Study On The Linkage Map Construction And Genome Consolidation In Nelumbo Nucifera Gaertn

Nelumbo nucifera Gaertn.,or sacred lotus,has been cultivated for over thousand years in Asia for the edible and medicinal value of its rhizomes,seeds and leaves.This plant has survived since the Late Cretaceous period,occupies an important position in understanding the origin of eudicots and ancient polyploidization events.Currently,two draft assemblies of Nelumbo nucifera nuclear genome,the genomes of ’China Antique’and ’Chinese Tai-zi’,as well as a precise chloroplast genome of N.nucifera,have been released.However,the insufficient information of genetic and physical maps in N.nucifera hindered the assembly of N.nucifera whole genome sequencing results into chromosomes,and resulted in many fragmented sequences in these two nuclear genome assemblies.To anchor scaffolds of the N.nucifera draft assembly into chromosomes,a linkage map with higher resolution and a physical map are needed.Moreover,information on the N.nucifera mitochondrial genome are needed to complement the whole genome information of N.nucifera.Here,in order to dissect large-scale genomic molecular marker resources for sacred lotus,we re-sequenced a Thailand sacred lotus cultivar ’Thailand Chiang Mai wild’ and compared with the reported lotus genome ’Chinese Tai-zi’.A total of 2,750,585 SNPs,328,251 InDels and 14,191 SVs were found between the two genomes.Furthermore,14,502 EST-SSRs were also indentified using the available RNA-seq data of N.nucifera.A subset of 150 SSRs(genomic and EST-SSRs)was randomly selected for validation and genetic diversity analysis.The genotypes could be easily distinguished using these SSR markers and the ’Chiang Mai wild lotus’ was obviously differentiated from the other Chinese accessions.Using N.nucifera cultivar ’Chinese Tai-zi’ and ’Thailand Chiang Mai’ as parents,we have built a F2 segregate population and constructed a high resolution linkage map for N.nucifera using restriction-site associated DNA sequencing data of the 181 F2 individuals.A total of 217,577 high-quality polymorphic SNPs were genotyped and converted into 2,371 bin markers.The resulting linkage map clustered all 217,577 SNPs into 8 linkage groups,with a total genetic distance of 789.54 cM.To validate the accuracy of the linkage map,the LOD scores ’were estimated,the mapping algorithms were changed,additional 198 SSR markers were included and comparisons to previously reported maps were performed.The results showed consistent marker orders with the original map,indicated that the marker orders were consistent across replications of analysis and were competent to represent their actual distributions along the chromosomes.To refine the draft assembly of’Chinese Tai-zi’,An optical map spanning 86.20%of the ’Chinese Tai-zi’ assembly(645.12 Mb)was constructed using 211-fold coverage of BioNano Irys clean data.Based on the information of BioNano optical map,the draft assembly of ’Chinese Tai-zi’ was improved,showing a 1.5-fold increase of the scaffold N50.By comparing with the linkage map,33 chimeric scaffolds were identified and split.Using a combination of multiple maps,97.9%of the scaffolds in the ’Chinese Tai-zi’ draft assembly and 97.6%of the scaffolds in the ’China Antique’ draft assembly were anchored into chromosomes,and the general location of centromeres were inferred from both crossover-suppressed regions and the distribution of NnCenH3 ChIP-Seq reads.Based on the gene orders along N.nucifera chromosomes,we proposed that several chromosome rearrangements incluading 14 ancestral chromosome fissions,20 fusions and one inversion were required to reach the modern genome structure of 8 chromosomes of N.nucifera form the 8 protochromosomes of ancestral eudicot karyotype.We sequenced the N.nucifera mitochondrial genome using single molecule real-time sequencing technology,and constructed the mitochondrial genome map.The results showed that the 524,797-bp N.nucifera mitochondrial genome has a total of 63 genes,including 40 protein-coding genes,three rRNA genes and 20 tRNA genes.Approximately 700 RNA editing sites in the protein-coding genes were identified.Nineteen chloroplast-derived fragments were identified,and seven tRNAs were derived from the chloroplast.These results suggest that the N.nucifera mitochondrial genome retains evolutionarily conserved characteristics,including ancient gene content,high levels of RNA editing,and low levels of chloroplast-derived fragment insertions.The present study provides(1)series of molecular markers,(2)a reliable high-resolution linkage map,(3)a BioNano optical map,(4)chromosome level genome assemblies and(5)mitochondrial genome map for N.nucifera,which are valuable for the breeding and cultivation of N.nucifera and future studies of comparative and evolutionary genomics in angiosperms.