The transgenic papaya “SunUp” was developed in the 1990s and gained much publicity due to its ability to resist the papaya ring spot virus. Although researchers from the Ming group had identified the genomic sequence of SunUp in 2008, it was unclear where the transgenic deposits were and what effect they had. A new study has now identified these changes and how they affect the transgenic plants.
Papaya fruits are a rich source of potassium, magnesium and vitamins A and C, which drives a steady increase in their global production. Papaya originated and was domesticated in southern Mexico and Central America and is now grown in tropical and subtropical regions around the world. Wild papaya has small sagging fruits with very little edible flesh, while the domesticated version can weigh more than five pounds. However, there was a major problem: Papaya was susceptible to papaya ringworm virus, which resulted in inhibited plants that do not produce ripe fruit, and there is no resistance in papaya’s genetic code.
To counteract this problem, researchers developed the transgenic papaya SunUp, using a technique called particle bombardment-mediated transformation. Gold particles were covered with the fur protein gene from the virus and shot into the cells of the non-transgenic papaya “Sunset” using a gene gun. SunUp therefore contained gene sequences of the virus and was protected from infection via RNA-mediated gene silence.
“It took us 8 years to read each DNA nucleotide in the inserts and rearrangements, and we repeated the sequencing using different technologies to understand the nature of these transgenic inserts,” said Ray Ming (GEGC), a professor of plant biology. “The insert was so complex that even though we sequenced the genome in 2008, we did not know where the transgenic sequences were.”
In previous studies, researchers used Sanger DNA sequencing techniques that read short stretches of DNA, 500 to 600 bases, making it difficult to accurately place the transgenic sequences in the genome of the draft. In the current study, they used sequencing technologies from Pacific Biosciences and Oxford Nanopore technologies to read very long stretches of DNA. “These are the latest available technologies and they enabled us to read over 50 to 200,000 base pairs at a time,” Ming said.
The group discovered that SunUp had a deposit of 1.6 million base pairs, which consisted of DNA fragments not only from the gene gun, but also nuclear DNA sequences derived from chloroplasts and mitochondria. “There were 74 fragments in the insert: 42 were nuclear chloroplast fragments, 13 were nuclear mitochondrial fragments, 10 were from the chloroplast genome and 3 were from the mitochondrial genome,” Ming said. “The particle bombardment broke the double-stranded DNA and inserted all 74 fragments into a site in chromosome 5 of the genome.”
Surprisingly, even though there is such a large deposit, the transgenic manipulation did not cause any change in gene expression. “We looked at each gene sequence and there is no effect on genome function. When we compared SunUp and Sunset, they have only 20 genes that are differentially expressed, due to transposon-mediated rearrangement and not from the genetic manipulation done by particle bomb-mediated transformation. , “sa Ming. Transposon-mediated rearrangements occur naturally and lead to gradual changes over time, which is expected as SunUp and Sunset have grown and diverged for 30 years.
The researchers will look at other transgenic papaya lines to see if they have similar rearrangements. “We expected many more insertions and rearrangements and we were surprised that there were only two. In addition to the 1.6 Mb insertion caused by the 74 fragments, there was a 591 Kb deletion in chromosome 5 that was moved into 1, “6 Mb insertion. We still do not understand why there were nuclear mitochondrial and chloroplast fragments that flanked the three transgenic fragments and why they were all inserted in the same place. We will investigate other transgenic lines to see if there is any underlining mechanism in common.” Ming.
“Because transgenic papaya has such a strong resistance to papaya ringworm virus and thus saved the Hawaiian papaya industry, it was the poster child for transgenic crops. Transgenic papaya was approved by several countries that rejected other such crops,” Ming said. “This work will reinforce the message that even after three decades, we can still consume transgenic papaya safely and there is no negative effect on the papaya genome or consumers.”
The work was supported by the US National Science Foundation Plant Genome Research Program Award, the National Natural Science Foundation in China, the Natural Science Foundation in Fujian Province and the Science and Technology Innovation Fund at Fujian Agriculture and Forestry University.