Locus Mapping Research Articles

Inversions contribute disproportionately to parallel genomic divergence in dune sunflowers.

The probability of parallel genetic evolution is a function of the strength of selection and constraints imposed by genetic architecture. Inversions capture locally adapted alleles and suppress recombination between them, which limits the range of adaptive responses. In addition, the combined phenotypic effect of alleles within inversions is likely to be greater than that of individual alleles; this should further increase the contributions of inversions to parallel evolution. We tested the hypothesis that inversions contribute disproportionately to parallel genetic evolution in independent dune ecotypes of Helianthus petiolaris. We analysed habitat data and identified variables underlying parallel habitat shifts. Genotype-environment association analyses of these variables indicated parallel responses of inversions to shared selective pressures. We also confirmed larger seed size across the dunes and performed quantitative trait locus mapping with multiple crosses. Quantitative trait loci shared between locations fell into inversions more than expected by chance. We used whole-genome sequencing data to identify selective sweeps in the dune ecotypes and found that the majority of shared swept regions were found within inversions. Phylogenetic analyses of shared regions indicated that within inversions, the same allele typically was found in the dune habitat at both sites. These results confirm predictions that inversions drive parallel divergence in the dune ecotypes.

Mapping of resistance genes to powdery mildew based on DNA re-sequencing and bulk segregant analysis in Capsicum.

Powdery mildew caused by Leveillula taurica adversely affects the development and growth of pepper plants. However, there have been few reports on the fine mapping and quantitative trait locus (QTLs) gene cloning of resistance genes to powdery mildew in pepper. Herein, an F2 segregating population was constructed using the high resistance material "NuMex Suave Red" and the extremely susceptible material "c89" for bulked segregant analysis and DNA re-sequencing (BSA-seq). Molecular markers were used to achieve fine mapping, followed by expression verification. A major QTL located on chromosome 5 (Chr5, 7.20-11.75Mb) that is associated with resistance to powdery mildew in pepper was mapped using BSA-seq. A narrow interval of 64.86kb encompassing five genes was refined using InDel and KSAP molecular markers developed from the QTL region. Among them, the expression of the ubiquitin-conjugating enzyme E2 gene, Capana05g000392, was significantly upregulated in multiple resistant materials. In addition, there was a single nucleotide polymorphism (SNP) of A/G in the 241st position of the CDS sequence of Capana05g000392, which in turn leads to an amino acid polymorphism of M/V between susceptible parent and resistant parent. Overall, these results indicate that the Capana05g000392 gene may serve as a robust potential factor against powdery mildew in pepper. These findings further elucidate the genetic mechanism of resistance to powdery mildew in pepper and facilitate molecular marker-assisted breeding.

Genome-wide association study and quantitative trait loci mapping of fruit-related traits using recombinant inbred lines in watermelon

Genome-wide association study and quantitative trait loci mapping of fruit-related traits using recombinant inbred lines in watermelon

Highly Efficient Methods with a Generalized Linear Mixed Model for the Quantitative Trait Locus Mapping of Resistance to Columnaris Disease in Rainbow Trout (Oncorhynchus mykiss)

Linear mixed models (LMMs) are commonly used in genome-wide association studies (GWASs) to evaluate population structures and relatedness. However, LMMs have been shown to be ineffective in controlling false positive errors for the analysis of resistance to Columnaris disease in Rainbow Trout. To solve this problem, we conducted a series of studies using generalized linear mixed-model association software such as GMMAT (v1.4.0) (generalized linear mixed-model association tests), SAIGE (v1.4.0) (Scalable and Accurate Implementation of Generalized mixed model), and Optim-GRAMMAR for scanning a total of 25,853 SNPs. Seven different SNPs (single-nucleotide polymorphisms) associated with the trait of resistance to Columnaris were detected by Optim-GRAMMAR, four SNPs were detected by GMMAT, and three SNPs were detected by SAIGE, and all of these SNPs can explain 8.87% of the genetic variance of the trait of resistance to Columnaris disease. The heritability of the trait of resistance to Columnaris re-evaluated by GMMAT was calibrated and was found to amount to a total of 0.71 other than 0.35, which was seriously underestimated in previous research. The identification of LOC110520307, LOC110520314, and LOC110520317 associated with the resistance to Columnaris disease will provide us more genes to improve the genetic breeding by molecular markers. Finally, we continued the haplotype and gene-based analysis and successfully identified some haplotypes and a gene (TTf-2) associated with resistance to Columnaris disease.

