Development of a sex-linked PCR marker for non-invasive and early sex determination in largemouth bass (Micropterus salmoides)

Largemouth bass (Micropterus salmoides L.) is generally considered to comprise two subspecies, Florida bass (M. floridanus) and Northern Largemouth bass (M. salmoides), which have biological characteristic differences because of their geographical distribution. In this study, whole-genome re-sequencing was performed among 10 Florida and 10 Northern largemouth bass, respectively. In total, 999,793 SNPs and 227,797 InDels were finally identified, and 507,401 SNPs (50.75%) and 116,213 InDels (51.01%) were successfully mapped to annotated 18,629 genes and 14,060 genes, respectively. KEGG classification indicated that most of these genes were focused on the pathways including signal transduction, transport and catabolism, and endocrine system. Genetic diversity analysis indicated that Florida largemouth bass had higher genetic diversity than Northern largemouth bass, indicating that the germplasm quality of Northern largemouth bass had markedly reduced in China. To examine the accuracies of the identified markers, 23 SNPs and eight InDels (the insertions or deletions of more than 45 bp) were randomly selected and detected among Florida largemouth bass, Northern largemouth bass, and their F1 hybrids. The detection efficiencies of all the markers were higher than 95%; nineteen SNPs and three InDels could accurately distinguish the two subspecies and their F1 hybrids with 100% efficiencies. Moreover, the three InDel markers could clearly distinguish the two subspecies and their F1 hybrids with a PCR-based agarose gel electrophoresis. In conclusion, our study established a simple PCR-based method for the germplasm identification of largemouth bass, which will be useful in the germplasm protection, management, and hybridization breeding of largemouth bass.

Sex locus and sex markers identification using whole genome pool-sequencing approach in the largemouth bass (Micropterus Salmoides L.)

Largemouth bass (Micropterus Salmoides) is an economically important fish species in China. Most research has focused on its growth, disease resistance, and nutrition improvement. However, the sex-determining genes in largemouth bass are still unclear. The transforming growth factor-beta (TGF-β) gene family, including amh, amhr2 and gsdf, plays an important role in the sex determination and differentiation of various fishes. These genes are potentially involved in sex determination in largemouth bass. We performed a systematic analysis of 5 sex-related genes (amh, amhr2, gsdf, cyp19a1, foxl2) in largemouth bass using sequence alignment, collinearity analysis, transcriptome, and quantitative real-time polymerase chain reaction (qRT-PCR). This included a detailed assessment of their sequences, gene structures, evolutionary traits, and gene transcription patterns in various tissues including gonads, and at different developmental stages. Comparative genomics revealed that the 5 sex-related genes were highly conserved in various fish genomes. These genes did not replicate, mutate or lose in largemouth bass. However, some were duplicated (amh, amhr2 and gsdf), mutated (gsdf) or lost (amhr2) in other fishes. Some genes (e.g., gsdf) showed significant differences in genomic sequence between males and females, which may contribute to sex determination and sex differentiation in these fishes. qRT-PCR was applied to quantify transcription profiling of the 5 genes during gonadal development and in the adult largemouth bass. Interestingly, amh, amhr2 and gsdf were predominantly expressed in the testis, while cyp19a1 and foxl2 were mainly transcribed in the ovary. All 5 sex-related genes were differentially expressed in the testes and ovaries from the 56th day post-fertilization (dpf). We therefore speculate that male/female differentiation in the largemouth bass may begin at this critical time-point. Examination of the transcriptome data also allowed us to screen out several more sex-related candidate genes. Our results provide a valuable genetic resource for investigating the physiological functions of these 5 sex-related genes in sex determination and gonadal differentiation, as well as in the control of gonad stability in adult largemouth bass.

IntroductionCompared to mammals and birds, sex-determining genes differ in most fish species. Largemouth bass (Micropterus Salmoides) is one of the most important cultured fish species in China, and there are growth differences between males and females. However, its sex-determining genes and mechanisms currently remain unknown.MethodsWe explored the sex-determination mechanism by integrating whole-genome sequencing, resequencing and comparative genomics approaches.ResultsIn this study, we employed HiFi and Hi-C sequencing technologies to construct a chromosome-level haplotypic genome assembly for male largemouth bass, with a genome size of 875.69 Mb. The assembled genome contains 23 chromosomes, covering 95.31% of the complete sequences with a high scaffold N50 of 35.93 Mb. A genome-wide association study (GWAS) of sex was performed with four populations consisting of 62 males and 58 females. For the sex trait, a total of 3,838 SNP loci were identified to be significantly associated with sexual discrepancy. Interestingly, almost all these significant SNPs (3,825) were clustered on chromosome 10 (Chr10), within a 3.5-Mb sex-determination region (SDR). They were homozygous in females while heterozygous in males. We therefore speculate that largemouth bass owns a XX/XY sex determination system. By comparing genomics data and examining coverage depth of resequencing reads, we revealed a ~51-kb male-specific region (MSR) on Chr10. Gene annotation discovered a coding sequence (msy) within MSR-1, which may contribute to sex determination of largemouth bass. By differential expression analysis, two candidate sex-determining genes (ccdc103 and jockey) were predicted within the target SDR. Moreover, we applied two male-specific non-coding fragments (within MSR-2 and MSR-3) to design specific sex markers, successfully obtaining universal gender identity in examined largemouth bass.DiscussionOverall, our findings improve our understanding of the molecular basis for sex determination in largemouth bass, which will thereby promote the mono-sexual breeding progress in the aquaculture industry.

