New method for sugarcane genome analysis

Technology could contribute to genetic improvement and the development of new, more productive varieties of the plant.

 By Elton Alisson

Agência FAPESP – Despite the economic importance of sugarcane in countries such as Brazil and the series of investments and efforts toward the genetic improvement of this crop that have been made in this country since the 1970s, we still have only a limited understanding of the sugarcane genome, say specialists in the area.

This is because sugarcane is polyploid. Unlike humans, who have two copies of each of the 23 pairs of chromosomes (one from the mother and one from the father) and therefore have two variants of each gene inherited from their parents, sugarcane has a much more complex genetic arrangement, with several copies of each chromosome and numerous variants of each gene.

For this reason, it is difficult to understand how genetic characteristics are transferred and how the multiple variants of each gene work within the plant, which makes the improvement and development of newer, more productive varieties of sugarcane challenging.

A group of researchers from the Universidade Estadual de Campinas Luiz de Queiroz School of Agriculture at the Universidade de São Paulo (ESALQ-USP), Universidade Federal de São Carlos in Araras (UFSCar) and the Agronomy Institute of Campinas (IAC) worked in collaboration with colleagues in Australia and the United States to develop a methodology for analyzing the genomes of polyploid plants (i.e., those with more than two sets of chromosomes of the same type and origin), which could help to uncover the complex structure of the sugarcane genome.

The result of a Thematic Project conducted under the auspices of the FAPESP Bioenergy Research Program (BIOEN), this new methodology has been utilized in a study analyzing the genome of sugarcane. The results were published in the December edition of Scientific Reports, which is an open-access magazine edited by Nature Publishing Group.

“The new methodology represents a watershed in the history of genetic and genomic improvement for sugarcane,” commented Anete Pereira de Souza, professor at the Molecular Biology and Genetic Engineering Center at Unicamp and one of the authors of the study.

“It is as if before, with the tools we had available, we could look at the sugarcane genome with a lens that magnified it 10 times, but now, with the methodology that we developed, we can analyze it with an electronic microscope that magnifies it 100 million times and has a much greater resolution,” she explains.

The new methodology utilizes a combination of single nucleotide polymorphism (SNP) detection with an innovative statistical genetic analysis to determine the genetic and genomic structure of complex polyploids, such as sugarcane.

Already used to study several human diseases, such as cancer, SNPs allow the interpretation of gene variation in polyploid plant species as two single alleles (variants of the same gene) for each SNP, explained the researcher.

Using a mass spectrometer and the statistical genetic analysis method they developed (based on algorithms and software created specifically for this purpose), the researchers identified these sugarcane gene variants and estimated the doses of each variant that are found in the plant.

The results of this genetic and genomic analysis revealed that sugarcane has high levels of ploidy and variants of genes. For example, the number of copies of each gene in the plant could vary from 6 to 14, as noted in the study. 

“The ploidy of sugarcane and other plants is the result of its evolution and domestication over thousands of years, which made it more productive and better adapted to different planting conditions,” explained Souza.

Possible contributions

The researchers’ evaluations indicate that the new method represents a reliable means of genetic analysis in polyploid plants and may contribute to the generation of molecular genetic maps that enable the identification of the exact location of genes of interest in the chromosomes of these agricultural crops, which represent approximately 70% of the extant plant species on the planet.

Although over the last few years, advances in the development of instrumentation and biochemical manipulation techniques have allowed for sequencing and analysis of the genomes of diverse organisms in record time, this has not resulted in a significant increase in the knowledge regarding and efficiency of genetic improvement of complex polyploid plants such as sugarcane.

One of the reasons for these lingering obstacles is that it is still not possible to perfectly understand how the different gene variants found in these plant species function, Souza noted.

“One of the challenges to genetic improvement of sugarcane, for example, is identifying which gene variant is responsible for a given characteristic that is of agricultural interest, such as disease resistance or greater sugar production, and determining how much of that variant is present in the plant’s genome,” she explained.

“Through this new methodology, it will be possible to identify regions of interest in the plant genome and clone them from a library containing more than 400,000 clones of sugarcane gene variants that we have built in parallel with the development of the study. This library can also be utilized for genetic transformation if there is interest,” said Souza.

The method also aids the assembly of the reference genome sequences of sugarcane and other polyploid agricultural crops of economic interest, such as cotton, wheat and strawberry.

Owing to a poor understanding of genes and their functions in these polyploid agricultural crops, it is still not possible to order and correctly identify the sequences or to have a complete picture of their genomes, noted the researchers. 

“The study indicated for the first time that it is possible to genotype complex polyploid plant species, such as sugarcane,” highlights Antonio Augusto Franco Garcia, researcher at the Genetics Department within ESALQ-USP and one of the authors of the study.

Next steps

In addition to sugarcane, the method is now being utilized to study the genome of forage plants that are of major significance for Brazilian livestock, such as Brachiaria and Guinea grass.

Like sugarcane, these plant species are used to feed cattle, and we also lack the methodology and the appropriate efficient tools for studying their genomes. Such tools are available for diploid plants (with two copies of each chromosome), such as beans, rice, soybeans and oranges, and they allow for the improvement of these crops such that they are better adapted and more productive.

“The genetic improvement programs for corn, soybean and rice, for example, routinely utilize molecular markers that are not used in sugarcane due to its genetic complexity,” said Garcia.

“The study that we conducted also paves the way for efficient incorporation of information obtained from molecular markers into Brazil’s existing sugarcane genetic improvement programs in the near future,” she stated.

Researchers participating in the project have dedicated themselves to building a genetic sugarcane map, which should be concluded in 2014. The next step is to identify the plant’s characteristics of interest using the new methodology.

“The idea is to connect the genomic information identified on the genetic map that is being developed with characteristics of interest for plant improvement programs,” explained Marcello Molinari, a FAPESP fellow who is currently doing his post-doctorate at ESALQ in the Genetics Department under the mentorship of Garcia.

Mollinari is travelling to Purdue University in the United States where he will complete a Research Internship Abroad (BEPE), also granted by FAPESP, to conclude this stage of research.

“With this study, we want to know which positions in the sugarcane genome are associated with variations in the plant’s fiber content, for example, which is an important characteristic for second-generation ethanol production,” said Mollinari.

The article SNP genotyping allows an in-depth characterization of the genome of sugarcane and other complex autopolyploids (doi: 10.1038/srep03399), by Garcia et al, can be read by accessing Scientific Reports at: www.nature.com/srep/2013/131202/srep03399/full/srep03399.html.