A Genetic Map for the Only Self-Fertilizing Vertebrate

Abstract

The mangrove killifish Kryptolebias marmoratus, and its close relative Kryptolebias hermaphroditus, are the only vertebrate species known to reproduce by self-fertilization due to functional ovotestis development. To improve our understanding of their genomes, we constructed a genetic map. First, a single F1 fish was made by artificial fertilization between K. marmoratus and K. hermaphroditus strains. F2 progeny were then obtained by self-fertilization of the F1 fish. We used RAD-seq to query genomic DNAs from the two parental strains, the F1 individual and 49 F2 progeny. Results identified 9904 polymorphic RAD-tags (DNA markers) that mapped to 24 linkage groups, corresponding to the haploid chromosome number of these species. The total length of the map was 1248 cM, indicating that about one recombination occurred for each of the 24 homologous chromosome pairs in each meiosis. Markers were not evenly distributed along the chromosomes: in all chromosomes, many markers (> 8% of the total markers for each chromosome) mapped to chromosome tips. Centromeres suppress recombination, and this uneven distribution is probably due to the species' acrocentric chromosomes. Mapped marker sequences were compared to genomic sequences of medaka and platyfish, the next most closely related species with sequenced genomes that are anchored to genetic maps. Results showed that each mangrove killifish chromosome corresponds to a single chromosome of both platyfish and medaka, suggesting strong conservation of chromosomes over 100 million years of evolution. Our genetic map provides a framework for the K. marmoratus/K. hermaphroditus genome sequence and an important resource for understanding the biology of hermaphroditism.

Keywords: centromeres and recombination; conserved chromosomes; genetics of sex; hermaphrodite; phylogeny by RAD-seq; teleost.

Figure 1

Phylogenetic relationships of two hermaphroditic mangrove killifish species, Kryptolebias marmoratus (strains DAN and VOL) and K. hermaphroditus (strains PAN-RS and HY), and a sister gonochoristic species, K. caudomarginatus (Kc). Trees were constructed by neighbor-joining (NJ) based on the presence or absence of RAD-tags (A), concatenated SNPs (53,544 polymorphic, informative bases) identified from RAD-tags (B), and from mitochondrial DNA sequences (a part of 12S and 16S rRNA combined, total 1431 bases) (C), respectively. The tree topology did not change when analyzed with maximum likelihood (ML) or maximum parsimony (MP) methods (data not shown). Numbers at the nodes represent percentage recovery of those nodes per 10,000 bootstrap replicates (NJ/MP for A and NJ/ML/MP for B and C). Scale bars indicate genetic distance.

Figure 2

A Genetic linkage map of K. marmoratus/K. hermaphroditus based on RAD sequencing. The map coalesced into 24 linkage groups (LGs). LGs were named in decreasing order of total number of markers. For each LG, numbers to the left of the vertical bars represent map distances (cM, using the Kosambi function). Numbers to the right of the vertical bars give the names of representative markers with bootstrap values in the parenthesis (10,000 bootstrap replicates with AntMap). The horizontal bars at far right represent the total number of markers mapped to the same bin. The percentage of markers mapped to the same bins is shown in a parenthesis if ≥ 8.0% of the total markers on the LG. Markers showing segregation distortion are indicated by asterisks (*, P < 0.05; **, P < 0.01). Note that the concentration of markers is high at the tips of all LGs. Bins with the two highest numbers of markers, 119 and 118 markers, mapped to the middle of LG1 and the tip of LG5, respectively. Data are provided in File S1.