Exploiting light energy utilization strategies in Populus simonii through multitrait-GWAS: insights from stochastic differential models.

The photosynthetic phenotype of trees undergoes changes and interactions that reflect their abilities to exploit light energy. Environmental disturbances and genetic factors have been recognized as influencing these changes and interactions, yet our understanding of the underlying biological mechanisms remains limited, particularly in stochastic environments. Here, we developed a high-dimensional stochastic differential framework (HDSD) for the genome-wide mapping of quantitative trait loci (QTLs) that regulate competition or cooperation in environment-dependent phenotypes. The framework incorporates random disturbances into system mapping, a dynamic model that views multiple traits as a system. Not only does this framework describe how QTLs regulate a single phenotype, but also how they regulate multiple phenotypes and how they interact with each other to influence phenotypic variations. To validate the proposed model, we conducted mapping experiments using chlorophyll fluorescence phenotype data from Populus simonii. Through this analysis, we identified several significant QTLs that may play a crucial role in photosynthesis in stochastic environments, in which 76 significant QTLs have already been reported to encode proteins or enzymes involved in photosynthesis through functional annotation. The constructed genetic regulatory network allows for a more comprehensive analysis of the internal genetic interactions of the photosynthesis process by visualizing the relationships between SNPs. This study shows a new way to understand the genetic mechanisms that govern the photosynthetic phenotype of trees, focusing on how environmental stochasticity and genetic variation interact to shape their light energy utilization strategies.

Quantitative trait locus mapping and OsFLOq12 identification for rice grain hardness: towards improved rice flour for wheat substitution.

Recent shifts in consumer dietary preferences have led to a significant decline in rice consumption in Korea, resulting in surplus rice production. To address this issue, rice flour has been proposed as a substitute for wheat flour. However, the physical, chemical and structural differences between rice and wheat, particularly in grain hardness, pose challenges in using rice flour as an alternative. Understanding the genetic factors that influence rice grain hardness is crucial for improving the milling process and producing high-quality rice flour suitable for wheat flour substitution. In this study, various grain traits, including length, width, thickness, length-to-width ratio and hardness, were measured in a population of brown and milled rice. Quantitative trait locus (QTL) analysis revealed a significant association between grain hardness and thickness, with QTLs for grain hardness mapped on chromosomes 1 and 12 for brown and milled rice, respectively. A total of 20 candidate genes related to grain hardness were identified through QTL analysis. Among them, OsFLOq12 (LOC_Os12g43550) was identified as a key gene influencing grain hardness, which encodes a Ras small GTPase. Phenotypic analysis showed differences in endosperm appearance and particle size between lines with low and high grain hardness. The genetic analysis of OsFLOq12 revealed a single nucleotide polymorphism associated with grain hardness. This study provides valuable insights into the genetic background of grain hardness, offering a foundation for breeding rice varieties optimized for flour production as a viable substitute for wheat flour. © 2024 Society of Chemical Industry.

QTL mapping and BSR-seq revealed loci and candidate genes associated with the sporadic multifoliolate phenotype in soybean (Glycine max)

Key messageThe QTLs and candidate genes governing the multifoliolate phenotype were identified by combining linkage mapping with BSR-seq, revealing a possible interplay between genetics and the environment in soybean leaf development.Soybean, as a legume, is typified by trifoliolate leaves. Although multifoliolate leaves (compound leaves with more than three leaflets each) have been reported in soybean, including sporadic appearances in the first compound leaves in a recombinant inbred line (RIL) population from a cross between cultivated soybean C08 and wild soybean W05 from this study, the genetic basis of this phenomenon is still unclear. Here, we integrated quantitative trait locus (QTL) mapping with bulked segregant RNA sequencing (BSR-seq) to identify the genetic loci associated with the multifoliolate phenotype in soybean. Using linkage mapping, ten QTLs related to the multifoliolate trait were identified. Among these, a significant and major QTL, qMF-2-1 on chromosome 2 and consistently detected across biological replicates, explained more than 10% of the phenotypic variation. Together with BSR-seq analyses, which analyzed the RILs with the highest multifoliolate frequencies and those with the lowest frequencies as two distinct bulks, two candidate genes were identified: Glyma.06G204300 encoding the transcription factor TCP5, and Glyma.06G204400 encoding LONGIFOLIA 2 (LNG2). Transcriptome analyses revealed that stress-responsive genes were significantly differentially expressed between high-multifoliolate occurrence lines and low occurrence ones, indicating environmental factors probably influence the appearance of multifoliolate leaves in soybean through stress-responsive genes. Hence, this study offers new insights into the genetic mechanism behind the multifoliolate phenotype in soybean.

