Pairs Of Loci Research Articles

Extensive Association Analysis Between Polymorphisms of PON Gene Cluster, Haplotypes, and Enzyme Activity with Myocardial Infarction in a Tunisian Population

Polymorphisms of paraoxonase gene (PON) cluster have been investigated in numerous studies for their association with myocardial infarction (MI) but the results have been inconsistent. The present study was designed to explore the distribution of PON1, PON2 and PON3 polymorphisms, including genotyping, lipid profile, and PON1 activity, and their association with PON1 activity and MI in a Tunisian population. We conducted a case-control study, including 380 MI cases and 350 controls. Genotyping for PON1, PON2, and PON3 variants was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) techniques. The enzymatic activity of PON1 was measured by a kinetic method using paraoxon as a substrate. We reported that of the 12 genotyped variants two were monomorphic (PON1-R160G and PON3-D107D), and ten showed allelic frequency ≥ 5%. Statistical analyzes identified a protective variant (PON1-824G/A) and four susceptibility variants for the risk of IDM (PON1: Q192R, −108 C/T and 162G/A), PON2-S311C. The study of the lipid profile according to the genotypes revealed that only the S311C variant was associated with apo-B and HDL-c levels. We also reported low activity of PON1 in IDM patients compared to controls. This activity was affected by the Q192R polymorphism, is negatively correlated with age and positively with HDL-c and apo-A1 concentrations. Measurement of the linkage disequilibrium (LD) between pairs of loci revealed that D’ varied between (0 and 0.53), whereas the correlation coefficient between alleles r 2 varied from (0 to 0.18), reflects low interaction and high recombination between alleles. Analysis of the haplotypes per gene showed that the QLTAAG and RLTGGA combinations were aggravating the risk of MI, while the QLTGAA combination was protective. Our finding suggested that genetic polymorphisms of PON may play a role in the susceptibility to MI among Tunisian population.

Generating a Three-Dimensional Genome from Xenopus with Hi-C.

Hi-C is a sequencing-based method that captures three-dimensional (3-D) genome interactions by counting the interaction frequencies of pairs of genomic loci. This protocol describes the application of in situ Hi-C to the Xenopus embryo. Briefly, after fixing embryos with formaldehyde, nuclei are isolated and chromatin is digested with a restriction enzyme. Restriction sites are filled in with a biotinylated nucleotide and the blunted ends are re-ligated in place, all while still contained in the nuclei (i.e., in situ). Subsequently, the re-ligated genomic DNA is isolated and fragmented by sonication. Biotinylated ligation junctions are captured with streptavidin-coated beads, and DNA fragments are amplified by ligation-mediated polymerase chain reaction (LM-PCR). The PCR product is isolated and sequenced from both ends (paired-end), and informatics methods are then applied to align the two sides of the ligation junctions to the reference genome. Because ligation occurs much more frequently intra- than interchromosomally, and with generally decreasing frequency the further away DNA loci are from each other on the linear chromosome, interaction frequency information can be used to assist in assembling genomes and to phase haplotypes, which is especially useful in the case of a tetraploid organism such as X. laevis Our streamlined version of in situ Hi-C was optimized for high throughput and low cost, and enables generation of high-quality Hi-C libraries from small cell numbers (down to ∼10,000 cells) in 2 d.

Characterizing human genomic coevolution in locus-gene regulatory interactions

BackgroundCoevolution has been used to identify and predict interactions and functional relationships between proteins of many different organisms including humans. Current efforts in annotating the human genome increasingly show that non-coding DNA sequence has important functional and regulatory interactions. Furthermore, regulatory elements do not necessarily reside in close proximity of the coding region for their target genes.ResultsWe characterize coevolution as it appears in locus-gene interactions in the human genome, focusing on expression Quantitative Trait - Locus (eQTL) interactions. Our results show that in these interactions the conservation status of the loci is predictive of the conservation status of their target genes. Furthermore, comparing the phylogenetic histories of intra-chromosomal pairs of loci and transcription start sites, we find that pairs that appear coevolved are enriched for cis-eQTL interactions. Exploring this property we found that coevolution might be useful in prioritizing association tests in cis-eQTL detection.ConclusionsThe relationship between the conservation status of pairs of loci and protein coding transcription start sites reveal correlations with regulatory interactions. Pairs that appear coevolved are enriched for intra-chromosomal regulatory interactions, thus our results suggest that measures of coevolution can be useful for prediction and detection of new interactions. Measures of coevolution are genome-wide and could potentially be used to prioritize the detection of distant or inter-chromosomal interactions such as trans-eQTL interactions in the human genome.

