Animals

M. rosenbergii BC male donors (40 ± 5 g) were reared in 600-L tanks at 28 ± 2 °C with constant aeration and a light regime of 14:10 (L:D) at the R&D facilities of Enzootic Holdings, Ltd. The prawns were fed ad libitum with shrimp pellets containing 30% protein. WW M. rosenbergii post-larvae (PL), obtained by cross breeding WW females with WZ neo-males, were reared in a 3.5 m3 U-shaped tank.

Sex reversal of WW females into WW neo-males

The M. rosenbergii male donors were endocrinologically manipulated, leading to androgenic gland (AG) hypertrophy18,55,56. Ten days post manipulation, the AGs were dissected from the manipulated animals under a dissecting microscope, and the hypertrophied AG (hAG) cells were separated by enzymatic dissociation, as previously described18. An aliquot of cell suspension was evaluated for viability and concentration, by using Trypan blue staining and counting of the cells on a hemocytometer under a light microscope. These hAG cells were transplanted (using a micro-injector under a light microscope), in an amount of ~3 × 103 hAG cells per female prawn, into the abdomens of WZ and WW females, at an age of <PL 60 (n = 300). The injected prawns were reared in earthen ponds (each ~140 m2 with a water depth of 0.9 m and a water temperature of 26–28 °C) at the Mevo Hama aquaculture facility, Israel.

Examination of masculine development

Two months post transplantation, the prawns injected with AG cells were examined for the development of male gonopores. Each animal was placed on its dorsal side, and the bases of the fifth pereiopods (walking legs) were examined for the presence or absence of male gonopores. Animals that had developed male gonopores were returned to the ponds for additional grow-out, and the other animals were removed from the ponds. Four months post injection, the prawns were examined for morphotypic differentiation, which constitutes a milestone in M. rosenbergii masculine development8.

Crossing WW neo-males with WW females

Eight months post injection, the above animals were taken out of the ponds, and their genotypes were determined using specific W- and Z-linked genomic sex markers, as previously described4,18. WW animals with male gonopores were considered WW neo-males and were transferred to a 4-m3 tank together with WW females for breeding. The breeding tank was examined on a weekly basis, and each berried female was removed and placed in an individual glass tank. Upon hatching, progeny was genetically tested (using the above-mentioned sex markers) to verify that the larvae did indeed bear the WW genotype. A workflow representing the process from obtaining a WW female to the achievement of WW all-female progeny is shown in Fig. 1.

Fecundity measurements

To assess the fecundity of the WW females that had been fertilized by WW neo-males, berried WW females (n = 11) were weighed before and after hatching of the larvae. The ratio of egg mass to body weight (BSI16,18) was calculated. A meta-analysis was conducted to compare these results with those previously obtained for WW females fertilized by ZZ males (n = 15), WZ females fertilized by ZZ males (n = 11)18, and ZZ ‘neo-females’ fertilized by ZZ males (n = 8)16. Since according to the Shapiro-Wilk test, the residuals of the BSI measurements were not normally distributed, differences between the measurements were tested by the non-parametric Kruskal-Wallis test using Statistica v9.0 (StatSoft, Tulsa, OK).

Genotyping all possible phenotypes in M. rosenbergii

The second pleopods were dissected from prawns bearing different chromosome combinations: a ZZ normal male, a sex-reversed normal female (expected to be a WZ neo-male18), a sex-reversed super female (expected to be a WW neo-male), a WZ normal female, a super female from a WZ × WZ cross (expected to be WW18), and a sex-reversed neo-female that was obtained by silencing the Mr-IAG gene (expected to be ZZ32). Genomic DNA was extracted using REDExtract-N-Amp Tissue PCR Kit (Sigma, Rehovot, Israel) according to the manufacturer’s instructions, and the genotype of each animal was determined using sex-specific genomic markers for M. rosenbergii, as previously described18.

Evaluating M. rosenbergii genome size

Evaluation of M. rosenbergii genome size is a meaningful step prior to de novo sequencing of the genome. Therefore, the haploid M. rosenbergii genome size was empirically determined by using a previously described flow cytometry protocol57. Briefly, hemolymph was extracted from 12 prawns and pooled. Hemocytes were retrieved from the hemolymph and stained with PI. Peripheral blood mononuclear cells (PBMCs) from H. sapiens were used as a source for a reference haploid genome with the known size of 3.2 Gb58. Each sample was analyzed twice, and the fluorescence relative intensity of PI in each cell was measured. In each analysis at least 10,000 events were analyzed. The Geo mean fluorescence of each cell population was calculated. The following formula was used to calculate the genome size:

$$Study\,genome\,size=\frac{(Reference\,genome\,size)\times (Study\,fluoresence\,mean)}{(Reference\,fluoresence\,mean)}$$