Figure 2

A Genetic linkage map of K. marmoratus/K. hermaphroditus based on RAD sequencing. The map coalesced into 24 linkage groups (LGs). LGs were named in decreasing order of total number of markers. For each LG, numbers to the left of the vertical bars represent map distances (cM, using the Kosambi function). Numbers to the right of the vertical bars give the names of representative markers with bootstrap values in the parenthesis (10,000 bootstrap replicates with AntMap). The horizontal bars at far right represent the total number of markers mapped to the same bin. The percentage of markers mapped to the same bins is shown in a parenthesis if ≥ 8.0% of the total markers on the LG. Markers showing segregation distortion are indicated by asterisks (*, P < 0.05; **, P < 0.01). Note that the concentration of markers is high at the tips of all LGs. Bins with the two highest numbers of markers, 119 and 118 markers, mapped to the middle of LG1 and the tip of LG5, respectively. Data are provided in File S1.

Figure 2

A Genetic linkage map of K. marmoratus/K. hermaphroditus based on RAD sequencing. The map coalesced into 24 linkage groups (LGs). LGs were named in decreasing order of total number of markers. For each LG, numbers to the left of the vertical bars represent map distances (cM, using the Kosambi function). Numbers to the right of the vertical bars give the names of representative markers with bootstrap values in the parenthesis (10,000 bootstrap replicates with AntMap). The horizontal bars at far right represent the total number of markers mapped to the same bin. The percentage of markers mapped to the same bins is shown in a parenthesis if ≥ 8.0% of the total markers on the LG. Markers showing segregation distortion are indicated by asterisks (*, P < 0.05; **, P < 0.01). Note that the concentration of markers is high at the tips of all LGs. Bins with the two highest numbers of markers, 119 and 118 markers, mapped to the middle of LG1 and the tip of LG5, respectively. Data are provided in File S1.

Figure 2

A Genetic linkage map of K. marmoratus/K. hermaphroditus based on RAD sequencing. The map coalesced into 24 linkage groups (LGs). LGs were named in decreasing order of total number of markers. For each LG, numbers to the left of the vertical bars represent map distances (cM, using the Kosambi function). Numbers to the right of the vertical bars give the names of representative markers with bootstrap values in the parenthesis (10,000 bootstrap replicates with AntMap). The horizontal bars at far right represent the total number of markers mapped to the same bin. The percentage of markers mapped to the same bins is shown in a parenthesis if ≥ 8.0% of the total markers on the LG. Markers showing segregation distortion are indicated by asterisks (*, P < 0.05; **, P < 0.01). Note that the concentration of markers is high at the tips of all LGs. Bins with the two highest numbers of markers, 119 and 118 markers, mapped to the middle of LG1 and the tip of LG5, respectively. Data are provided in File S1.

Figure 3

Representative conserved synteny of three K marmoratus/K. hermaphroditus linkage groups (Kma 12, 18, and 24) to platyfish (X. maculatus, Xma) and medaka (O. latipes, Ola) chromosomes suggesting, in general, a one-to-one relationship of K. marmoratus/K. hermaphroditus chromosomes to those of platyfish and medaka. Dotted lines indicate homology of mapped K. marmoratus/K. hermaphroditus markers to either Xma or Ola mapped genome sequences identified by the blastn program with default parameters. For platyfish, blast hits with a cut-off e-value of 1.0E-9 are shown. Kma 12 generally shares conserved syntenies to Xma 16 and Ola 8, including conserved order of sequences along the chromosomes (A). Some K. marmoratus/K. hermaphroditus markers show homology to sequences on non-orthologous chromosomes (indicated by red letters in parentheses). For example, a marker at 40.2 cM on Kma 12 has a homology to a sequence on Xma 10 at 5.5 Mb (A). Many K. marmoratus/K. hermaphroditus LGs show intrachromosomal rearrangements with respect to the other fish, probably due to inversions incurred after divergence from the last common ancestors of K. marmoratus/K. hermaphroditus and either platyfish or medaka (B). Results suggested a possible translocation involving Kma 18, which corresponds to Ola 23 or Xma 17 with a part of Ola 4 and Xma 9 attached, (B) and (C). Figure S7 provides data for all K. marmoratus/K. hermaphroditus LGs.