Identification of Sex-Specific Markers Through 2b-RAD Sequencing in the Sea Urchin (Mesocentrotus nudus)

Sex-specific markers play an important role in revealing sex-determination mechanism. Sea urchin (Mesocentrotus nudus) is an economically important mariculture species in several Asian countries and its gonads are the sole edible parts for people. However, growth rate and immunocompetence differ by sex in this species, sex-specific markers have not been identified, and the sex-determination mechanism of sea urchin remains undetermined. In this study, type IIB endonuclease restriction-site associated DNA sequencing (2b-RAD-seq) and a genome survey of M. nudus were performed, and three female-specific markers and three female heterogametic single nucleotide polymorphism (SNP) loci were identified. We validated these sex-specific markers via PCR amplification in a large number of individuals, including wild and artificially bred populations. Several open reading frames (ORFs) were predicted, although there are no potential genes known for sex determination and sex differentiation within the scaffold in which the sex-specific markers are located. Importantly, the female-specific sequences and female heterozygous SNP loci indicate that a female heterogametic and male homogametic ZW/ZZ sex-determination system should exist in M. nudus. The results provide a solid basis for revealing the sex-determination mechanism of this species, and open up new possibilities for developing sex-control breeding in sea urchin.

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A Novel Splice Variant of the Masculinizing Gene Masc with piRNA-Cleavage-Site Defect Functions in Female External Genital Development in the Silkworm, Bombyx mori

Biomolecules ◽

10.3390/biom9080318 ◽

2019 ◽

Vol 9 (8) ◽

pp. 318 ◽

Cited By ~ 3

Author(s):

Qin Zhao ◽

Juan Li ◽

Mao-Yu Wen ◽

He Wang ◽

Yao Wang ◽

...

Keyword(s):

Sex Determination ◽

Splice Variant ◽

Cleavage Site ◽

Specific Binding ◽

Sex Differentiation ◽

Specific Cell ◽

Over Expression ◽

Primary Signal ◽

Genital Development ◽

Female Specific

In the silkworm, the sex-determination primary signal Fem controls sex differentiation by specific binding of Fem-derived piRNA to the cleavage site in Masc mRNA, thus inhibiting Masc protein production in the female. In this study, we identified a novel splicing isoform of Masc, named Masc-S, which lacks the intact sequence of the cleavage site, encoding a C-terminal truncated protein. Results of RT-PCR showed that Masc-S was expressed in both sexes. Over-expression of Masc-S and Masc in female-specific cell lines showed that Masc-S could be translated against the Fem-piRNA cut. By RNA-protein pull-down, LC/MS/MS, and EMSA, we identified a protein BmEXU that specifically binds to an exclusive RNA sequence in Masc compared to Masc-S. Knockdown of Masc-S resulted in abnormal morphology in female external genital and increased expression of the Hox gene Abd-B, which similarly occurred by Bmexu RNAi. These results suggest that the splice variant Masc-S against Fem-piRNA plays an important role in female external genital development, of which function is opposite to that of full-length Masc. Our study provides new insights into the regulatory mechanism of sex determination in the silkworm.

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A ZZ/ZW Sex Chromosome System in Cephalochordate Amphioxus

Genetics ◽

10.1534/genetics.120.303051 ◽

2020 ◽

Vol 214 (3) ◽

pp. 617-622

Author(s):

Chenggang Shi ◽

Xiaotong Wu ◽

Liuru Su ◽

Chaoqi Shang ◽

Xuewen Li ◽

...

Keyword(s):