Circulating cell-free fetal DNA (cffDNA) provides the opportunity for noninvasive prenatal diagnosis. Early knowledge of the fetal sex is essential in cases with a risk of a sex-linked genetic disease. A reliable and highly sensitive sex determination test is required for first trimester testing because of the low amounts of cffDNA. First trimester blood samples from 326 pregnant women were analyzed by real-time quantitative polymerase chain reaction (qPCR) for the presence of Y chromosome repetitive sequence (YRS). Blood samples were collected from gestational weeks 4-12. Fetal sex was predicted on the basis of results from the YRS assay of cffDNA extracted from maternal plasma. The predicted sex was compared with the phenotypic sex of the newborn baby (n = 294). There was high concordance between the test results from the YRS assay and the actual sex at birth. There were no false-positive results, indicating agreement between male YRS results and male sex at birth. Two results were false negative (from gestational weeks 4 and 6) predicting female fetuses, when the actual sex at birth was male. Overall, the sensitivity of the YRS assay was 98.6% (95% CI, 95.1%-99.8%), specificity was 100% (95% CI, 97.5%-100%), and accuracy was 99.3% (95% CI, 97.5%-99.9%). From 7 weeks of gestation, sensitivity, specificity, and accuracy were 100%. This study shows that qPCR can be used to detect and quantify repetitive DNA sequences from 0.3 genome equivalents per milliliter of plasma. Prenatal sex determination by qPCR of YRS in cffDNA from maternal plasma was reliable and robust with cffDNA extracted from 1 mL of nonhemolyzed plasma, with a plasma equivalent per PCR of 167 μL. The YRS assay can be used for early noninvasive prenatal sex determination from a gestational age of 7 weeks.

Development and validation of a PCR-RFLP / TaqMan MGB probe method for rapid sex identification of largemouth bass (Micropterus salmoides)

A PCR-based method for genetic sex identification and evidence of the XX/XY sex determination system in largemouth bass (Micropterus salmoides L.)

Mapping sex-determination region and screening DNA markers for genetic sex identification in largemouth bass (Micropterus salmoides)

Sturgeon farming has high potential economic value, but the industry is characterized by late fish maturity, leading to a long payback period for sturgeon farms. Early sex determination (ESD) of fish is important for increasing the profitability of sturgeon farms due to early male separation, thus enabling growing more females and producing more caviar for the same farm area. The data for Russian sturgeons farmed in northern Israel and the results of molecular markers developed for ESD are used in this study. Two sturgeon sex determination technologies are examined and compared from an economic point of view: 1) using molecular DNA markers for ESD at the age of 0.5 years; and 2) using endoscopic methods at the age of 3.5 years, as is currently done in many sturgeon farms. For this purpose, a bio-economic model of a sturgeon farm was developed and an economic evaluation of a sturgeon farm was made to compare both technologies. The ESD technology results in a significant improvement in the economic performance of sturgeon farms. This improvement is greater when caviar prices are lower.

Carica papaya L. is a fruit tree species that is cultivated worldwide. Its production involves the use of hermaphrodite plants, which produce pear-shaped fruits with abundant pulp, making these fruits of high market value. Female plants are eliminated after the sex of the flowers is determined; this process occurs 5 months after sowing and increases the costs, labor, and time required for production. Here, we describe an effective method of early molecular sex determination from embryogenic callus that allows the propagation of only hermaphrodite emblings of C. papaya ‘Golden’ via somatic embryogenesis. This workflow reduces costs and the time required for C. papaya micropropagation. The present work provides a foundation for the future use of this approach for agronomic purposes. An effective method of early molecular sex determination from embryogenic callus of Carica papaya ‘Golden’ has been developed, allowing the propagation of only hermaphrodite emblings during somatic embryogenesis.

Genomic organization and sexually dimorphic expression of the Dmrt1 gene in largemouth bass (Micropterus salmoides).

