Construction of a high-density, high-quality genetic map of cultivated lotus (Nelumbo nucifera) using next-generation sequencing

Zhengwei Liu, Honglian Zhu, Yuping Liu, Jing Kuang, Kai Zhou, Fan Liang, Zhenhua Liu, Depeng Wang, Weidong Ke

The sacred lotus (Nelumbo nucifera) is widely cultivated in China for its edible rhizomes and seeds. Traditional plant breeding methods have been used to breed cultivars with increased yields and quality of rhizomes and seeds with limited success. Currently, the available genetic maps and molecular markers in lotus are too limited to be useful for molecular genetics based breeding programs. However, the development of next-generation sequencing (NGS) technologies has enabled large-scale identification of single-nucleotide polymorphisms (SNPs) for genetic map construction. In this study, we constructed an SNP-based high-density genetic map for cultivated lotus using double digest restriction site-associated DNA sequencing (ddRADseq). An F2 population of 96 individuals was derived from a cross between the rhizome lotus cultivar 'Juwuba' (male parent) and the seed lotus cultivar 'Mantianxing' (female parent). Genomic DNAs from this population were digested with the restriction enzymes EcoRI and MspI and then sequenced. In total, 133.65 Gb of raw data containing 1,088,935,610 pair-end reads were obtained. The coverage of reads on a reference genome was 7.2 % for the female parent, 6.56 % for the male parent, and 1.46 % for F2 individuals. From these reads, 10,753 valid SNP markers were used for genetic map construction. Finally, 791 bin markers (so-segregated adjacent SNPs treated as a bin marker), consisting of 8,971 SNP markers, were sorted into 8 linkage groups (LGs) that spanned 581.3 cM, with an average marker interval of 0.74 cM. A total of 809 genome sequence scaffolds, covering about 565.9 cM of the wild sacred lotus genome, were anchored on the genetic map, accounting for 70.6 % of the genome assembly. This study reports the large-scale discovery of SNPs between cultivars of rhizome and seed lotus using a ddRADseq library combined with NGS. These SNPs have been used to construct the first high-density genetic map for cultivated lotus that can serve as a genomic reference and will facilitate genetic mapping of important traits in the parental cultivars.