Sex Determination ◽

Mutant Strain ◽

Sex Chromosome ◽

Female Offspring ◽

Phylogenetic Position ◽

Z Chromosome ◽

Transcription Activator ◽

Sex Chromosome System ◽

Female Specific ◽

Determination Mechanism

Sex determination is remarkably variable among animals with examples of environmental sex determination, male heterogametic (XX/XY) and female heterogametic (ZZ/ZW) chromosomal sex determination, and other genetic mechanisms. The cephalochordate amphioxus occupies a key phylogenetic position as a basal chordate and outgroup to vertebrates, but its sex determination mechanism is unknown. During the course of generating Nodal mutants with transcription activator-like effector nucleases (TALENs) in amphioxus Branchiostoma floridae, serendipitously, we generated three mutant strains that reveal the sex determination mechanism of this animal. In one mutant strain, all heterozygous mutant offspring over three generations were female and all wild-type descendants were male. This pattern suggests the Nodal allele targeted is on a female-specific W chromosome. A second mutant showed the same W-linked inheritance pattern, with a female heterozygote passing the mutation only to daughters. In a third mutant strain, both male and female offspring could be heterozygous, but a female heterozygote passed the mutation only to sons. This pattern is consistent with the targeted allele being on a Z chromosome. We found an indel polymorphism linked to a Nodal allele present in most females, but no males in our cultured population. Together, these results indicate that Nodal is sex chromosome-linked in B. floridae, and that B. floridae has a ZZ/ZW sex chromosome system.

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Sex determination and Y chromosome constitutive heterochromatin

Current Research in Biochemistry and Molecular Biology ◽

10.33702/crbmb.2019.1.1.1 ◽

2019 ◽

Vol 1 (1) ◽

pp. 1-5

Author(s):

Abyt Ibraimov

Keyword(s):

Sex Determination ◽

Y Chromosome ◽

Constitutive Heterochromatin ◽

Sex Differentiation ◽

Secondary Sexual Characteristics ◽

Biological Meaning ◽

Heterochromatin Block ◽

Sexual Characteristics ◽

Open Question ◽

Efficient Elimination

In many animals, including us, the genetic sex is determined at fertilization by sex chromosomes. Seemingly, the sex determination (SD) in human and animals is determined by the amount of constitutive heterochromatin on Y chromosome via cell thermoregulation. It is assumed the medulla and cortex tissue cells in the undifferentiated embryonic gonads (UEG) differ in vulnerability to the increase of the intracellular temperature. If the amount of the Y chromosome constitutive heterochromatin is enough for efficient elimination of heat difference between the nucleus and cytoplasm in rapidly growing UEG cells the medulla tissue survives. Otherwise it doomed to degeneration and a cortex tissue will remain in the UEG. Regardless of whether our assumption is true or not, it remains an open question why on Y chromosome there is a large constitutive heterochromatin block? What is its biological meaning? Does it relate to sex determination, sex differentiation and development of secondary sexual characteristics? If so, what is its mechanism: chemical or physical? There is no scientifically sound answer to these questions.

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The Sex Determination Cascade in the Silkworm

Genes ◽

10.3390/genes12020315 ◽

2021 ◽

Vol 12 (2) ◽

pp. 315

Author(s):

Xu Yang ◽

Kai Chen ◽

Yaohui Wang ◽

Dehong Yang ◽

Yongping Huang

Keyword(s):

Sex Determination ◽

Molecular Mechanisms ◽

Future Research ◽

Model Species ◽

Male And Female ◽

Master Regulators ◽

Primary Signal ◽

Basic Understanding ◽

Female Specific ◽

Working Mechanisms

In insects, sex determination pathways involve three levels of master regulators: primary signals, which determine the sex; executors, which control sex-specific differentiation of tissues and organs; and transducers, which link the primary signals to the executors. The primary signals differ widely among insect species. In Diptera alone, several unrelated primary sex determiners have been identified. However, the doublesex (dsx) gene is highly conserved as the executor component across multiple insect orders. The transducer level shows an intermediate level of conservation. In many, but not all examined insects, a key transducer role is performed by transformer (tra), which controls sex-specific splicing of dsx. In Lepidoptera, studies of sex determination have focused on the lepidopteran model species Bombyx mori (the silkworm). In B. mori, the primary signal of sex determination cascade starts from Fem, a female-specific PIWI-interacting RNA, and its targeting gene Masc, which is apparently specific to and conserved among Lepidoptera. Tra has not been found in Lepidoptera. Instead, the B. mori PSI protein binds directly to dsx pre-mRNA and regulates its alternative splicing to produce male- and female-specific transcripts. Despite this basic understanding of the molecular mechanisms underlying sex determination, the links among the primary signals, transducers and executors remain largely unknown in Lepidoptera. In this review, we focus on the latest findings regarding the functions and working mechanisms of genes involved in feminization and masculinization in Lepidoptera and discuss directions for future research of sex determination in the silkworm.

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Identification of sex differentiation-related microRNA and long non-coding RNA in Takifugu rubripes gonads

Scientific Reports ◽

10.1038/s41598-021-83891-w ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Hongwei Yan ◽

Qi Liu ◽

Jieming Jiang ◽

Xufang Shen ◽

Lei Zhang ◽

...

