Identification of a genomic region linked with sex determination of Undaria pinnatifida (Alariaceae) through genomic resequencing and genetic linkage analyses of a segregating gametophyte family.

Different from the traditional knowledge about kelp, three sexual phenotypes (female, male, and monoecious) exist in the haploid gametophytes of Undaria pinnatifida. However, the sex-determining mechanisms remain unknown. Genetic linkage mapping is an efficient tool to identify sex-linked regions. In the present study, we resequenced a segregating gametophyte family based on the male genome of U. pinnatifida. A high-density genetic linkage map was constructed using 9887 SNPs, with an average distance of 0.41 cM between adjacent SNPs. On the basis of this genetic map and using the composite interval mapping method, we identified 62 SNPs significantly linked with the sexual phenotype. They were located at a position of 67.67 cM on the linkage group 23, corresponding to a physical range of 14.67 Mbp on the HiC_Scaffold_23 of the genome. Reanalysis of the previous specific length amplified fragment sequencing data according to the reference genome led to the identification of a sex-linked genomic region that encompassed the above-mentioned 14.67 Mbp region. Hence, this overlapped genomic range was likely the sex-determining region. Within this region, 129 genes were retrieved and 39 of them were annotated with explicit function, including the potential male sex-determining gene-encoding high mobility group (HMG) domain protein. Relative expression analysis of the HMG gene showed that its expression was higher in male gametophytes during the vegetative phase and monoecious gametophytes during both the vegetative and gametogenesis phases, but significantly lower in male gametophytes during the gametogenesis phase. These results provide a foundation for deciphering the sex-determining mechanism of U. pinnatifida.

Construction of a high-density genetic map and mapping of a sex-linked locus for the brown alga Undaria pinnatifida (Phaeophyceae) based on large scale marker development by specific length amplified fragment (SLAF) sequencing.

BACKGROUND: Undaria pinnatifida is an important economic brown alga in East Asian countries. However, its genetic and genomic information is very scarce, which hinders further research in this species. A high-density genetic map is a basic tool for fundamental and applied research such as discovery of functional genes and mapping of quantitative trait loci (QTL). In this study the recently developed specific length amplified fragment sequencing (SLAF-seq) technology was employed to construct a high-density genetic linkage map and locate a sex determining locus for U. pinnatifida. RESULTS: A total of 28.06 Gb data including 140.31 M pair-end reads was obtained. After linkage analysis 4626 SLAF markers were mapped onto the genetic map. After adding the sex linked simple sequence repeat (SSR) marker [GenBank:AY738602.1], the final genetic map was 1816.28 cM long, consisting of 30 linkage groups with an average distance of 0.39 cM between adjacent markers. The length of LGs ranged from 20.12 to 106.95 cM. A major sex associated QTL was mapped to LG22 within a window starting at 29.01 cM and ending at 68.81 cM with a total of 68 SLAF markers. The SSR marker and five SLAF markers (Marker6556, 19020, 43089, 60771 and 26359) were identified as tightly sex-linked markers, as indicated by the absence of recombination between them and the sex phenotype. These markers were located at the position of 59.50 cM, which was supposed to be the sex determining region. CONCLUSIONS: A high-density genetic linkage map was constructed using SLAF-seq technique and F1 gametophyte population for the first time in the economically important brown alga U. pinnatifida. For the first time, a major sex associated QTL suggesting a sex determining region was mapped to a single LG. This map will facilitate the further fundamental and applied research such as QTL mapping and map-based gene clone in U. pinnatifida and provide a reference for studies in other kelp species.

Development of PCR-Based Markers to Determine the Sex of Kelps.

Sex discriminating genetic markers are commonly used to facilitate breeding programs in economically and ecologically important animal and plant species. However, despite their considerable economic and ecological value, the development of sex markers for kelp species has been very limited. In this study, we used the recently described sequence of the sex determining region (SDR) of the brown algal model Ectocarpus to develop novel DNA-based sex-markers for three commercially relevant kelps: Laminaria digitata, Undaria pinnatifida and Macrocystis pyrifera. Markers were designed within nine protein coding genes of Ectocarpus male and female (U/V) sex chromosomes and tested on gametophytes of the three kelp species. Seven primer pairs corresponding to three loci in the Ectocarpus SDR amplified sex-specific bands in the three kelp species, yielding at least one male and one female marker for each species. Our work has generated the first male sex-specific markers for L. digitata and U. pinnatifida, as well as the first sex markers developed for the genus Macrocystis. The markers and methodology presented here will not only facilitate seaweed breeding programs but also represent useful tools for population and demography studies and provide a means to investigate the evolution of sex determination across this largely understudied eukaryotic group.

Complete Plastid Genome Sequence of the Brown Alga Undaria pinnatifida.

In this study, we fully sequenced the circular plastid genome of a brown alga, Undaria pinnatifida. The genome is 130,383 base pairs (bp) in size; it contains a large single-copy (LSC, 76,598 bp) and a small single-copy region (SSC, 42,977 bp), separated by two inverted repeats (IRa and IRb: 5,404 bp). The genome contains 139 protein-coding, 28 tRNA, and 6 rRNA genes; none of these genes contains introns. Organization and gene contents of the U. pinnatifida plastid genome were similar to those of Saccharina japonica. There is a co-linear relationship between the plastid genome of U. pinnatifida and that of three previously sequenced large brown algal species. Phylogenetic analyses of 43 taxa based on 23 plastid protein-coding genes grouped all plastids into a red or green lineage. In the large brown algae branch, U. pinnatifida and S. japonica formed a sister clade with much closer relationship to Ectocarpus siliculosus than to Fucus vesiculosus. For the first time, the start codon ATT was identified in the plastid genome of large brown algae, in the atpA gene of U. pinnatifida. In addition, we found a gene-length change induced by a 3-bp repetitive DNA in ycf35 and ilvB genes of the U. pinnatifida plastid genome.

Complete mitochondrial genome of Undaria pinnatifida (Alariaceae, Laminariales, Phaeophyceae).

Undaria pinnatifida is one of the most important economic marine algae and key components of coastal ecosystems. Undaria pinnatifida owns a typical heteromorphic, diplohaplontic life cycle. We present the complete sequence of mitochondrial genome of U. pinnatifida, focusing on genome organization and phylogenetic relationship between different brown algae lineages. The size of U. pinnatifida mitochondrial DNA is 37,402 bp, including 3 rRNAs, 25 tRNAs, 35 proteins, as well as 3 ORFs. No intron is found and most genes are encoded on the H-strand. The phylogenetic trees (BI) constructed on 35 protein-coding genes from 17 species proved that Saccharina has a closer relationship with Laminaria than that with Undaria. The results supported the conclusion that Alariaceae is sister genus to the Laminariaceae. Above researches will facilitate the understanding of evolutionary relationship within brown algae.