Isolation and characterization of a female-specific DNA marker in the giant freshwater prawn Macrobrachium rosenbergii

In this study, a female-specific DNA marker in the freshwater prawn Macrobrachium rosenbergii was identified through amplified fragment length polymorphism (AFLP). The AFLP-derived sequence-characterized amplified region (SCAR) marker was tested in over 200 individuals, giving reproducible sex identification. Further molecular characterization of the sex-marker's genomic region (～ 3 kb long) revealed the presence of tandem and inverted repeats. The ～ 3-kb sequence was identified both in male and female prawns, but with subtle differences: a deletion of 3 bp (present in female prawn but absent in male prawn) identified upstream of the SCAR marker sequence and two female-specific single-nucleotide polymorphisms, both indicating that male prawns are homozygous, whereas female prawns are heterozygous in this locus. Fluorescent in situ hybridization showed the ～ 3-kb sequence to be unique: to the best of our knowledge, this is the first report of a unique sex-specific sequence observed in situ in crustaceans. The sex-specific marker identified in M. rosenbergii may have considerable applied merit for crustacean culture in that it will enable the determination of genetic sex at early developmental stages when phenotypic differences are not identifiable.     

Genetic diversity of the cultured giant freshwater prawn (Macrobrachium rosenbergii) in China based on microsatellite markers

The Giant freshwater prawn (GFP), Macrobrachium rosenbergii, is one of the most important crustacean species cultured in China, but information on the genetic diversity of GFP in China is still limited. In this study we analyzed the genetic diversity of 6 cultured GFP populations collected in China, and 2 wild populations from Viet Nam. The wild populations were included as a reference so that the genetic diversity of the introduced cultured GFP populations could be evaluated. We used six polymorphic primer pairs to estimate genetic diversity. Results showed that the number of alleles per locus ranged from 6 to 19. The mean observed heterozygosity (0.364 ± 0.090) was less than the expected heterozygosity (0.893 ± 0.016); highly significant deficiencies in heterozygotes were detected (100%). The polymorphism information content (PIC) of 8 populations at 6 loci was greater than 0.5 (varying from 0.616 to 0.912). Pair-wise genetic differentiation (F_ST) varied from 0.006 to 0.129 among the eight populations. The dendrogram based on genetic distance showed six populations distributed on the same major branch, suggesting that they have closely genetic distance and two populations (ZJC and GDB) distributed on the other branch, so they also have closely genetic distance. The results of study provide a baseline assessment of genetic diversity in some populations in China and Viet Nam that will be useful for the development of breeding programmes in the future.     

Within‐population polymorphism of sex‐determination systems in the common frog (Rana temporaria)

In sharp contrast with birds and mammals, the sex chromosomes of ectothermic vertebrates are often undifferentiated, for reasons that remain debated. A linkage map was recently published for Rana temporaria (Linnaeus, 1758) from Fennoscandia (Eastern European lineage), with a proposed sex‐determining role for linkage group 2 (LG2). We analysed linkage patterns in lowland and highland populations from Switzerland (Western European lineage), with special focus on LG2. Sibship analyses showed large differences from the Fennoscandian map in terms of recombination rates and loci order, pointing to large‐scale inversions or translocations. All linkage groups displayed extreme heterochiasmy (total map length was 12.2 cM in males, versus 869.8 cM in females). Sex determination was polymorphic within populations: a majority of families (with equal sex ratios) showed a strong correlation between offspring phenotypic sex and LG2 paternal haplotypes, whereas other families (some of which with female‐biased sex ratios) did not show any correlation. The factors determining sex in the latter could not be identified. This coexistence of several sex‐determination systems should induce frequent recombination of X and Y haplotypes, even in the absence of male recombination. Accordingly, we found no sex differences in allelic frequencies on LG2 markers among wild‐caught male and female adults, except in one high‐altitude population, where nonrecombinant Y haplotypes suggest sex to be entirely determined by LG2. Multifactorial sex determination certainly contributes to the lack of sex‐chromosome differentiation in amphibians.     

Identification of sex-linked SNP markers using RAD sequencing suggests ZW/ZZ sex determination in Pistacia vera L.

Background

Pistachio (Pistacia vera L.) is a dioecious species that has a long juvenility period. Therefore, development of marker-assisted selection (MAS) techniques would greatly facilitate pistachio cultivar-breeding programs. The sex determination mechanism is presently unknown in pistachio. The generation of sex-linked markers is likely to reduce time, labor, and costs associated with breeding programs, and will help to clarify the sex determination system in pistachio.

Results

Restriction site-associated DNA (RAD) markers were used to identify sex-linked markers and to elucidate the sex determination system in pistachio. Eight male and eight female F₁ progenies from a Pistacia vera L. Siirt × Bağyolu cross, along with the parents, were subjected to RAD sequencing in two lanes of a Hi-Seq 2000 sequencing platform. This generated 449 million reads, comprising approximately 37.7 Gb of sequences. There were 33,757 polymorphic single nucleotide polymorphism (SNP) loci between the parents. Thirty-eight of these, from 28 RAD reads, were detected as putative sex-associated loci in pistachio. Validation was performed by SNaPshot analysis in 42 mature F₁ progenies and in 124 cultivars and genotypes in a germplasm collection. Eight loci could distinguish sex with 100% accuracy in pistachio. To ascertain cost-effective application of markers in a breeding program, high-resolution melting (HRM) analysis was performed; four markers were found to perfectly separate sexes in pistachio. Because of the female heterogamety in all candidate SNP loci, we report for the first time that pistachio has a ZZ/ZW sex determination system. As the reported female-to-male segregation ratio is 1:1 in all known segregating populations and there is no previous report of super-female genotypes or female heteromorphic chromosomes in pistachio, it appears that the WW genotype is not viable.