Quantitative Trait Loci Mapping for Powdery Mildew Resistance in Wheat Genetic Population.

Powdery mildew is a prevalent wheat disease that affects yield and quality. The characterization and fine mapping of genes associated with powdery mildew resistance can benefit marker-assisted breeding. In this study, quantitative trait loci (QTL) associated with powdery mildew were mapped using a high-density 35K DArT genetic linkage map developed from a population of double haploid lines (DHs) created by crossing "Jinmai 33 (a highly resistance line) with Yannong 19 (a highly susceptible line)". Three stable QTLs for powdery mildew were identified on chromosomes 1B, 2B, and 6A combined with the composite interval graphing method and multiple interval mapping, explaining phenotypic variations (PVE) that range from 4.98% to 13.25%. Notably, Qpm.sxn-1B and Qpm.sxn-2B were identified across three environments, with the PVE ranging from 9.37% to 13.25% and from 4.98% to 5.23%, respectively. The synergistic effects of these QTLs were contributed by the parental line "Jinmai 33". Qpm.sxn-1B was the major stable QTL, and Qpm.sxn-2B was close to Pm51. Furthermore, Qpm.sxn-6A was identified in two environments, accounting for PVE values of 7.13% and 7.65%, respectively, with the resistance effects originating from the male parent. Remarkably, this locus has not been reported previously, indicating that Qpm.sxn-6A represents a newly dis-covered QTL governing powdery mildew genes. Conclusions Five molecular markers available for mark-er-assisted selection were selected for tracking Qpm.sxn-1B and Qpm.sxn-2B in the program. The identification of this novel newly discovered QTL and markers reported in this study will be useful for marker-assisted selection of powdery mildew resistance.

Cleaved Amplified Polymorphic Sequence Markers in Horticultural Crops: Current Status and Future Perspectives

DNA markers have broad applications, including marker-assisted selection (MAS) for breeding new cultivars. Currently, single nucleotide polymorphisms (SNPs) have become a preferred choice of markers for molecular geneticists and breeders. They offer many advantages, such as high abundance and coverage in the genome, codominant inheritance, locus specificity, and flexibility for high-throughput genotyping/detection formats, and they are relatively inexpensive. The availability of reference genome sequences enables precise identification of candidate genes and SNPs associated with a trait of interest through quantitative trait loci (QTL) mapping and genome-wide association studies (GWAS). Such SNPs can be converted into markers for their application in MAS in crop breeding programs. Cleaved amplified polymorphic sequence (CAPS) markers amplify short genomic sequences around the polymorphic endonuclease restriction site. This review provides insight into the recent advancements made in the development and application of CAPS markers in several horticultural plants. We discussed many new tools that aid faster and more accurate design of CAPS markers from the whole genome resequencing (WGRS) data. The developed CAPS markers offer immense application in germplasm screening and field trials, genomic loci mapping, identifying candidate genes, and MAS of important horticultural traits such as disease resistance, fruit quality and morphology, and genetic purity.

Research Progress on the Genetic and Molecular Mechanisms of Cucumber (Cucumis sativus L.) Powdery Mildew Resistance

As a common fungal disease, powdery mildew (PM) is one of the main diseases that harm the growth and development of cucumbers. Understanding the types of pathogenic fungus and analysis of the genetic and molecular mechanisms of cucumber resistance to PM at the molecular level are important when breeding disease-resistant varieties. The present review summarizes the hazards, prevention, and control of PM, and it discusses resistance inheritance rules, molecular markers, quantitative trait locus (QTL) mapping, gene cloning, omics, and gene editing technology, providing research insights on cucumber breeding varieties resistant to PM.