Isolation and characterization of microsatellite loci from three widespread tropical sea cucumbers of the genus Holothuria (Echinodermata, Holothuroidea), and cross-amplification among them.

Holothuria (Microthele) fuscogilva (Cherbonnier, 1980), Holothuria sp. type "Pentard" and Holothuria (Microthele) nobilis (Selenka, 1867) are three tropical sea cucumber taxa that are heavily fished worldwide for the beche-de-mer trade market. In order to investigate the population genetic structure, diversity and connectivity of these taxa, 16, 19 and 25 microsatellite loci were isolated from H. fuscogilva, Holothuria sp. type "Pentard" and H. nobilis DNA libraries, respectively. These loci were tested on 94, 60 and eight individuals of the respective species, collected from the Seychelles. The number of alleles per locus ranged from 2 to 30. The observed heterozygosity ranged from 0.245 to 0.890 for H. fuscogilva and from 0.200 to 0.950 for Holothuria sp. type "Pentard", while the expected heterozygosity ranged from 0.231 to 0.952 and from 0.504 to 0.951, respectively. Several loci were at Hardy-Weinberg equilibrium and linkage disequilibrium was detected in only three pairs of loci. Cross-amplification was also tested and almost all loci (49 over 60) were polymorphic for at least two of the three studied taxa, showing high transferability among them. These loci represent useful tools for assessing genetic diversity and population structure of these three taxa in fishery areas, and therefore providing relevant knowledge for resource management.

Development and characterization of 20 polymorphic microsatellite loci in the deep sea squat lobster, Munida isos Ahyong and Poore, 2004 and cross-amplification in two congeneric species

Munida isos is a deep sea squat lobster species that is widely distributed across the New Zealand and east Australian region, and is often associated with deep sea vulnerable marine ecosystems. To investigate its population genetic structure and patterns of regional connectivity, microsatellite loci were developed for M. isos from two genomic libraries using the Illumina HiSeq 2500 sequencing platform. Twenty-six loci amplified consistently in M. isos from the Tasman Sea, among which 20 were polymorphic and selectively neutral. Evidence of null alleles was observed at eight loci. Most loci exhibited moderate to high levels of polymorphism, with an average polymorphic information content value of 0.482. The mean number of alleles per locus was 7.45, with a mean expected heterozygosity of 0.520. Thirteen loci exhibited significant deviation from Hardy-Weinberg equilibrium, while only one locus pair was in linkage disequilibrium after false discovery rate correction for multiple testing (P < 0.05). Cross-species amplification tests revealed that the transferability of 14 loci (70%) was positive for the two congeners M. endeavourae and M. gracilis. The accessibility to new polymorphic microsatellite loci will facilitate population genetic studies and aid in developing conservation and management strategies for vulnerable marine ecosystems.

Abstract P6-05-08: LINC02273 interacts with hnRNPL and promotes metastasis through directly activating AGR2 in breast cancer

