bstract:【Purpose】Plant polyploidization is the evolution of adapting to environmental changes and protecting their own population development. The polyploidization of kiwifruit will double its chromosome number and affect the structure and function of its genome, thereby enriching the genetic diversity of kiwifruit. 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 (Actinidia arguta) and AcD2303 (Actinidia valvata) with reference to the diploid Actinidia chinensis Hongyang. The samples were analyzed by flowcytometry 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 conducted by 0.8% agarose gel. DNA quality was detected with UV spectrophotometer. Use the second-generation sequencing technology Illumina NovaSeq sequencing platform to perform double end sequencing on the sample library. Use software such as FastP to view the distribution of base quality, average error rate distribution of reads, and base content distribution of reads sequencing. Filter the raw data with adapters and low-quality reads to obtain high-quality sequences, and compare them with nucleic acid databases. 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, which have been published in the NGDC and NCBI databases, were compared with the reference genome Actinidia chinensis 'Hongyang' v4.0. SNP calling was performed using GATK software, and the Maximum likelihood algorithm in fastTree software was used to construct phylogenetic trees of 15 kiwifruit species, including Actinidia chinensis, Actinidia arguta and Actinidia valvata.【Result】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 AcD2301 and AcD2302 were both tetraploid, while the chromosome ploidy of AcD2303 was hexaploid. The subsequent genome survey analysis results were consistent with this. The total genomic DNA of the sample was extracted by CTAB. After being detected, the AcD2301, AcD2302 and AcD2303 gene DNA were sequenced by the Illumina NovaSeq sequencing platform. 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 AcD2301 were 96.95% and 91.91%, respectively. The Q20 and Q30 values of AcD2302 were 97.09% and 92.07%, respectively. The Q20 and Q30 values of AcD2303 were 96.80% and 91.43%, respectively; The GC contents are approximately 37.20%, 36.77%, and 36.15%, respectively. The sequencing data quality values are all greater than 35, and the base error rates are all less than 0.045, indicating that the genome reads have high quality and can be used for subsequent analysis. Randomly select reads from the sequencing data of AcD2301, AcD2302, and AcD2303 and compare them with the nucleic acid library (NT library). The results show that the randomly selected reads can all be compared with the genome of kiwifruit plants, indicating that there is no contamination in the sequencing data. Through K-mer analysis of the kiwifruit genome data after quality control, AcD2301 estimated the genome size to be 626 Mb, heterozygosity to be 3.00%, and repeat sequence ratio to be 43.70%; The estimated size of the AcD2302 genome is 668 Mb, with a heterozygosity of 3.30% and a repeat sequence ratio of 45.30%; The estimated genome size of AcD2303 is 585Mb, with a heterozygosity of 8.06% and a repeat sequence ratio of 40.7%. In addition, the support rates for homologous tetraploids of AcD2301 and AcD2302 were 97% and 96.7%, respectively. To analyze the evolutionary relationship of kiwifruit plants, SNP sequences were screened from the second-generation sequencing data of 15 kiwifruit, including 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 forming one evolutionary branch. Actinidia chinensis var. deliciosa in the third evolutionary branch is a small evolutionary branch, while the other 12 kiwifruit species form a small evolutionary branch. For the latter, Actinidia hubeiensis is a separate group; The remaining 11 kiwifruit species are grouped together, with the 6 kiwifruit species in the net fruit group clustered into a small evolutionary branch, while the 9 kiwifruit species in the remaining branches are all part of the spotted fruit group. From this, it can be seen that AcD2302, which belongs to the net fruit group, is closely related to AcD2303, and both have evolved independently from AcD2301 in the spotted fruit group.【Conclusion】The chromosome ploidy, genome size, and phylogenetic relationships of AcD2301, AcD2302 and AcD2303 were analyzed, providing reference for future polyploid kiwifruit whole genome sequencing and theoretical support for further research on kiwifruit polyploidization and phylogenetic relationships.
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