Genome Assembly,annotation And Evolutionaryanalysis Of Asparagus Setaceus

Asparagus setaceus,a hermaphrodite herb within the genus Asparagus,is an important ornamental plant.However,this specieshas been poorly studied,especially at the molecular level.As a close relative of the Asparagus officinalis,an important economic vegetable,the analysis of the genome of A.setaceus can not only lay a foundation for its own molecular and genetic research,but also facilitate further studies on the comparative genomics of A.setaceus and A.officinalis even on the genus Asparagus.Therefore,we combined different sequencing and assembly strategies to assemble the nuclear genome and chloroplast genome sequences of A.setaceus,and carried out a comprehensive annotation and evolutionary analysis of the genome,finally,acomparative analysis of cytogenetics of A.setaceus and A.officinalis was performed using fluorescence in situ hybridization.The main findings are as follows:1.We constructed a chromosome-scale reference genome of A.setaceus to facilitate the investigation of its genome feature and evolution.A total of 112.52 Gb of long reads was yielded from Nanopore platform,based on 156.28× depth coverage of a predicted genome size of 720 Mb.By means of sequence assembly,calibration of Illumina-short reads,and analysis of data such as10×Genomics linked reads and Hi-C,we generated a high-quality assemblies of A.setaceus covering 710.15 Mb,accounting for 98.63% of the predicted genome size.96.85% of the sequences were anchored to 10 super-scaffolds,corresponding to the 10 chromosomes.The genome of A.setaceus was predicted to have 28,410 genes,and 25,649(90.28%)of them were functionally annotated.65.59% of the genome were occupied by repetitive sequences,of which long terminal repeats were predominant(42.51% of the whole genome).Evolutionary analysis estimated that A.setaceus and its close relative A.officinalis have diverged from ～9.66 million years ago,and A.setaceus underwent two rounds of whole genome duplication events.In addition,762 specific gene families,96 positively selected genes,and 76 resistance R genes in A.setaceus were detected and functionally predicted.2.According to the results of long-read sequencing,we assembled and annotated the chloroplast(cp)genome of A.setaceus with the reference sequence of A.officinalis.The complete cp genome of A.setaceus is 156,978 bp in length,contains a large single copy region of 85,311 bp and a small single copy region of 18,641 bp,and a pair of inverted repeat regions of 26,513 bp.A total of 132 genes were annotated,including 38 t RNA,6 r RNA,and 88 protein coding genes.According to the evolutionary analysis of chloroplast sequences,the chloroplast genome of A.setaceus is clustered with the chloroplast genomes of two other asparagus species,A.officinalis and Asparagus schoberioides,and is closely related to the chloroplast genome of other plants in the liliaceae family.3.We used bioinformatics to analyze NUPTs of nuclear integrants of plastid DNA in A.setaceus.The results indicate that there were 1,232 NUPT sequences inserted into the nucleargenome of A.setaceus,with a total length of 467,208 bp,accounting for 0.066% of the nuclear genome.Analysis of the length distribution and distribution on chromosomes of NUPTs shows that about 68.8% of NUPTs are below 299 bp in size,the number of NUPTs between 100-199 bp is up to 374,and there are 89 NUPTs over 1000 bp.Of which the longest insert is 12,114 bp in length and is located on assembled chromosome 2.NUPT is distributed on each chromosome.The number of NUPTs inserted on chromosome 1 is the largest,at 195,and the total length of NUPTs inserted on chromosome 5 is the longest,which is 997 bp /100 Mb.NUPT is distributed unevenly on the chromosomes,and there are some hot spots on chromosomes 1 and 5.Analysis of chloroplast genome sources found that the insertion densities of NUPTs from LSC,IR,and SSC were 8/kb,14/kb,and 8/kb,respectively,indicating that NUPTs sequences from the IR region were more frequently transferred to the nuclear genome.4.To further investigate the evolutionary relationship between A.setaceus and A.officinalis,we performed fluorescence in situ hybridization on the mitotic metaphase and interphase chromosomes of A.setaceus using a series of single chromosome specific probes from the A.officinalis genome.The result showed that among the 10 single-chromosome-specific probes,7 of them can be specifically located on the chromosome of A.setaceus,and the signal was clear and stable.Comparing the hybridization results of A.setaceus and A.officinalis:the chromosome 1,3,4,5,7,8 and 9 pairs of A.setaceus karyotype correspond to the chromosome 4,5,2,3,8,7 and 10 pairs of A.officinalis karyotype,respectively.These findings not only enables us to distinguish the chromosomes of A.setaceus in cytology to establish a standard karyotype,but also lays a foundation for the future cytogeneticstudies on A.setaceus and the genus Asparagus,such as the evolution of the sex chromosome of A.officinalis,the evolution and chromosome rearrangement of the species of the genus Asparagus.