members of the community think we should harness the brainpower of thousands of biology undergraduate students by assigning annotations for class. I like the idea of getting students involved, and hope they follow through.Diversity of people to represent the maize genetics community.
A panel discussion followed, where a lot of great new ideas for annotation were brought up (unfortunately I don’t have the names of some of the people that spoke).
One panel member said we need “Zeazomics” – a collection of information including genomics, metabolimics, proteomics, and whatever else we can come up with – to fill in gaps in our knowledge. being able to link all of this information together will lead to stronger explanations of the phenotypes we see. He said this process will not be definitive, it will create a series of hypothesis that will lead to more hypotheses. The hypothesis testing will lead to functional biolgoy, from physiology to biochemistry to cell biology and more. Additional genome sequencing is necessary to capture the entire diversity of maize. Maize is the model for grasses, for crops, for future applications like biofuels. Now is the time to push maize research to a much higher level.
To accomplish all this, we’ll need to take care of a few things, as the other panel members and members of the community brought up:

• Need to have reciprocal links from genes from MaizeGDB to NCBI Entrez Gene. Currently, about 20,000 NCBI Entrez Genes need links back to MaizeGDB.
• To help with annotation, Lisa Harper, curator of MaizeGDB, will do a movie that shows the common problems of using the databases, including how the genome changes over time as the contigs are reordered, etc. This is needed because people are often working off of older copies of the information for a given gene, as it might not be updated frequently enough.
• There is also a need to integrate microarray data into the databases. Particularly complicated are those microarrays that are specific to a particular tissue and/or developmental stage. Volker says that this problem is common and new technologies with new ways to visualize data are necessary.
• MaizeGDB needs a forum such that people working on the same genes can coordinate their work.
• iPlant is organizing a workshop in St. Louis in June to help coordinate the various genome annotation groups.

• There is a plan to create outreach information that any member of the maize community will be able to download and use to communicate the needs and accomplishments to the public and to government officials.

Monsanto’s press releases on Roundup Ready 2 Yield uses the term “advanced gene mapping and insertion process”. This sounds impressive, but what does it mean? A colleague asks: “can advanced gene mapping and insertion tech improve yield of the plant or would other factors like selection and crop physiology really be what’s improving yields?”
From the press release:

[David Nothmann, Monsanto’s Soybean Agronomic Trait Lead,] said Roundup Ready 2 Yield technology is based on an advanced gene-mapping and insertion process. “Through gene mapping, Monsanto has identified specific DNA regions in soybeans that have a positive impact on yield,” he explained. “Using these new insertion and selection technologies, the Roundup Ready 2 Yield gene is situated in one of these DNA regions.”

There has undoubtedly been decreased yield in Roundup Ready crops when compared to conventionally bred crops. This has two possible causes: lag “a temporary or transient problem associated with the introduction of a new technology” or drag “an inherent yield reduction associated with the technology itself”. There is a lot of evidence that the problem is in fact lag, but more research must be done. Some of the issues are discussed in Challenges in Comparing Transgenic and Nontransgenic Soybean Cultivars.
I covered the topic of yield lag/drag somewhat in my post Exposed, Indeed.

GM seeds are often “one hit wonders” that excel in one specific trait, but not particularly for increased yield. Non-GM lines, on the other hand, are improved every year, with the best yielding plants being used to produce the next year’s seed. I recently attended a seminar presented by a scientist from Pioneer where he said that they were working to develop better yielding lines that would work in conjunction with their primary transgenic traits. The companies are aware that this is a problem with their products, and are of course working to solve it, to avoid losing sales.

Back to the question at hand – as I understand it, advanced gene mapping is a selection tool.  The companies start with huge experimental fields (much larger than what an academic lab can afford) in multiple locations with different climates that include many varieties of the crop in question. They measure yield and determine genetic markers* for each variety/location combo, using known markers for yield as the starting point. The researchers are then able to see which varieties do and do not have certain markers. They cross varieties that have different markers, with the goal of a super high yielding plant that has all of the markers that are positively correlated with yield and none of the markers that are negatively correlated with yield. They have fields in multiple locations so they can choose the markers that confer an advantage in a variety of climates – ensuring that the plants will perform no matter what the location or conditions. There are a lot of benefits of this method over blind selection, the biggest of which (in my opinion) is that you don’t have to know what’s happening physiologically in the plant. Knowing what each gene does (and what each mutation to each gene does) is nice, but really not necessary for the purpose of breeding bigger better plants.
As for the “insertion process” part, I admit that I’m not 100% sure why positioning the insertion in an area of the genome that is correlated with high yield would matter (any readers who know, feel free to enlighten me!). I can think of a few reasons why the specific position of the transgene insertion does matter, but all of them are part of the normal process running up to a marketable genetically engineered crop. In fact, I’m in the process of some of those stages right now. Once the gene of interest is chosen, a compatible promoter must also be chosen. I imagine that a constitutive promoter (always on, in every cell) would be used for the glyphosate resistance trait. The gene construct is introduced into many plant cells that are then grown into individual adult plants. Each introduction is called an event. Each event is treated separately because the position of insertion is different each time. When the insertion lands in the middle of a gene, it can stop the gene’s normal expression – so many events are investigated to see which ones have the least effect on the plant’s normal gene expression while at the same time producing the desired trait. A video on Monsanto’s website says that they used Agrobacterium instead of biolistic transformation in RR2 because it is “gentler”, causing less damage to the surrounding DNA. They then screened many events using genetic markers to find the best ones – an expensive process that (to my knowledge) has not been done before. They say that having the insertion in one of the areas near a marker for high yield increases yield an additional 7 to 11 percent.
Edit: I don’t know why I was having a mental block on this! What I said in the last paragraph stands but I’ve figured out why having the insertion in a high yield correlated area would matter. If the insertion is near an allele for a gene that is correlated with poor yield, selecting for the trait of interest would bring along the area that you don’t want. Having the insertion in a  “good” area of the genome (assuming that it isn’t actually interrupting any genes) eliminates this problem.
*Here, markers basically correspond to alleles of a gene. Wikipedia has a decent explanation of genetic markers, but unfortunately requires the understanding of much more jargon. If you’d like a more detailed explanation of genetic markers, please let me know, and I will be happy to write a post on the subject.

