Understanding Genomic Modifications in Transgenic Papaya

The transgenic papaya “SunUp” was developed in the 1990s and received a lot of publicity for its ability to resist the papaya ringspot virus. Although researchers from the Ming group had identified SunUp’s genomic sequence in 2008, it was unclear where the transgenic inserts 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, steadily increasing their worldwide production. Papaya originated and domesticated in southern Mexico and Central America, and is now grown worldwide in tropical and subtropical regions. Wild papaya has small seedy fruits with very little edible flesh, while the domesticated version can weigh more than five pounds. However, there was one major problem: Papaya was susceptible to the papaya ringspot virus, which resulted in immature plants that failed to produce mature fruit, and there is no resistance in the papaya’s genetic code.

To counter this problem, researchers developed the transgenic papaya SunUp, using a technique called particle bombardment-mediated transformation. Gold particles were coated with the virus’s coat protein gene and fired into the cells of the non-transgenic papaya “Sunset” with a gene gun. SunUp therefore contained gene sequences from the virus and was protected from infection via RNA-mediated gene silencing.

“It took us 8 years to read every DNA nucleotide in the insertions and rearrangements, and we repeated the sequencing using different technologies to understand the nature of these transgenic insertions,” said Ray Ming (GEGC), a professor of plant biology. “The insertion was so complex that even though we sequenced the genome in 2008, we didn’t know where the transgenic sequences were.”

In previous studies, the researchers used Sanger DNA sequencing technology that reads out short stretches of DNA, 500 to 600 bases, making it difficult to accurately place the transgenic sequences in the draft genome. 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 newest techniques available and they allowed us to read more than 50 to 200 thousand base pairs at once,” Ming said.

The group found that SunUp had a 1.6 million base pair insert, which consisted not only of DNA fragments from the gene gun, but also nuclear DNA sequences 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 mitochondrial genome,” Ming said. “The particle bombardment broke the double-stranded DNA and placed all 74 fragments in one location in chromosome 5 of the genome.”

Surprisingly, although there is such a large insertion, the transgene manipulation caused no change in gene expression. “We looked at every gene sequence and there is no impact on genome function. When we compared SunUp and Sunset, they have only 20 genes that are differentially expressed, which are due to transposon-mediated rearrangements and not the genetic manipulation done by particle bombardment-mediated transformation,” Ming said. realignments occur naturally and lead to gradual changes over time, which is expected as SunUp and Sunset have been growing and diverging for 30 years.

The researchers will look at other transgenic papaya lines to see if they have similar rearrangements. “We expected a lot more insertion locations and realignments and were surprised that there were only two. In addition to the 1.6 Mb insert caused by the 74 fragments, there was a 591 Kb deletion in chromosome 5 that was moved to the 1.6 Mb insert. We still do not understand why there were nuclear mitochondrial and chloroplast fragments flanking the three transgenic fragments and why they were all introduced in the same location. We will explore other transgenic lines to see if there is a common underlining mechanism,” Ming said.

“Because transgenic papaya has such a strong resistance to the papaya ringspot virus and thus saved the Hawaiian papaya industry, it was the epitome of transgenic crops. Transgenic papaya has been approved by several countries that have rejected other such crops,” Ming said. “This work will reinforce the message that even after three decades we can still safely consume transgenic papaya and that 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, National Natural Science Foundation of China, Natural Science Foundation of Fujian Province and the Science and Technology Innovation Fund of Fujian Agriculture and Forestry University.

Reference:

  1. Jingjing Yue, Robert VanBuren, Juan Liu, Jingping Fang, Xingtan Zhang, Zhenyang Liao, Ching Man Wai, Xiuming Xu, Shuai Chen, Shengchen Zhang, Xiaokai Ma, Yaying Ma, Hongying Yu, Jing Lin, Ping Zhou, Yongji Huang, Ban Deng, Fang Deng, Xiaobing Zhao, Hansong Yan, Mahpara Fatima, Dessireé Zerpa-Catanho, Xiaodan Zhang, Zhicong Lin, Mei Yang, Nancy J. Chen, Eric Mora-Newcomer, Patricia Quesada-Rojas, Antonio Bogantes, Víctor M. Jiménez , Haibao Tang, Jisen Zhang, Ming-Li Wang, Robert E. Paull, Qingyi Yu, Ray Ming. SunUp and Sunset genomes revealed the impact of particle bombardment-mediated transformation and domestication history in papaya. Natural Genetics, 2022; DOI: 10.1038/s41588-022-01068-1
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