Abstract The prognosis of breast cancer patients with metastasis is still poor even improved by current treatment modalities. Unveiling new biomarkers and molecular mechanisms that underlie metastasis are of vital importance for the treatment of breast cancer. The recent discovery of long noncoding RNAs (lncRNAs) has gained widespread attention. To identify critical lncRNAs that contributed to breast cancer metastasis, we profiled their expression in 5 pairs of primary tumors and lymph nodes metastasis loci by HTA2.0 microarray. LINC02273 is significantly upregulated in metastasis loci and its high expression is associated with poor diseases free survival in a validation set of 254 patients. LINC02273 was mainly located in the cell nucleus . RACE-PCR showed two isoforms and the longest one was the most abundant isoform in breast cancer. Through transwell assay, 3D spheroid invasion assay and mice xenograft metastasis model, we found that LINC02273 promoted breast cancer cell migration, invasion and metastasis. Via mass spectrometry, hnRNPL was found to interact with LINC02273 to enhance its stability, which was further confirmed by Actinomycin D inhibition assay and luciferase reporter assay. Furthermore, ChIRP-seq and ChIP-seq showed that LINC02273 stimulated oncogene AGR2 expression by directly binding to the AGR2 promoter region and increasing H3K27ac modification. Triplex formation assay was performed for verification. We demonstrated that the expression level and oncogenic ability of AGR2 were regulated by hnRNPL through LINC02273. Clinical data and mouse xenograft tumors also revealed the positive correlation of AGR2 and LINC02273. In conclusion, LINC02273, which is stabilized by hnRNPL can promote breast cancer metastasis through upregulation of AGR2 and may serve as a prognostic biomarker for breast cancer. Citation Format: Xiu B, Chi Y, Ji W, Zhang Q, Wu J. LINC02273 interacts with hnRNPL and promotes metastasis through directly activating AGR2 in breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-05-08.

DNA recovery from fired hollow point ammunition

ABSTRACTFirearm-related exhibits are often found at crime scenes. These exhibits may include the firearm, cartridges, cartridges cases or bullets. As ammunition needs to be handled to load the weapon, regardless of the action or loading type, DNA may be deposited onto the ammunition via touch. As reproducible DNA profiles have been obtained from fired cartridge casings and Improvised Explosive Device (IED) fragments, it is possible that quantifiable amounts of DNA could be recovered from fired bullets. A series of 40 Winchester PowerPoint 22LR 42 grain HP Copper Plated bullets were loaded with serially diluted cell suspensions obtained from a female donor. These were shot into 500 sheet reams of A3 paper for capture and returned to a sterile DNA laboratory for removal, extraction and quantification of DNA. Repeatable partial profiles with five reportable loci pairs consistent with the cell donor were obtained in one replicate of the neat sample. Weak partial profiles were also present in 1:2, 1:5 and 1:10 dilutions. To our knowledge, this is the first reported evidence of DNA surviving the cycle of fire and being recovered from a fired bullet under controlled conditions.

Screening for epistatic selection signatures: A simulation study

Detecting combinations of alleles that diverged between subpopulations via selection signature statistics can contribute to decipher the phenomenon of epistasis. This research focused on the simulation of genomic data from subpopulations under divergent epistatic selection (ES). We used D’IS2 and FST statistics in pairs of loci to scan the whole-genome. The results showed the ability to identify loci under additive-by-additive ES (ESaa) by reporting large statistical departures between subpopulations with a high level of divergence, while it did not show the same advantage in the other types of ES. Despite this, limitations such as the difficulty to distinguish between the quasi-complete fixation of one locus by ESaa from other events were observed. However, D’IS2 can detect loci under ESaa by defining a minimum boundary for the minor allele frequency on a multiple subpopulation analysis where ES only takes place in one subset. Even so, the major limitation was distinguishing between ES and single-locus selection (SS); therefore, we can conclude that divergent locus can be also a result of ES. The test conditions with D-statistics of both Ohta (1982a, 1982b) and Black and Krafsur (1985) did not provide evidence to differentiate ES in our simulation framework of isolated subpopulations.

Genetic polymorphism of 27 X-chromosomal short tandem repeats in an Egyptian population.