Early sex determination methods are not only crucial in the worldwide massive poultry industry, but also for small-holder producers. The profitability of sexing techniques must be accounted for when aiming to boost management, nutrition, and conservation practices in endangered poultry breeds. This becomes pivotal when the local breed dealt with belongs to an understudied species, such as the turkey. So, the main objective of this study is to identify which method combination may report a higher likelihood of successful sex determination in poults across the three-pattern varieties of the Andalusian turkey breed. A total of 84 one to two days old Andalusian turkey poults (42 black, 28 black-roan, and 14 bronze-roan) were evaluated in this study. Sex determination was performed using 15 methods, which included testing external egg metrics and eggshell color, poult morphological appraisal and phaneroptics, and behavioral traits. Possible differences across plumage varieties and the interaction between sex and plumage were observed when external egg quality was measured. Sex determination through behavioral methods in black base feathered (black and black-roan) male sex individuals showed seven times higher sensitivity when compared to the rest of the studied individuals (χ2 = 7.14, df = 1, P < 0.01). In contrast, for the black-roan plumage females, the method based on the color of down feathers was approximately four times more sensitive (χ2 = 3.95, df = 1, P ≤ 0.05). For the bronze-roan pattern, none of the sexing techniques was reported to efficiently predict sex itself. However, the most proper method combination to determine sex, independent of plumage color, was physical external egg characteristics, the color of down feathers, and behavioral approaches (“English method” and “slap technique”). The specificity values were found to be 49.12, 93.33, and 100%, while the sensitivity values were observed to be 74.64, 91.03, and 100%, which translated into accuracy of 63.10, 92.26, and 100% in black, black-roan, and bronze-roan poults, respectively. Our results suggest that the method combination tested in this study could be considered a highly accurate, simple, and affordable alternative for sex determination in turkeys. This could mean a pivotal advance for small producers of turkeys, as early sex detection can help to plan timely conservational management strategies, which is of prominent importance in the context of endangered poultry breeds.

Abstract. Laily AN, Daryono BS, Purwantoro A, Purnomo. 2022. Various macro and micro-morphological characters of three sex types of highland papaya (Vasconcellea pubescens) in Java, Indonesia. Biodiversitas 23: 6238-6246. Highland papaya (Vasconcellea pubescens A.DC.), locally called karika, is a fruit plant successfully introduced to Java, Indonesia. V. pubescens plays an essential role as it contains various substances that can be used as ingredients in the food, pharmaceutical, and cosmetic industries. This plant is dioecious and monoecious as it contains three sex types, namely female, male, and monoecious. It grew from seed and began to bear fruit two years after sowing. The consumer preference for fruit is produced from female plants because female plants produce fruit faster, more abundantly, and with maximum fruit size under suitable environmental conditions. Sex differentiation is important for farmers to ensure the growth of female plants. Therefore, this research aimed to investigate the variation in macro and micro-morphological characters. The results showed that leaf and flower differ in macromorphological characters among the three sex types. Similarly, there were considerable differences in the trichomes of female and monoecious plants based on micro-morphological characteristics. There was no significant difference in seed shape between those. Seeds from female and monoecious plants differed in macro and micro-morphological characters. Macro and micro-morphological characters might have potential applications in early sex determination in V. pubescens. Macro and micro-morphological characters have many potential applications in early sex determination in V. pubescens.

Testicular type Sox9 is not involved in sex determination but might be in the development of testicular structures in the medaka, Oryzias latipes

The largemouth bass is an important species, and its culture has risen sharply with the surge in fish aquaculture in China. Due to the lack of selective breeding technology for the largemouth bass, the growth rate and disease resistance are low, its sexual maturation is slow, and other serious problems are contributing to a sharp decline in the safety and quality of largemouth bass products in recent decades. Therefore, comprehensive breeding programs to improve the economic performance and promote the modern industrial development of largemouth bass must be considered a priority. Here, a total of 152 adult largemouth bass, including two parents and 150 progenies, were selected to produce the genetic mapping family. Then, a high-density linkage map was constructed based on restriction site–associated DNA sequencing using 6,917 single-nucleotide polymorphisms (SNPs) located in 24 linkage groups (LGs). The total genetic length of the linkage map was 1,261.96 cM, and the length of each LG varied from 24.72 cM for LG02 to 117.53 cM for LG16, with an average length of 52.58 cM and an average SNP number of 286. Thirteen significant quantitative trait loci (QTLs) for sex determination were located on LG04, LG05, LG08, LG12, LG15, LG21, and LG23. An informative QTL cluster that included six QTLs was detected on LG12. However, one notable QTL, which accounted for 71.48% of the total phenotypic variation, was located in the region of 1.85 cM on LG05. In addition, 32 identified QTLs were related to growth, including body weight, body length, body height, and head length. The QTLs for these growth-related traits are located in 13 LG regions and have little effect on phenotypic variation. This high-density genetic linkage map will enable the fine-mapping of economic traits and support the future genome assembly of the largemouth bass. Additionally, our study will be useful for future selective culture of largemouth bass and could potentially be used in molecular-assisted breeding of largemouth bass for aquaculture.