A New Standard for Crustacean Genomes: The Highly Contiguous, Annotated Genome Assembly of the Clam Shrimp Eulimnadia texana Reveals HOX Gene Order and Identifies the Sex Chromosome

Abstract

Vernal pool clam shrimp (Eulimnadia texana) are a promising model system due to their ease of lab culture, short generation time, modest sized genome, a somewhat rare stable androdioecious sex determination system, and a requirement to reproduce via desiccated diapaused eggs. We generated a highly contiguous genome assembly using 46× of PacBio long read data and 216× of Illumina short reads, and annotated using Illumina RNAseq obtained from adult males or hermaphrodites. Of the 120 Mb genome 85% is contained in the largest eight contigs, the smallest of which is 4.6 Mb. The assembly contains 98% of transcripts predicted via RNAseq. This assembly is qualitatively different from scaffolded Illumina assemblies: It is produced from long reads that contain sequence data along their entire length, and is thus gap free. The contiguity of the assembly allows us to order the HOX genes within the genome, identifying two loci that contain HOX gene orthologs, and which approximately maintain the order observed in other arthropods. We identified a partial duplication of the Antennapedia complex adjacent to the few genes homologous to the Bithorax locus. Because the sex chromosome of an androdioecious species is of special interest, we used existing allozyme and microsatellite markers to identify the E. texana sex chromosome, and find that it comprises nearly half of the genome of this species. Linkage patterns indicate that recombination is extremely rare and perhaps absent in hermaphrodites, and as a result the location of the sex determining locus will be difficult to refine using recombination mapping.

Keywords: HOX genes; genome assembly; genome biology; genomics; invertebrate genetics; sex chromosomes.

Fig. 1.

—A male clam shrimp (left), and a hermaphrodite clam shrimp (right). Both are exemplars of the E. texana species. Note the presence of clasping arms on the male—these are required for nonself-fertilized sex, and the presence of a brood pouch along the dorsal surface of the hermaphrodite.

Fig. 2.

—Top: The D. melanogaster HOX regions hand-aligned against the E. texana HOX regions. The ortholog identities of the E. texana HOX genes are established via bootstrap consensus maximum likelihood trees in MEGA. Note the similarity between the Antennapedia complex and Contig 7, and note that Contig 2 appears to be a combination of a copy of the Antennapedia complex and a portion of the Bithorax complex. Bottom: a visualization of the genome regions identified above. In this bottom panel, genes have been renamed for clarity. Genes that correspond to a hox gene are renamed in the figure as “DrosophilaName_E.texana name” with the Drosophila gene name prefixed to the E. texana gene name. Each instance of a given Drosophila name is numbered. To extract the correspond gene from E texana annotation files the Drosophila prefix should be removed.

Fig. 3.

—A plot of cumulative genome coverage of the E. texana genome assembly by contig. As the plot progresses from left to right, the contig lengths are added to the cumulative coverage in order from largest to smallest. A high-quality assembly should achieve a high cumulative coverage with a small number of contigs. Here ∼80% of the assembly is contained in contigs larger than ∼5 Mb.

Fig. 4.

—An illustration of Hox gene loss in the crustaceans. Tree branch lengths are not informative. As shown here, the loss of AntP and Ubx is uncommon in crustaceans.

Fig. 5.

—A heat map of expression for genes differentially expresses between males and hermaphrodites (adjusted P<0.05). Note the small portion of genes that have nearly zero expression in males, and high expression in hermaphrodites.