Genetic sequencing technology expands wheat research

A new genotyping platform automates the preparation of DNA samples at Embrapa Trigo (RS) and expands the capacity for genetic analysis of wheat crops. The technology reduces the time required for this step from days to about 12 hours, reducing research costs and accelerating the identification of genetic markers linked to productivity and disease resistance.

This advancement was made possible thanks to the evolution of the genotyping process—a technique that allows the identification of variations in an individual's DNA. "In two decades, the protocol has been modernized, with the replacement of manual gel analyses by electrophoresis systems—which use an electric field to separate molecules—and, more recently, by sequencing, which allows for large-scale genotyping with high precision," explains laboratory technician Jordalan Muniz.

Second-generation sequencing has been incorporated into the routine of the Biotechnology Laboratory at Embrapa Trigo with the arrival of two new pieces of equipment: the ION Chef, responsible for sample preparation, and the ION GeneStudio S5 Plus (photo below), which performs the sequencing. "We can say that a year's worth of work can now be done in just one week. It's a gain in efficiency that reduces costs and accelerates the acquisition of molecular data in support of the wheat genetic improvement program at Embrapa," explains researcher Luciano Consoli.

Potential uses of the platform

The wheat genome is 5 times larger than the human genome. With the sequencing of this species' genome in 2018, by an international consortium of research institutions, it became possible to advance new tools to support biotechnology work.

Currently, the Molecular Genetics team at Embrapa Trigo has information on 5 molecular markers for wheat and 3.500 markers for barley compatible with this platform. According to laboratory technician Tatiane Crespi, the identification of molecular markers associated with genes of interest contributes to the selection of lines with desirable agronomic characteristics.

This knowledge resulted in the development of a panel of markers for use in wheat cultivation. Using a second-generation sequencing platform, it is possible to simultaneously analyze 384 samples with 202 markers, generating approximately 80 data points.

Furthermore, the genotyping platform makes it possible to evaluate specific regions in DNA sequences. “The first result of using this technology to generate molecular genotyping data is the increased efficiency of the process of generating new cultivars and identifying alleles or gene fragments of interest for breeding programs. If before we had a cartographic representation, now we have something more detailed, with streets and road signs,” says Consoli.

Advances in genetic improvement

The analyzed wheat cultivars or lines, which possess the molecular markers of interest, can be used in the crossing blocks of breeding programs. In the case of wheat, these analyses are already being conducted, such as in the work to expand the base of genetic resistance to diseases, such as blast, one of the consolidated work fronts using molecular analyses and assisted selection at Embrapa Trigo:

“Once a molecular marker associated with resistance, such as to blast disease, is identified in a particular cultivar, its plants can be incorporated into the breeding program for crossbreeding,” points out researcher Gisele Torres. However, she reminds us that, during field evaluations, it is essential to analyze the plants' reaction under conditions of exposure to the fungus that causes the disease.

“The mere presence of a particular marker, even when DNA fragments are known to confer resistance, is not enough to make the plants that possess it resistant. The work of biotechnology is complementary to the work of breeding. Thus, to verify the effectiveness of a particular molecular marker, it is essential that breeders evaluate the reaction of plants to the pathogen in the field,” adds Torres.

The team is exploring other potential practical uses of the second-generation sequencing platform. In soils, for example, it is possible to extract DNA from a sample to identify microorganisms that can benefit wheat cultivation in a given region. In other words, a molecular profile of that soil is identified, which will then be correlated with production factors.

In the challenge of developing wheat varieties for celiac patients, it is possible to characterize molecular markers associated with genes encoding proteins related to the immune response to gluten. Researchers also intend to explore other possibilities for gene expression analysis using RNAseq, small genome sequencing (microorganisms), and other methods.

Consoli highlights the enthusiasm for the tool's potential uses: “In the past, we used a magnifying glass, then moved on to a microscope, and evolved to see what's inside plants. Now, we can identify all kinds of variations present in plant DNA fragments that are associated with defense and the use of environmental resources.”