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Second-Gen Sequencers Help Monsanto Find SNPs, Analyze Variation for Breeding

Providing an example of how second-generation sequencing technologies are gaining a foothold in agricultural-biotechnology research, Monsanto said last week that its scientists have used several undisclosed high-throughput sequencing technologies to assemble de novo the genome of a “top-performing elite female corn line.”
The new technologies are helping Monsanto researchers to analyze genetic variation between different corn lines, information they expect will enable them to develop better corn hybrids. The platforms have also helped Monsanto develop SNP markers for a number of other crop species.
The female inbred corn line the company has sequenced de novo to draft quality is a distant offspring of the B73 line, which was developed at Iowa State University. Earlier this year, researchers led by Washington University St. Louis announced that they had used Sanger sequencing to generate a draft sequence of the line (see In Sequence 2/26/2008). Monsanto said it contributed sequence data to that draft genome.
According to Bob Reiter, vice president of breeding technology at Monsanto, for their latest project, he and his colleagues used a blend of “more than one” second-generation technology and no Sanger sequencing. He would not disclose which specific platforms the company is using or how many it has available in-house, but confirmed that Monsanto has been using both its own instruments and has been outsourcing some sequencing.
The reason the company decided to forgo Sanger sequencing for this project, at least for now, is the speed and relatively low cost of the new technologies, according to Reiter.
“Being able to generate a draft sequence in a couple of months versus something that in the past would take several years, and probably 10 times the amount of money, is very exciting,” he said. Sanger sequencing might come into play later on, when the company completes the genome, he added.
The greatest challenge for now is the de novo assembly of the data, which is especially tricky because of the large amount of repetitive sequence in the corn genome.
“That’s why it is important that it’s done with a blend of technologies to be successful,” Reiter said, adding that “once you have a good reference sequence, the strategy changes. But for de novo assembly, I don’t think you can just do it with one platform.”

“The new sequencing platforms really have been a great tool for us to accelerate the development of SNP markers very much for many crop species.”

The researchers hope that the genome of their corn line will reveal the origin of some of its properties. “[We will] really be able to start looking at what the special and unique genetic components of that line are that make it stand out relative to some of the other lines that we have in our collection,” Reiter said. “We are very interested in seeing how some things have been selected, and which genes have been accumulated in that line.”
Until now, he and his colleagues have mainly used SNP-genotyping to characterize lines. Having their complete genomes “really now allows us to fully understand all of the sequence differences that exist,” he said.
He said Monsanto will probably sequence more corn lines in the future, or at least specific regions of interest in their genomes.
Second-generation sequencing has also helped Monsanto in other areas besides genetically characterizing corn lines. For example, it has enabled company researchers to develop SNP markers in species for which no such markers were available, according to Reiter.
When Monsanto acquired Seminis, a vegetable and fruit seed provider, in early 2005, it obtained a large number of species that it wanted to breed based on DNA markers, said Reiter.
“The new sequencing platforms really have been a great tool for us to accelerate the development of SNP markers very much for many crop species,” he said. “Whereas in the past, with Sanger sequencing, we would spend an entire year working on developing a reasonable number of SNP markers for one species, [now,] using these new technologies, we literally are tackling seven species at once.”
In addition, Reiter said he is interested in using second-generation sequencing for gene-expression analysis. “My expectation is that it is very likely that these new platforms will ultimately become the standard for transcriptome analysis as well,” he said. “We just need to have enough data to be comfortable that we are really getting results that are consistent with past experience with arrays.”

Monsanto is not the only firm that is using second-generation sequencing to help improve crop breeding. Another is Netherlands-based Keygene, a plant breeding-technology company that has been using both 454’s and Illumina’s sequencing technologies to discover and detect polymorphisms (see In Sequence 1/23/2007).

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