Inherited infertility: Mapping loci associated with impaired female reproduction

Female infertility is a common and complex health problem affecting millions of women worldwide. While multiple factors can contribute to this condition, the underlying cause remains elusive in up to 15%-30% of affected individuals. In our large genome-wide association study (GWAS) of 22,849 women with infertility and 198,989 control individuals from the Finnish population cohort FinnGen, we unveil a landscape of genetic factors associated with the disorder. Our recessive analysis identified a low-frequency stop-gained mutation in TATA-box binding protein-like 2 (TBPL2; c.895A>T [p.Arg299Ter]; minor-allele frequency [MAF]= 1.2%) with an impact comparable to highly penetrant monogenic mutations (odds ratio [OR]= 650, p= 4.1×10-25). While previous studies have linked the orthologous gene to anovulation and sterility in knockout mice, the severe consequence of the p.Arg299Ter variant was evidenced by individuals carrying two copies of that variant having significantly fewer offspring (average of 0.16) compared to women belonging to the other genotype groups (average of 1.75 offspring, p= 1.4×10-15). Notably, all homozygous women who had given birth had received infertility therapy. Moreover, our age-stratified analyses identified three additional genome-wide significant loci. Two loci were associated with early-onset infertility (diagnosed before age 30), located near CHEK2 and within the major histocompatibility complex (MHC) region. The third locus, associated with late-onset infertility, had its lead SNP located in an intron of a long non-coding RNA (lncRNA) gene. Taken together, our data highlight the significance of rare recessive alleles in shaping female infertility risk. The results further provide evidence supporting specific age-dependent mechanisms underlying this complex disorder.

Identification of quantitative trait locus and positional candidate loci influencing chicken egg quality under tropical conditions.

Egg quality is a vital factor in the poultry industry. High-quality eggs not only meet consumer expectations for appearance, taste, and nutritional value but also have high marketability, profitability, and consumer satisfaction. Accordingly, we executed our research with the purpose of determining chromosomal regions and genetic markers associated with egg quality in an F2 cross-bred chicken population under tropical conditions; we determined these through a genome-wide association study and quantitative trait locus (QTL) mapping. This population was created by cross-breeding the L2 line of Taiwan Country chickens, which is adapted to local conditions in Taiwan, with an experimental line (R-line) of Rhode Island Red layer chickens, which was developed by the French National Research Institute for Agriculture, Food and the Environment. A 60K single nucleotide polymorphism (SNP) genotyping array for chickens was employed to execute the analysis. Our analysis revealed 40 QTLs associated with egg quality under tropical conditions, namely 20 QTLs with genome-wide statistical significance and 20 QTLs with chromosome-wide statistical significance. Furthermore, we identified 93 SNPs exerting discernible effects on egg quality, with 10 of these effects exhibiting genome-wide significance and 83 exhibiting potential significance. The majority of the detected QTL regions and SNPs agreed with those identified as having an association with egg quality or production traits in previous studies, thus supporting the interrelationships determined between the studied characteristics. The findings of this study enhance the understanding regarding the genetic regulation governing chicken egg quality, thereby serving as a valuable reference for future functional investigations.

Genome-wide mapping of quantitative trait loci conferring resistance to stripe rust in spring wheat line PI 660072.

Two major QTL for resistance to stripe rust were mapped on chromosome 2BL and 4BL in spring wheat PI 660072, and their KASP markers were developed. Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most devastating diseases of wheat worldwide. Identifying resistance genes is crucial for developing resistant cultivars to control the disease. Spring wheat PI 660072 (Triticum aestivum) has been identified to possess both adult-plant resistance (APR) and all-stage resistance (ASR) to stripe rust. To elucidate the genetic basis of the resistance in PI 660072, a mapping population consisting of 211 F5-F7 recombinant-inbred lines (RILs) was developed from a cross of PI 660072 with susceptible spring wheat Avocet S. The mapping population was phenotyped for stripe rust responses across five field environments from 2020 to 2022 and genotyped using the 15K SNP (single nucleotide polymorphism) array to map stripe rust resistance loci. The mapping population was also tested at the seedling stage with predominant Chinese Pst races CYR31, CYR32, CYR34 and PST-YX1-3-1 in the greenhouse. Stripe rust resistance genes were identified using the quantitative trait locus (QTL) mapping approach. Two QTL were identified with QYrPI660072.swust-2BL mapped on the long arm of chromosome 2B for ASR and QYrPI660072.swust-4BL on the long arm of chromosome 4B for APR. To facilitate marker-assisted selection breeding, Kompetitive allele specific PCR (KASP) markers, KASP-1269 for QYrPI660072.swust-2BL and KASP-3209 for QYrPI660072.swust-4BL, were developed. These markers could be used to introgress the effective resistance QTL into new wheat cultivars.

Mapping of quantitative trait loci (QTL) in Brassica napus L. for tolerance to water stress.

