Understanding genomic modifications in transgenic papaya

The transgenic “SunUp” papaya was developed in the 1990s and has been widely publicized for its ability to resist papaya ringspot virus. Although Ming Group researchers identified the genomic sequence of SunUp in 2008, it was not known where the transgenic insertions were and what effect they had. A new study has now identified these changes and their influence on transgenic plants.

Papayas are a rich source of potassium, magnesium and vitamins A and C, propelling a steady increase in their production worldwide. The papaya originated and was domesticated in southern Mexico and Central America, and is now grown in tropical and subtropical regions around the world. The wild papaya has small, grainy fruits with very little edible flesh, while the domesticated version can weigh over five pounds. However, there was one major problem: the papaya was susceptible to papaya ringspot virus, resulting in stunted plants that do not produce ripe fruit, and there is no resistance in the papaya genetic code. .

To counter this problem, the researchers developed the SunUp transgenic papaya, using a technique called particle bombardment-mediated transformation. Gold particles were coated with the virus coat protein gene and injected into the cells of the non-transgenic “Sunset” papaya using a gene gun. SunUp therefore contained gene sequences from the virus and was protected from infection by 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), Professor of plant biology. “The insertion was so complex that although we sequenced the genome in 2008, we didn’t know where the transgenic sequences were.”

In previous studies, researchers have used Sanger DNA sequencing technology which reads short stretches of DNA, 500 to 600 bases, making it difficult to accurately place 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 latest techniques available and they have allowed us to read over 50 to 200,000 base pairs at a time,” Ming said.

The group found that SunUp had a 1.6 million base pair insert, which consisted of DNA fragments not only 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 the mitochondrial genome,” Ming said. “The particle bombardment broke the double-stranded DNA and inserted the 74 fragments at one location on chromosome 5 of the genome.”

Surprisingly, even though there is such a large insertion, the transgenic manipulation did not cause any changes in gene expression. “We have looked at every gene sequence and there is no impact on genome function. When we compared SunUp and Sunset, they only have 20 differentially expressed genes, which are due to transposon-mediated rearrangements and not the genetic manipulation performed by particle bombardment-mediated transformation,” said Ming. Transposon-mediated rearrangements occur naturally and lead to gradual changes over time, which is expected since SunUp and Sunset have been developing and diverging for 30 years.

Researchers will examine other transgenic papaya lines to see if they exhibit similar rearrangements. “We expected many more insertion sites and rearrangements and were surprised that there were only two. In addition to the 1.6 Mb insertion caused by the 74 fragments, there are had a 591 Kb deletion in chromosome 5 which was moved into the 1.6 Mb insertion. We still do not understand why there were nuclear fragments of mitochondria and chloroplasts flanking the three transgenic fragments and why they have all were inserted in the same place. We will look at other transgenic lines to see if there is a common underlying mechanism,” Ming said.

“Since transgenic papaya has such strong resistance to papaya ringspot virus and thus saved the Hawaiian papaya industry, this was the poster child for transgenic crops. Transgenic papaya was approved by several countries who 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 there are no negative effects on papaya genome or consumers.”

The work was supported by the US National Science Foundation Plant Genome Research Program Award, the National Natural Science Foundation of China, the 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, Desireé 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. The SunUp and Sunset genomes revealed the impact of particle bombardment-mediated transformation and domestication history in papaya. Genetics of Nature, 2022; DOI: 10.1038/s41588-022-01068-1
/Public release. This material from the original organization/authors may be ad hoc in nature, edited for clarity, style and length. The views and opinions expressed are those of the author or authors. See in full here.

Leave a Comment