gDNA extraction for second- and third-generation sequencing

M. rosenbergii high molecular weight gDNA was extracted from a WZ female by using the phenol-chloroform method as follows: 200 mg of muscle tissue was flash frozen in liquid nitrogen and then ground in a mortar and pestle. Using a clean metal spatula, the powdered tissue homogenate was transferred to a 50-ml tube preloaded with 10 ml of Proteinase K buffer (20 mg/ml) in 50 mM Tris (pH 8) and calcium acetate (1.5 mM) and incubated at 45 °C overnight. After complete digestion of the tissue, 10 ml of TE saturated phenol was added, and the sample was incubated for 1 h at room temperature. Following incubation, the lower phase was discarded using a serological pipette. The latter step was repeated twice more, and then the sample was incubated overnight (without removing the lower phase in the final repetition). Next, the lower phase was discarded, 10 ml of phenol-chloroform suspension was added to the aqueous phase, and the sample was incubated at room temperature for 1 h. Thereafter, the phenol-chloroform phase was discarded, and 100% chloroform was added, followed by a 1 h incubation at room temperature. This step was repeated twice, and after adding the chloroform for the third time, the sample was incubated overnight. Next, the lower, chloroform phase was discarded. The remaining aqueous phase, retrieved from the previous step, was supplemented with 10% v/v sodium acetate (3 M) and two volumes of ice cold ethanol (100%). The sample was mixed by gentle rotation, 3 times. Using a glass shepherd’s rod, the DNA precipitate was transferred to 30 ml of cold ethanol (70%) for 5 min. Finally, the DNA was removed from the 70% ethanol using the glass shepherd’s rod and air dried for 5 min. The dry DNA precipitate was reconstituted in 100 μL of TE buffer (0.1 M).

Sequencing and de novo assembly of the M. rosenbergii genome

The M. rosenbergii gDNA samples were sequenced by NRGene (Ness-Ziona, Israel) with a second-generation sequencing technology having a total depth (coverage) of ×261 [based on an estimated total (diploid) genome size of 8.2 Gb] by using Illumina (San Diego, CA) technologies. PCR-free Pair-End (PE) and Mate-Pair (MP) libraries were used to provide accurate and precise raw data. In addition, third-generation sequencing libraries were prepared and sequenced using Illumina machines, including 10X chromium, creating additional sequencing data with a depth (coverage) of ×61. A detailed description of the sequencing data is given in Table 4. The sequencing data was processed and assembled using the DeNovoMAGIC assembler application version 3.0 (NRGene, Ness-Ziona, Israel). Contig assembly, scaffolding and gap filling were performed as previously described43. In addition, DeNovoMAGIC was used to assemble, independently, phased and unphased genomes. Phased genome assembly aims at assembling a heterozygous genome, in which each heterozygous region of the genome should be covered by two separate scaffolds, one of maternal origin and the other of paternal origin. The unphased genome is comprised of longer scaffolds representing the longest possible sequence per locus in the genome, and each region of the genome is covered by a single scaffold. Therefore, the N50 of the unphased assembly is expected to be higher than the N50 of the phased assembly. The integrity of the assemblies was verified with several quality-assurance procedures including the independent BUSCO benchmark59,60, against the “Arthropoda_odb9” database with default parameters. BUSCO is used to specifically indicate the genic region integrity, ploidy and zygosity characteristics of the assembled genome.

Table 4 Sequencing strategy description. Full size table

Mining for W/Z-associated scaffolds

Upon sequencing and assembly of the M. rosenbergii genome, we aligned the sequence of our previously described W- and Z-associated markers14,18 to the phased genome. The scaffolds that matched the W- and Z-associated markers were compared using two independent approaches, as follows: (1) Using the Mauve genome aligner61, the scaffolds were aligned and visualized using the progressive Mauve algorithm of Mauve desktop application version 20150226. (2) Using MUMmer 3.0 genome aligner62, the scaffolds were compared with the nucmer script (version 3.1), and reports were created with the dnadiff script (version 1.3). “mcoords”, “qdiff” and “rdiff” reports were converted to bed format with a modified version of the script, as described in: https://sequencingforever.wordpress.com/2016/12/09/view-nucmer-alignments-in-igv/. The bed files, describing regions of similarity and dissimilarity between the scaffolds, were visualized using the Integrative Genomics Viewer (IGV)63.

In vitro validation of the putative W and Z scaffolds

Sex-linked genomic markers derived from the putative W or Z scaffolds were tested and verified on prawn individuals bearing every possible genotype. DNA was extracted from a WZ female, a WW female, a ZZ neo-female, a WZ neo-male, a WW neo-male and a ZZ male by using PCR (94 °C for 3 min, followed by 35 cycles of 94 °C for 30 s, 57 °C for 30 s, and 72 °C for 45 s, and then by a final elongation step of 72 °C for 5 min) with the Ready Mix REDTaq kit of Sigma Aldrich (St. Louis, MO), used according to the manufacturer’s instructions. The PCR products were separated on 1.5% agarose gel.

Extending W and Z scaffolds

To extend validated W and Z scaffolds and to obtain a higher coverage of the sex chromosomes, each scaffold from the phased assembly was realigned with the unphased assembly. Some of the scaffolds matched in the unphased assembly were longer than those in the phased assembly and had an extended ‘tail’ that was not part of the scaffold in the phased assembly. Then, the tail was aligned with the phased assembly, and a new candidate W/Z-associated scaffold was found in some cases. A scheme and illustration of the process of searching and extending the W/Z-associated scaffold are shown in Figs 5 and S1.