Keyword(s):

Sex Determination ◽

High Throughput Sequencing ◽

Sex Differentiation ◽

Mature Mirnas ◽

Takifugu Rubripes ◽

Sequencing Technology ◽

Non Coding Rna ◽

Differentially Expressed Mirnas ◽

Tiger Pufferfish ◽

Long Non Coding Rna

AbstractAlthough sex determination and differentiation are key developmental processes in animals, the involvement of non-coding RNA in the regulation of this process is still not clarified. The tiger pufferfish (Takifugu rubripes) is one of the most economically important marine cultured species in Asia, but analyses of miRNA and long non-coding RNA (lncRNA) at early sex differentiation stages have not been conducted yet. In our study, high-throughput sequencing technology was used to sequence transcriptome libraries from undifferentiated gonads of T. rubripes. In total, 231 (107 conserved, and 124 novel) miRNAs were obtained, while 2774 (523 conserved, and 2251 novel) lncRNAs were identified. Of these, several miRNAs and lncRNAs were predicted to be the regulators of the expression of sex-related genes (including fru-miR-15b/foxl2, novel-167, novel-318, and novel-538/dmrt1, novel-548/amh, lnc_000338, lnc_000690, lnc_000370, XLOC_021951, and XR_965485.1/gsdf). Analysis of differentially expressed miRNAs and lncRNAs showed that three mature miRNAs up-regulated and five mature miRNAs were down-regulated in male gonads compared to female gonads, while 79 lncRNAs were up-regulated and 51 were down-regulated. These findings could highlight a group of interesting miRNAs and lncRNAs for future studies and may reveal new insights into the function of miRNAs and lncRNAs in sex determination and differentiation.

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Intercistronic heterogeneity of the 16S–23S rRNA spacer region among Pseudomonas strains isolated from subterranean seeds of hog peanut (Amphicarpa bracteata)

Microbiology ◽

10.1099/mic.0.028274-0 ◽

2009 ◽

Vol 155 (8) ◽

pp. 2630-2640 ◽

Cited By ~ 7

Author(s):

J. T. Tambong ◽

R. Xu ◽

E. S. P. Bromfield

Keyword(s):

Root Zone ◽

Phylogenetic Analyses ◽

Melting Curve ◽

Pcr Amplification ◽

23S Rrna ◽

Melting Curve Analysis ◽

Its1 Region ◽

A Genome ◽

Pure Cultures ◽

Intercalating Dye

Intercistronic heterogeneity of the 16S–23S rRNA internal transcribed spacer regions (ITS1) was investigated in 29 strains of fluorescent pseudomonads isolated from subterranean seeds of Amphicarpa bracteata (hog peanut). PCR amplification of the ITS1 region generated one or two products from the strains. Sequence analysis of the amplified fragments revealed an ITS1 fragment of about 517 bp that contained genes for tRNAIle and tRNAAla in all 29 strains; an additional smaller ITS1 of 279 bp without tRNA features was detected in 15 of the strains. The length difference appeared to be due to deletions of several nucleotide blocks between the 70 bp and 359 bp positions of the alignment. The end of the deletions in the variant ITS1 type coincided with the start of antiterminator box A, which is homologous to box A of other bacteria. Phylogenetic analyses using the neighbour-joining algorithm revealed two major phylogenetic clusters, one for each of the ITS1 types. Using a single specific primer set and the DNA-intercalating dye SYBR Green I for real-time PCR and melting-curve analysis produced highly informative curves with one or two recognizable melting peaks that readily distinguished between the two ITS1 types in pure cultures. The assay was used to confirm the presence of the variant ITS1 type in the Pseudomonas community in total DNA from root-zone soil and seed coats of hog peanut. Heterogeneity of the ITS1 region between species has potential for studying molecular systematics and population genetics of the genus Pseudomonas, but the presence of non-identical rRNA operons within a genome may pose problems.

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PrimerPooler: automated primer pooling to prepare library for targeted sequencing

Biology Methods and Protocols ◽

10.1093/biomethods/bpx006 ◽

2017 ◽

Vol 2 (1) ◽

Cited By ~ 7

Author(s):

Silas S. Brown ◽

Yun-Wen Chen ◽

Ming Wang ◽

Alexandra Clipson ◽

Eguzkine Ochoa ◽

...

