Survey analysis and phylogenetic study of three polyploid kiwifruit genomes

Abstract: 【Objective】Plant polyploidization is the evolution of adaption to environmental changes and protection of their own population development. The polyploidization of kiwifruit could double its chromosome number and affect the structure and function of its genome, thereby enriching the genetic diversity of the species. The study aimed to comprehensively understand the chromosomal ploidy and genomic characteristics of polyploid kiwifruit germplasm resources, and analyze their systematic evolutionary relationships in kiwifruit.【Methods】This study analyzed the chromosome ploidy of AcD2301 (Actinidia chinensis), AcD2302 (A. arguta) and AcD2303 (A. valvata) with reference to the diploid A. chinensis‘Hongyang’. The samples were analyzed by flow cytometry on the CyFlow Space flow cytometer after being lysed by CyStar UV Precise P kit and dyed by DAPI fluorescent dye in the dark. The total genomic DNA of kiwifruit was extracted by CTAB method, and then electrophoresis was con-ducted with 0.8% agarose gel. The DNA quality was detected with UV spectrophotometer. The secondgeneration sequencing technology Illumina NovaSeq sequencing platform was used to perform double end sequencing on the sample library. The softwares such as FastP were used to view the distribution of base quality, average error rate distribution of reads, and base content distribution of reads sequencing. The raw data with adapters and low-quality reads were filtered to obtain high-quality sequences, and the sequences were compared with nucleic acid databases. The high quality sequencing data was generated using Jellyfish (version 2.3.0) software k-mer19 to generate K-mer frequency tables, and genome size, heterozygosity, and repeatability were estimated using the GenomeScope 2. The next- generation sequencing data of kiwifruit, published in the NGDC and NCBI databases, were compared with the reference genome A. chinensis Hongyang v4.0. The SNP calling was performed using GATK software, and the Maximum likelihood algorithm in fast Tree software was used to construct phylogenetic trees of the 15 kiwifruit species, including A. chinensis, A. arguta and A. valvata.【Results】The samples were subjected to flow cytometry analysis, and the peak values of the diploid Hongyang kiwifruit were compared with the reference species. The chromosome ploidy of the AcD2301 and AcD2302 were both tetraploid, while the chromosome ploidy of the AcD2303 was hexaploid. The subsequent genome survey analysis results were consistent with this. The AcD2301, AcD2302 and AcD2303 gene DNA were sequenced by the Illumina NovaSeq sequencing platform. The sequencing yielded raw data of 162.91 Gb, 139.74 Gb, and 142.44 Gb, followed by filtering to obtain high-quality data of 160.64 Gb, 138.16 Gb, and 140.73 Gb. The sequencing quality assessment showed that the Q20 and Q30 values of the AcD2301 were 96.95% and 91.91% , respectively. The Q20 and Q30 values of the AcD2302 were 97.09% and 92.07%, respectively. The Q20 and Q30 values of the AcD2303 were 96.80% and 91.43%, respectively; The GC contents were approximately 37.20%, 36.77%, and 36.15%, respectively. The sequencing data quality values were all greater than 35, and the base error rates were all less than 0.045, indicating that the genome reads had high quality and could be used for subsequent analysis. The reads from the sequencing data of the AcD2301, AcD2302, and AcD2303 were randomly selected and compared with the nucleic acid library (NT library). The results showed that all the randomly selected reads could be compared with the genome of kiwifruit plants, indicating that there was no contamination in the sequencing data. Through K-mer analysis of the kiwifruit genome data after quality control, the genome size of the AcD2301 was estimated to be 626 Mb, heterozygosity to be 3.00% , and repeat sequence ratio to be 43.70%; The estimated size of the AcD2302 genome was 668 Mb, with a heterozygosity of 3.30% and a repeat sequence ratio of 45.30%; The estimated genome size of the AcD2303 was 585 Mb, with a heterozygosity of 8.06% and a repeat sequence ratio of 40.70%. In addition, the support rates for homologous tetraploids of the AcD2301 and AcD2302 were 97% and 96.7%, respectively. To analyze the evolutionary relationship of kiwifruit plants, the SNP sequences were screened from the second-generation sequencing data of the 15 kiwifruit, including the AcD2301, AcD2302, and AcD2303. The Maximum likelihood algorithm was used to construct a phylogenetic tree. The results showed that the 15 kiwifruit plants were divided into three major evolutionary branches, with Actinidia chinensis AcD2301 as an independent branch, Actinidia chinensis‘Donghong’as another independent branch, and the remaining 13 kiwifruit species as an evolutionary branch. Actinidia chinensis var. deliciosa in the third evolutionary branch was a small evolutionary branch, while the other 12 kiwifruit species formed a small evolutionary branch. For the latter, Actinidia hubeiensis was a separate group; The remaining 11 kiwifruit species were grouped together, and the 6 kiwifruit species in the net fruit group were clustered into a small evolutionary branch, while the 9 kiwifruit species in the remaining brancheswere all part of the spotted fruit group. From this, it could be seen that the AcD2302, which belonged to the net fruit group, was closely related to the AcD2303, and both had evolved independently from the AcD2301 in the spotted fruit group.【Conclusion】The chromosome ploidy, genome size, and phylogenetic relationships of the AcD2301, AcD2302 and AcD2303 were analyzed, which could provide reference for the whole genome sequencing of the polyploid kiwifruit in the future.