Cytonuclear Evolution Analysis Of Allotetraploid Cucumis ×hytivus

Allopolyploids undergo "genome shock",resulting in a large number of genetic and epigenetic modifications.Previous studies mainly focused on nuclear changes,and little is known about organelle-associated genetic variation during allopolyploidization.Allopolyploids are often faced with the challenge of maintaining well coordination between nuclear and cytoplasmic genes inherited from different species.The synthetic allotetraploid Cucumis ×hytivus,which has a clear genetic background,with ancestors C.hystrix and C.sativus,is a useful model system to study cytonuclear variation.The creation of allotetraploid marks the break of existing cytonuclear interactions and the establishment of new cytonuclear interactions.It is necessary to investigate the evolution of cytonuclear after allopolyploidization to elucidate the species formation and stabilization mechanism of allopolyploids.In this study,C.sativus,C.hystrix,allotetraploid C.×hytivus,and allotriploid were used as test materials.Sequencing and comparative analysis of allotetraploid C.×hytivus and its diploid parents using high-throughput sequencing,genome-wide identified nuclear genes targeting organelle and analyzed their expression bias,analysis the sequence and expression bias of the cytonuclear co-encoded complex RuBisCO enzyme after allopolyploid and the phenotypic variation associated with cytonuclear interactions were carried out to investigate the cytonuclear evolutionary characteristics during the formation of allotetraploid C.×hytivus.The main research contents and results are as follows:1.Sequencing and comparative analysis of the chloroplast genomes of C.×hytivus and its diploid parentsThree chloroplast genome sequences were obtained after sequencing（C.sativus L.’BeijingJietou’:MH424440;C.×hytivus Chen and Kirkbride:NC₀33871;C.hystrix Chakr:MH427087）,with genome lengths ranging from 154,673 to 155,760 bp,had similar gene content,gene order,and GC content.The comparative genomic analysis supported chloroplast maternal inheritance,i.e.,C.×hytivus chloroplasts inherited from its female parent C.hystrix.However,sequence alignment and variation analysis between C.×hytivus and C.hystrix identified 51 Indel and 292 SNPs as genetic variants,indicating that the chloroplast genome has been affected by the allopolyploidization based on maternal inheritance,and some mutations have occurred.Nine intergenic regions with rich variation were identified through comparative chloroplast genome analysis in the Cucumis subgenus.A molecular phylogeny based on the chloroplast genome sequences clarified the evolution and taxonomic position of the synthetic allotetraploid C.×hytivus.2.Analysis of the effects of hybridization and genome doubling on organelle genomesTo distinguish the accumulation period of organelle genome variation,the whole genome data of the interspecific hybrid F1 and the early generation of allotetraploids（S0,S4-S13）were used to identify SNP and Indel with reference to maternal C.hystrix chloroplasts and paternal C.sativus mitochondria,respectively.The results showed that F1 hybrids caused 79.5%of chloroplasts and 75.11%of mitochondrial SNP mutations,as well as 55.81%of chloroplasts and 71.65%of mitochondrial Indel mutations,indicating that the effect of hybridization on organelle genome was far greater than the effect of genome doubling.3.Evolutionary analysis of nuclear genes targeting organellesUsing TargetP and LOCALIZER software,the subcellular localization analysis of 46845 nuclear genes of allotetraploid C.×hytivus was performed,and 4461（9.5%）and 2203（4.7%）genes encoding proteins were identified to target chloroplasts and mitochondria,respectively.Expression bias analysis of the homologous gene pairs indicated that the nuclear genes targeting the organelles showed parental C.sativus（C）subgenomic dominant expression.4.Analysis of cytonuclear co-encoding complex RuBisCOThe cytonuclear co-encoding complex RuBisCO was used as a window to investigate cytonuclear interaction during allopolyploidization,the maternal plastid may interact with the biparentally inherited rbcS alleles.The expression of the rbcS gene of C-homoeologs（paternal）was significantly higher than that of H-homoeologs（maternal）in C.×hytivus.Protein interaction prediction analysis showed that the rbcL protein has stronger binding affinity to the paternal copy of rbcS protein than that of maternal copy in C.×hytivus,which might explain the transcriptional bias of the rbcS homoeologs.Moreover,both the activity and content of RuBisCO in C.× hytivus showed mid-parent heterosis.5.Phenotypic variation related to cytonuclear interactionThe RuBisCO enzyme activity in the leaves of allotetraploid and cucumber C.sativus after high temperature treatment was measured.The results showed that the RuBisCO enzyme activity of allotetraploid was not significantly different from that of normal temperature,while the RubsiCO enzyme activity of C.sativus was significantly reduced under high temperature treatment,indicating that the RuBisCO enzyme of allotetraploids had a greater stability than cucumber under high temperature.The analysis of organelle components of the reciprocal allotriploids showed that the differences were mainly in the source of the chloroplast genome.The forward and reverse triploids have obvious differences in phenotype,and the forward hybrids have obvious advantages over the reverse hybrids in microscopic and photosynthetic characteristics,which may be speculated to be related to the greater coordination of its nuclear and cytoplasm.