Raymer, PL and TL Grey (2003). Challenges in Comparing Transgenic and Nontransgenic Soybean Cultivars. Crop Science, 43, 1584-1589.

In India and other Southeast Asian countries, large areas of the bedrock naturally contain arsenic (As), which leaches into the groundwater. The FAO estimates that up to 500 million people are at risk of being exposed to dangerous levels of arsenic in both drinking water and in the crops that were irrigated with the groundwater. The problem was investigated by the FAO in Bangladesh in 2006. They found that:

[A]rsenic levels in the grain of different varieties of rice in Bangladesh were as high as 1.8 parts per million, compared to levels of just 0.05 parts per million in Europe and the US. Contamination was even greater in leafy vegetables – in amaranthus and spinach, arsenic content can be double or triple the levels found in rice. For drinking water, WHO recommends a maximum arsenic level of 0.01 parts per million, which indicates that for some people, staple food crops such as rice may be an important source of exposure to arsenic.

Until now, the farmers essentially have three options: leave the fields fallow, plant rice and hope it doesn’t have too much arsenic, or attempt to plant a crop that doesn’t need as much water.
Om Parkash (photo and story from Newswise) of the University of Massachusetts Amherst primarily works on bioremediation, which aims to remove pollutants from the soil by binding it up in plants. His recent work branches into the opposite direction, using genetic engineering to produce rice plants that take up less As. The work is in the process of patenting, so technical details are scarce. For now, I’ll have to be content with the following:

“By increasing the activity of certain genes, we can create strains of rice that are highly resistant to arsenic and other toxic metals,” says Parkash, a professor of plant, soil and insect sciences. “Rice plants modified in this way accumulate several-fold less arsenic in their above-ground tissues, and produce six to seven times more biomass, making the rice safer to eat and more productive.” This could help alleviate the current world-wide rice shortage.

I’m really looking forward to learning more about the genetics, and hope that Dr. Parkash is able to move forward with this exciting crop improvement.
While As is actually a necessary mineral in small amounts and only becomes dangerous to health when consumed in high levels (as in Bangladesh), decreasing As in the food supply is definitely a worthy cause. Dr. Parkash says that As can accumulate in all parts of the rice plant, including grain and straw. High As levels in rice not only affect people, but can sicken animals who eat the straw and contaminate their meat (think bioaccumulation). See GreenFacts for a good summary of arsenic as it relates to human health and the environment (incidentally, they also have some of the most levelheaded information on GM crops that I’ve ever seen).
Other recently published work on arsenic levels in rice by Yamily Zavala and John Duxbury of Cornell was reported in the 2 May 2008 ISAAA Crop Biotech Update. For a summary of the articles, see the press release from the American Chemical Society. Disclosure: I wasn’t able to access these two articles themselves as ISU’s library site is down while I write this.
In Arsenic in Rice: I. Estimating Normal Levels of Total Arsenic in Rice Grain, they showed that mean As concentrations in samples of commercial rice in Europe and the US (0.198 mg/kg) were higher than in samples from Asia (0.07 mg/kg). The concentrations varied greatly by region, but not by farming method. Their data confirmed that irrigation with As contaminated groundwater in Bangladesh is correlated with higher As concentrations in grain. In the US, where groundwater is not contaminated with As, the authors suggest that historical contamination of soil is a likely cause. Note: mg/kg and ppm are equivalent units.
In Arsenic in Rice: II. Arsenic Speciation in USA Grain and Implications for Human Health, they showed that the As in some rice varieties accumulates in a less toxic form than inorganic As (inorganic = molecules do not contain carbon). Arsenic in rice grown in the US is bound into mostly into dimethyl arsinic acid (DMA), which . This data is in agreement with previous studies done by Andrew Meharg of the University of Aberdeen in the UK. There is evidence that DMA is safer than inorganic As, which means that US rice may be safer than European or Asian rice. The authors hypothesize that 30 years of breeding in the US for straighthead disorder resistant rice could have caused US varieties to acquire this As metabolic pathway.
Huge phenotypic variance is present in rice grains across varieties. It’s easy to imagine that metabolic pathways vary widely from variety to variety as well.

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