Impact Of Biolistic-mediated Transformation On Papaya Genome Structure And Function

Papaya is a productive and nutritious tropical fruit and is a model system for studying sex chromosome evolution in plants.Papaya Ringspot Virus(PRSV)is the most devastating pathogen threatening papaya production worldwide.Transgenic papaya is the most publicized success story that saved the papaya industry from destruction by ringspot virus in Hawaii and other regions.It was developed by particle bombardment that breaks the double strand nuclear DNA,and randomly integrating the target genes and vector fragments during the DNA repair process,which could cause chromosomal rearrangements,deletions,and mutations.Up to date,there is no genome wide assessment of the impact of particle bombardment on genome structure and function.The objective of this study is to apply high throughput genome re-sequencing and RNA-Seq approaches to compare genome-wide structural and functional differences between transgenic SunUp and non-transgenic progenitor Sunset,providing supporting evidence to assess the safty of transgenic papaya and other crops.Besides the direct effects of biolistic transformation,such as transgene copy numbers and integration position effects,other factors such as somaclonal variations caused by tissue culture and spontaneous mutations during meiosis of over twenty generations might induce unintended genetic and epigenetic changes in transgenic papaya cultivar,which would lead to the divergence of gene expression between genetically engineered(GE)and non-GE papaya cultivars.Separating the changes induced by transformation from other types of genetic variations can be a challenge.In order to address the aforementioned problems,we conducted in-depth studies in the following aspects based on traditional methods,high throughput sequencing technology and bioinformatic analysis.At first,we evaluated the type,frequency,distribution,and molecular basis of spontaneously mutated SSR markers in papaya genome,which can improve our understanding of the rate of spontaneous mutation in papaya,and the mechanisms of genomic modification that shape the genome and contributed to increased genetic diversity.Subsequently,we performed whole-genome resequencing on papaya transgenic progenitor Sunset to identify genome-wide structural changes between genomes of transgenic SunUp and non-transgenic progenitor Sunset.Lastly,large scale RNA-Seq experiment was carried out in young healthy leaves of transgenic SunUp and its progenitor Sunset.These data allow for comparisons of genomic variation in transgenic papaya to the genomic variation observed in standing accessions.The results provide new insight towards the complex molecular and cellular events during various biological pathways in these two cultivars and give evidence at genome-wide structural and transcriptional level that transgenic papaya are not harmful concerning the biosafety of genetically modified organisms(GMOs).The main results and conclusions are as follows.1.Molecular basis of off-type microsatellite markers in papayaFrom a simple sequence repeat(S SR)mapping project in papaya,37 SSRs displayed non-parental alleles in the F2 population,known as off-type markers.They all belong to Class ?(?20bp,hypervariable markers).To investigate the molecular basis of these non-parental alleles,seven SSRs with off-type alleles were cloned and sequenced from two parents and selected F2 individuals.SSR repeat motif variation was the major cause for the generation of off-type alleles and single nucleotide polymorphisms(SNPs)and Insertion/deletion(InDels)also contributed to off-type polymorphism.Off-type SSRs arose after one meiosis at 3%frequency.Two of the seven markers had no polymorphisms between the parents but non-parental alleles segregated among the F2 individuals.Four of the seven markers exhibited SNPs between parents with transition and transversion at 2:1 ratio.Six markers showed additional SNPs between F2 offspring and parents with transition and transversion at about 1:1 ratio,suggesting higher than expected level of transversion mutations at the F2 population after one meiosis event between the parental genomes.These off-type SSRs were analyzed in a second F2 population and 43%of them were conserved.The major cause of off-type SSRs in papaya is the variation of repeat length in Type ? SSRs,and the underlying mechanism might be DNA replication slippage of repeat motifs.The presence and high conservation of these off-type markers demonstrated that hypervariable class I SSRs could generate genetic variation in one generation and increase genetic diversity to for natural