Keyword(s):

Variant Calling ◽

Pcr Amplification ◽

Illumina Miseq ◽

Degenerate Primers ◽

Cross Hybridization ◽

Targeted Next Generation Sequencing ◽

Target Sequencing ◽

A Genome ◽

Experimental Protocols ◽

Minimal Depth

Abstract Targeted next-generation sequencing based on PCR amplification involves pooling of hundreds to thousands of primers, for preamplification and subsequent parallel single/multiplex PCR. It is often necessary to allocate the set of primers into subpools, a common issue being potential cross-hybridization. For smaller numbers of primers, pool division can be done manually using trial and error to minimize potential hybridization, but this becomes inefficient and time consuming with increasing numbers of primer pairs. We developed PrimerPooler that automates swapping of primer pairs between any user-defined number of subpools to obtain combinations with low-potential interactions. PrimerPooler performs inter-/intra-primer hybridization analysis to identify the adverse interactions, as well as simultaneous mapping of all primers onto a genome sequence in a single run without requiring a prior index of the genome. This allows detection of overlapping primer pairs and allocation of these primer pairs into separate subpools where tiling approaches are used. Using PrimerPooler, 1153 primer pairs were assigned to three preamplification pools (388, 389 and 376 primer pairs each), then 144 subpools of six- to nine-plex PCR for Fluidigm Access Array PCR, followed by Illumina MiSeq sequencing. With optimized experimental protocols, an average of 3269 reads was achieved for the targeted regions, with 95% of targets covered by at least 50 reads, the minimal depth of reads for confident variant calling. PrimerPooler provides a fast and highly efficient stratification of primer pairs for targeted enrichment, thus ensuring representative amplification of the targeted sequences. PrimerPooler is also able to analyse degenerate primers, and is thus also useful for microbiological identification and related target sequencing.

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Induction of female Sex-lethal RNA splicing in male germ cells: implications for Drosophila germline sex determination

Development ◽

10.1242/dev.124.24.5033 ◽

1997 ◽

Vol 124 (24) ◽

pp. 5033-5048 ◽

Cited By ~ 7

Author(s):

J.H. Hager ◽

T.W. Cline

Keyword(s):

Sex Determination ◽

Germ Cells ◽

Rna Splicing ◽

Feedback Loop ◽

Dose Effect ◽

Male Germ Cells ◽

Control Female ◽

Sex Lethal ◽

Specific Regulation ◽

Female Specific

With a focus on Sex-lethal (Sxl), the master regulator of Drosophila somatic sex determination, we compare the sex determination mechanism that operates in the germline with that in the soma. In both cell types, Sxl is functional in females (2X2A) and nonfunctional in males (1X2A). Somatic cell sex is determined initially by a dose effect of X:A numerator genes on Sxl transcription. Once initiated, the active state of SXL is maintained by a positive autoregulatory feedback loop in which Sxl protein insures its continued synthesis by binding to Sxl pre-mRNA and thereby imposing the productive (female) splicing mode. The gene splicing-necessary factor (snf), which encodes a component of U1 and U2 snRNPs, participates in this RNA splicing control. Here we show that an increase in the dose of snf+ can trigger the female Sxl RNA splicing mode in male germ cells and can feminize triploid intersex (2X3A) germ cells. These snf+ dose effects are as dramatic as those of X:A numerator genes on Sxl in the soma and qualify snf as a numerator element of the X:A signal for Sxl in the germline. We also show that female-specific regulation of Sxl in the germline involves a positive autoregulatory feedback loop on RNA splicing, as it does in the soma. Neither a phenotypically female gonadal soma nor a female dose of X chromosomes in the germline is essential for the operation of this feedback loop, although a female X-chromosome dose in the germline may facilitate it. Engagement of the Sxl splicing feedback loop in somatic cells invariably imposes female development. In contrast, engagement of the Sxl feedback loop in male germ cells does not invariably disrupt spermatogenesis; nevertheless, it is premature to conclude that Sxl is not a switch gene in germ cells for at least some sex-specific aspects of their differentiation. Ironically, the testis may be an excellent organ in which to study the interactions among regulatory genes such as Sxl, snf, ovo and otu which control female-specific processes in the ovary.

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