Brassica napus L. plants are sensitive to water stress conditions throughout their life cycle from seed germination to seed setting. This study aims at identifying quantitative trait loci (QTL) linked to B. napus tolerance to water stress mimicked by applications of 10% polyethylene glycol-6000 (PEG-6000). Two doubled haploid populations, each consisting of 150 genotypes, were used for this research. Plants at the two true leaf stage of development were grown in the absence (control) or presence (stress) of PEG-6000 under controlled environmental conditions for 48h, and the drought stress index was calculated for each genotype. All genotypes, along with their parents, were genotyped using the Brassica Infinium 90K SNP BeadChip Array. Inclusive composite interval mapping was used to identify QTL. Six QTL and 12 putative QTL associated with water stress tolerance were identified across six chromosomes (A2, A3, A4, A9, C3, and C7). Collectively, 2154 candidate genes for water stress tolerance were identified for all the identified QTL. Among them, 213 genes were identified as being directly associated with water stress (imposed by PEG-6000) tolerance based on nine functional annotations. These results can be incorporated into future breeding initiatives to select plant material with the ability to cope effectively with water stress.

Discovery of key volatile compound and regulatory gene governing the ‘taro-like’ aroma in wax gourd (Benincasa hispida)

The distinctive ‘taro-like’ aroma in wax gourd has become a crucial flavor characteristic, significantly enhancing its market value. Despite its importance, the volatile compounds responsible for this aroma and their genetic regulatory mechanisms have not been sufficiently studied. Utilizing headspace-gas chromatography-quadrupole time of flight-mass spectrometry (HS-GC-QTOF-MS), this research examined the volatile compounds in wax gourd germplasms with and without the ‘taro-like’ aroma, identifying 2-acetyl-1-pyrroline (2-AP) as the primary volatile responsible for this aroma. To uncover the genetic basis of this trait, segregating populations and recombinant inbred lines (RILs) were developed from MY-1 x GX-71 germplasms, which exhibit significant differences in aroma expression. Genetic analysis revealed that a single recessive gene governs the ‘taro-like’ aroma trait in wax gourd. Through bulked segregant analysis sequencing (BSA-seq) and quantitative trait loci (QTL) mapping, the candidate region for this aroma gene was localized to a 285.29 kb interval on Chromosome 2. Further examination of candidate gene expression and sequence variations highlighted the BhBADH gene's critical role in the formation of the ‘taro-like’ aroma. Additionally, RNA-seq analysis revealed significant enrichment of differentially expressed genes (DEGs) associated with metabolic pathways involved in environmental information processing, which contribute to 2-AP synthesis in wax gourd with a ‘taro-like’ aroma. Our research provides a theoretical foundation for the molecular breeding to enhance the aroma quality of wax gourd.

Quantitative Trait Locus Mapping Combined with RNA Sequencing Identified Candidate Genes for Resistance to Powdery Mildew in Bitter Gourd (Momordica charantia L.)

Improving the powdery mildew resistance of bitter gourd is highly important for achieving high yield and high quality. To better understand the genetic basis of powdery mildew resistance in bitter gourd, this study analyzed 300 lines of recombinant inbred lines (RILs) formed by hybridizing the powdery mildew-resistant material MC18 and the powdery mildew-susceptible material MC402. A high-density genetic map of 1222.04 cM was constructed via incorporating 1,996,505 SNPs generated by resequencing data from 180 lines, and quantitative trait locus (QTL) positioning was performed using phenotypic data at different inoculation stages. A total of seven QTLs related to powdery mildew resistance were identified on four chromosomes, among which qPm-3-1 was detected multiple times and at multiple stages after inoculation. By selecting 18 KASP markers that were evenly distributed throughout the region, 250 lines and parents were genotyped, and the interval was narrowed to 207.22 kb, which explained 13.91% of the phenotypic variation. Through RNA-seq analysis of the parents, 11,868 differentially expressed genes (DEGs) were screened. By combining genetic analysis, gene coexpression, and sequence comparison analysis of extreme materials, two candidate genes controlling powdery mildew resistance in bitter gourd were identified (evm.TU.chr3.2934 (C3H) and evm.TU.chr3.2946 (F-box-LRR)). These results represent a step forward in understanding the genetic regulatory network of powdery mildew resistance in bitter gourd and lay a molecular foundation for the genetic improvement in powdery mildew resistance.

Development of a high-density genetic linkage map and identification of quantitativetrait loci (QTLs) associated with Botrytis cinerea resistance in strawberry(Fragaria × ananassa Duch.)