selection to enhance fitness.2.Comparative analysis between PRSV resistant transgenic papaya and its progenitor cultivar reveals structural variations induced by biolistic based transformationUsing paired-end short-read next-generation sequencing(NGS),a total of more than 74 million reads for nontransgenic papaya Sunset were obtained and mapped onto transgenic papaya SunUp reference genome.In total,310,364 polymorphisms(SNPs),34,071 small InDels(Inserts/deletions)and 1,200 structural variations(SVs)were found between Sunset and SunUp.Those variations have an uneven distribution across the 9 chromosomes of papaya.Regarding the effects of the mutations on gene function,>90%of the mutations were located in intergenic regions,while only 0.27%were predicted to be high-impact mutations.Gene ontology(GO)enrichment analysis revealed that ATP-related categories were highly enriched among these high-impact genes.The SNP mutation rate was about 8.4×10-4per site,highly comparable with the rate induced by spontaneous mutation over numerous generations.The transition-to-transversion ratio was 1.439 and the predominant mutations were C/G to T/A transitions.We inferred that spontaneous mutation was the leading cause of SNPs in transgenic papaya SunUp.We also studied the integration of organelle DNA into the nuclear DNA of SunUp and Sunset.A total of 3,430 nuclear plastid DNA(NUPT)and 2,764 nuclear mitochondrial DNA(NUMT)junction sites had been found in SunUp,which is proportionally higher than the predicted total NUPT and NUMT junction sites in Sunset.Among all nuclear organelle DNA(norgDNA)junction sites,95%junction sites were shared by SunUp and Sunset,suggesting norgDNA are abundant in papaya nuclear genomes and highly conserved after transgene insertion.NUPT/NUMT junction sites exclusively in SunUp were significantly different from those in Sunset.The average identity between SunUp specific norgDNA and corresponding organelle genomes was higher than the identity between norgDNA shared by SunUp and Sunset and corresponding organelle genomes.BLAST between the six SunUp organelle-like borders of transgenic insertions and corresponding organelle genomes brought us to percent identities of 98.18-100%,which were higher than the identities between five NUPT borders and Sunset norgDNA.All paired-end spans of mapped Sunset reads were shorter than SunUp transformation plasmid derived inserts.Those hints suggested that many new DNA transferred from organelles to the nuclear genome during bombardment,including these six organelle-like borders.Taken together,the present study of comparative whole-genome analyses between SunUp and Sunset using NGS provides a reliable estimate of genome-wide discrepancy.Development of SNP/InDel markers that occurred in high-impact genes could facilitate marker-assisted PRSV disease resistance breeding in papaya.The newly integrated norgDNA induced by particle bombardment implied the mechanisms underlying the process of foreign gene transformation.3.Transcriptome profiling revealed stress-induced and disease resistance genes up-regulated in PRSV resistant transgenic papayaWe conducted transcriptome sequencing of PRSV resistant transgenic papaya SunUp and its PRSV susceptible progenitor Sunset to compare the transcriptional changes in young healthy leaves prior to infection with PRSV.In total,20,700 transcripts were identified,and 842 differentially expressed genes(DEGs)randomly distributed among papaya chromosomes.Gene ontology(GO)category analysis revealed that microtubule-related categories were highly enriched among these DEGs.Numerous DEGs related to various transcription factors,transporters and hormone biosynthesis showed clear differences between the two cultivars,and most were up-regulated in transgenic papaya.Many known and novel stress-induced and disease-resistance genes were most highly expressed in SunUp,including MYB,WRKY,ERF,NAC,nitrate and zinc transporters,and genes involved in the abscisic acid,salicylic acid,and ethylene signaling pathways.We also identified 67,686 alternative splicing(AS)events in Sunset and 68,455 AS events in SunUp,mapping to 10,994 and 10,995 papaya annotated genes,respectively.GO enrichment for the genes displaying AS events exclusively in Sunset was significantly different from those in SunUp.Transcriptomes in Sunset and transgenic SunUp are very similar with noteworthy differences,which increased PRSV-resistance in transgenic papaya.No detrimental pathways and allergenic or toxic proteins were induced on a genome-wide scale in transgenic SunUp.Our results provide a foundation for unraveling the mechanism of PRSV resistance in transgenic papaya.