1. One approach to investigate the functional capability of a microbial community is through genomic assignment methods. In these methods, one first determines which microbial species are present in a community (for example, by sequencing the bacterial 16S rRNA gene), and then attempts to assign functional genes to these species by comparing them to a database of fully sequenced microbial genomes
A. For what kind of microbial communities and ecosystems do you think this approach will work well (meaning, give an accurate estimate of the true metagenome)? In what situations do you think it will work poorly? Explain why. (8 points)
B. The value of genomic assignment approaches grows as we continue to add reference genomes to our databases, especially for unculturable species. Describe one way in which scientists can gain full genome sequences from organisms that they cannot grow in culture. (8 points)
2. An alternative approach to investigating metagenomes is with shotgun sequencing, in which small segments of DNA from all the microbial cells in the community are sequenced randomly, and then assigned to functional gene categories.
A. Propose a scientific question/study for which a shotgun metagenomic approach would be the most justified approach, and explain why this question could not be answered with methods that only characterize the composition of the community (such as 16S or ITS metabarcoding, as we have done in our lab) (8 pts)
B. Describe two reasons that the functional capability of a microbial community represented in the metagenome may NOT reflect the actual function of that community measured at any particular point in time. (8 points)
3. Numerous researchers have posited that modern crop varieties may have a reduced genetic capacity to benefit from rhizosphere microbial interactions as a result of intense
eeding for maximal yields under modern, high input agricultural settings.
A. (16 points) Explain the reasoning behind this hypothesis. Include in your answer a discussion of:
1) How agricultural management can affect the abundance, diversity, and function of microbial communities
2) How agricultural management can affect the expected costs and benefits of microbial mutualists for crop plants
B. Briefly, design an experiment to test this hypothesis for one particular crop species. In particular, be sure to include what plant species/types you will compare (if more than one), what microbial communities you would compare (if more than one), and what experimental conditions you would impose (if more than one). Be clear as to what measurements you would make, and what specific patterns or comparisons would constitute evidence for or against the hypothesis (16 pts)
4. Many companies are now marketing biological inoculant products, in which they sell viable spores, etc. of a single microbial species, which farmers would then add to their soils to establish a population in their field. One of your farmer friends has asked your opinion on whether she should buy a particular product, which is an associative diazatroph known to associate with grasses (Azospirillum
asilense).
Unfortunately, the company is not willing to send you the product, so you cannot do any experiments involving this particular organism
However, you do have access to
1) a complete genome sequence for the organism.
2) Your friend’s field, to collect any samples of soil, plant roots, etc.
3) all the laboratories at UW-Madison, so you can perform any biochemical, DNA-based, or other molecular biology techniques in order to measure the composition and function of the microbial communities in her field.
4) Soil science laboratories to measure soil nutrient levels and any other soil physical or chemical properties
Thinking about all that we have learned about 1) measuring microbial composition and function, 2) the process of microbial community assembly, and 3) how nutrient-based mutualisms respond to environmental conditions, especially nutrient levels in soils, describe what techniques and measurements you would like to make in order to make the best possible prediction of whether adding this microbial inoculant is likely to lead to an improvement in crop health (assume unlimited funds).
A complete answer will include a) measurements of relevant environmental variables, b) measures of microbial community composition, and c) measures of microbial community functional ability.
It is unlikely that you will be able to make a perfect prediction. Also explain what questions you cannot answer from these approaches that might be important for making this prediction. (36 pts)