Conclusion

Sex-linked SNP markers were identified and validated in a large germplasm and proved their suitability for MAS in pistachio. HRM analysis successfully validated the sex-linked markers for MAS. For the first time in dioecious pistachio, a female heterogamety ZW/ZZ sex determination system is suggested.     

The evolution of sex‐determining mechanisms: lessons from temperature‐sensitive mutations in sex determination genes in Caenorhabditis elegans

Sexual reproduction is one of the most taxonomically conserved traits, yet sex‐determining mechanisms (SDMs) are quite diverse. For instance, there are numerous forms of environmental sex determination (ESD), in which an organism’s sex is determined not by genotype, but by environmental factors during development. Important questions remain regarding transitions between SDMs, in part because the organisms exhibiting unique mechanisms often make difficult study organisms. One potential solution is to utilize mutant strains in model organisms better suited to answering these questions. We have characterized two such strains of the model nematode Caenorhabditis elegans. These strains harbour temperature‐sensitive mutations in key sex‐determining genes. We show that they display a sex ratio reaction norm in response to rearing temperature similar to other organisms with ESD. Next, we show that these mutations also cause deleterious pleiotropic effects on overall fitness. Finally, we show that these mutations are fundamentally different at the genetic sequence level. These strains will be a useful complement to naturally occurring taxa with ESD in future research examining the molecular basis of and the selective forces driving evolutionary transitions between sex determination mechanisms.     

A sex‐linked SCAR marker in Bryonia dioica (Cucurbitaceae), a dioecious species with XY sex‐determination and homomorphic sex chromosomes

Genetic crosses between the dioecious Bryonia dioica (Cucurbitaceae) and the monoecious B. alba in 1903 provided the first clear evidence for Mendelian inheritance of dioecy and made B. dioica the first organism for which XY sex‐determination was experimentally proven. Applying molecular tools to this system, we developed a sex‐linked sequence‐characterized amplified region (SCAR) marker for B. dioica and sequenced it for individuals representing the full geographic range of the species from Scotland to North Africa. For comparison, we also sequenced this marker for representatives of the dioecious B. cretica, B. multiflora and B. syriaca, and monoecious B. alba. In no case did any individual, male or female, yield more than two haplotypes. In northern Europe, we found strong linkage between our marker and sex, with all Y‐sequences being identical to each other. In southern Europe, however, the linkage between our marker and sex was weak, with recombination detected within both the X‐ and the Y‐homologues. Population genetic analyses suggest that the SCAR marker experienced different evolutionary pressures in northern and southern Europe. These findings fit with phylogenetic evidence that the XY system in Bryonia is labile and suggest that the genus may be a good system in which to study the early steps of sex chromosome evolution.     

Sex‐linked microsatellite marker detected in the female gametophytes of Undaria pinnatifida (Phaeophyta)

In our microsatellite analysis of three male and three female gametophytes of Undaria pinnatifida (Harv.) Suringar, a microsatellite marker (part of the locus Up‐AC‐2A8, GenBank accession no. AY738602.1) was only polymerase chain reaction‐amplified in three female gametophytes. This putative female‐specific marker was further tested by the use of 32 male and 21 female gametophytes maintained in the Marine Biological Culture Collection Centre, China. In addition, three sporophytes were included for confirmation. Results showed that the marker was present in all of the female gametophytes and sporophyte cultures, but absent in all of the male gametophytes. To our knowledge, this is the first sex‐related marker ever reported in U. pinnatifida. The discovery of this marker will accelerate gender identification and shed light on our understanding of the mechanisms of sex determination at a molecular level in this commercially important seaweed.     

The detection of sex‐linked heteroplasmy in Pseudocardium sachalinense (Bivalvia: Mactridae) and its implications for the distribution of doubly uniparental inheritance of mitochondrial DNA

Although mitochondrial inheritance in metazoans is typically strictly maternal, doubly uniparental inheritance (DUI) is probably the major exception to this widespread rule. DUI has been found in many species of bivalve molluscs, belonging to several different families. Based on current understanding, the detection of DUI generally relies on the detection of two distinct mitochondrial DNA lineages: a female‐transmitted one, that dominates somatic tissues in males and females and eggs, and a male‐transmitted one, that dominates the male germline and sperm. When a new species with DUI is identified, novel data are available to make a better inference on the evolution of this phenomenon within the Bivalvia. In this study, mitochondrial heteroplasmy in Pseudocardium sachalinense (Schrenck, 1862) is described. This species belongs to the family of Mactridae, in which DUI has not been previously demonstrated: this finding allowed to upgrade the present knowledge about the distribution of DUI.