Development of a high-density genetic linkage map and identification of quantitativetrait loci (QTLs) associated with Botrytis cinerea resistance in strawberry(Fragaria × ananassa Duch.)

SNP-Based and Kmer-Based eQTL Analysis Using Transcriptome Data.

Traditional expression quantitative trait locus (eQTL) mapping associates single nucleotide polymorphisms (SNPs) with gene expression, where the SNPs are derived from large-scale whole-genome sequencing (WGS) data or transcriptome data. While WGS provides a high SNP density, it also incurs substantial sequencing costs. In contrast, RNA-seq data, which are more accessible and less expensive, can simultaneously yield gene expressions and SNPs. Thus, eQTL analysis based on RNA-seq offers significant potential applications. Two primary strategies were employed for eQTL in this study. The first involved analyzing expression levels in relation to variant sites detected between populations from RNA-seq data. The second approach utilized kmers, which are sequences of length k derived from RNA-seq reads, to represent variant sites and associated these kmer genotypes with gene expression. We discovered 87 significant association signals involving eGene on the basis of the SNP-based eQTL analysis. These genes include DYNLT1, NMNAT1, and MRLC2, which are closely related to neurological functions such as motor coordination and homeostasis, play a role in cellular energy metabolism, and function in regulating calcium-dependent signaling in muscle contraction, respectively. This study compared the results obtained from eQTL mapping using RNA-seq identified SNPs and gene expression with those derived from kmers. We found that the vast majority (23/30) of the association signals overlapping the two methods could be verified by haplotype block analysis. This comparison elucidates the strengths and limitations of each method, providing insights into their relative efficacy for eQTL identification.

Quantitative trait locus mapping for water-soluble protein content in soybean across six environments

Quantitative trait locus mapping for water-soluble protein content in soybean across six environments

8610 The Role of Adenylyl Cyclase 2 Gene-Dosage Changes in Congenital Male Genitourinary Anomalies

Abstract Disclosure: D.J. Lamb: Advisory Board Member; Self; Ro Health, Inc. Stock Owner; Self; Fellow Health, Inc. M.A. O'Neill: None. Collectively, congenital genitourinary (GU) birth defects are the most common birth defects in males, yet relatively little is known about their cause. In part, this reflects the current clinical perspective that, because most of these birth defects can be surgically repaired, there is nothing to be gained by understanding the cause. Yet our studies show that seemingly simple birth defects like hypospadias or cryptorchidism, may be associated with other more significant underappreciated defects, such as those affecting the kidney, heart, eyes or behavior. We tested the hypothesis that chromosome microdeletions and microduplications (called copy number variations, CNVs) result in gene-dosage changes that are important regulators of GU development and when haploinsufficient or duplicated normal genitourinary development is impacted causing birth defects of the upper and/or lower genitourinary tract. We successfully used this strategy to identify 15 different previously unrecognized genes that when microduplicated or microdeleted result in conditions such as cryptorchidism, hypospadias, disorders of sexual differentiation (DSD) and other anomalies. Remarkably, gene-dosage changes affected major signaling pathways and post-translational modifications in novel, previously unrecognized ways. Herein, we focused on defining the role of a candidate gene commonly microdeleted or microduplicated in patients with cryptorchidism, hypospadias and/or micropenis. Locus mapping allowed identification of adenylate cyclase 2 (ADCY2) as a candidate gene at the 5p15.31 Cri-du-Chat region in patients with male genital tract anomalies. ADCY2 CNVs were more common in non-syndromic patients with micropenis, cryptorchidism, and/or hypospadias (1.6%) than the general population (0.07%; p< 0.001). Whole exome sequence studies revealed a range of phenotypic anomalies in patients with damaging and potentially damaging ADCY2 variants in addition to the male GU anomalies, including congenital anomalies of the kidney and urinary tract. It is our hypothesis that ADCY2 duplications result in a de-sensitization of the LH receptor diminishing testosterone biosynthesis in the testis, whereas deletions diminish LH receptor signaling. ADCY2 CNVs were assessed in 100 men with spermatogenic failure who were born with cryptorchidism. The ADCY2 CNV frequency was the same as in our cohort of boys with GU anomalies, but as adults they also have hypergonadotropic hypogonadism, which we predicted if ADCY2 over-expression was impacting adult Leydig cell steroidogenesis through partial desensitization of the LH receptor. These studies identified an unrecognized cause of GU anomalies. In the future, such knowledge may lead to improved diagnosis and therapeutic approaches to ameliorate these common birth defects. Presentation: 6/2/2024