We presented allele frequencies of 27 X-chromosomal short tandem repeats (DXS6807, DXS9902, DXS6795, DXS6810, DXS10076, DXS10077, DXS10078, DXS10162, DXS10163, DXS10164, DXS7132, DXS981, DXS6800, DXS6803, DXS6809, DXS6789, DXS6799, DXS7424, DXS101, DXS7133, GATA172D05, DXS10103, HPRTB, GATA31E08, DXS8377, DXS10147, and DXS7423) obtained from 352 unrelated individuals in Egypt. No deviation from Hardy-Weinberg equilibrium was detected. Two pairs of adjacent loci showed significant linkage disequilibrium. In the principal component analysis plot, the Egyptian data were located between Europe and sub-Saharan Africa, away from Asia.

Characterization of 36 insert/deletion mutations by STR genotyping method in grass carp, Ctenopharyngodon idella

Grass carp is one of the most important freshwater aquaculture fish in China. Though a few molecular markers had been reported, it was not enough for grass carp germplasm improvement and protection. In order to verify the effectiveness and practicability of insert/deletion mutations reported in grass carp genome, 85 insert/deletion mutations were randomly selected and analyzed. After simple sequence analysis and attempting primer design, 49 markers were removed due to being simple sequence repeats or unable to design primer for them. Finally, 36 insert/deletion mutations were successfully characterized by STR genotyping method in 32 wild grass carp (collected from Pearl River). The mean effective number of alleles (Ne), observed heterozygosity (Ho), expected heterozygosity (He) and polymorphic information content (PIC) were 1.4976, 0.3190, 0.2970 and 0.2349, respectively. Among all the 36 insert/deletion mutations, three deviated significantly from Hardy–Weinberg equilibrium (P < 0.05). In addition, three pairs of loci (ID-13F with ID-24H, ID-20F with ID-21H and ID-39F with ID-64H) had significant linkage disequilibrium (P < 0.05). Those insert/deletion mutations may lay the foundation for germplasm improvement and protection in grass carp.

Development and characterization of novel microsatellite loci for the Blue-fronted Amazon (Amazona aestiva, Psittaciformes, Aves) and cross-species amplification for other two threatened Amazona species.

Parrots are among the most threatened avian groups of the world, with illegal pet trade being a major threat to some Amazon (Amazona genus) and macaw (Ara and Anodorhynchus genera) species. Population genetic studies and effective control of commercial breeders are important actions for the conservation of these parrot species; however, few microsatellite loci are available for most Amazona species to date. In this study, 25 polymorphic microsatellite loci were identified in silico and characterized for the Blue-fronted Amazon [Amazona aestiva (Aa)]. Loci were tested in 24 Blue-fronted Amazons from wild population from central Brazil with cross-species amplified in two individuals of Amazona vinacea (Av) and Amazona pretrei (Ap) from northeastern Minas Gerais and Rio Grande do Sul, in southeastern and south of Brazil, respectively. The number of alleles per locus for Aa ranged from 5 to 24 with an average of 13.1. Twenty-four and 25 loci were successfully amplified for Av and Ap, respectively. The observed and expected heterozygosities for Aa ranged from 0.27 to 1.00 and from 0.35 to 0.94, with averages of 0.75 and 0.85, respectively. Nine loci significantly deviated from Hardy-Weinberg equilibrium after Bonferroni correction (likely due to null alleles) and no significant linkage disequilibrium between loci pairs was detected. The combined paternity exclusion probability was very high, and the probability of identity was extremely low. This new set of microsatellite loci will be useful for analyzing population genetic structure and making conservation and management decisions, as well as for parentage analysis and the control of commercial breeding of Aa and potentially other Amazona species.

Most chromatin interactions are not in linkage disequilibrium.

Chromatin interactions and linkage disequilibrium (LD) are both pairwise measurements between genomic loci that show block patterns along mammalian chromosomes. Their values are generally high for sites that are nearby in the linear genome but abruptly drop across block boundaries. One function of chromatin boundaries is to insulate regulatory domains from one another. Since recombination is depressed within genes and between distal regulatory elements and their promoters, we hypothesized that LD and chromatin contact frequency might be correlated genome-wide with the boundaries of LD blocks and chromatin domains frequently coinciding. To comprehensively address this question, we compared chromatin contacts in 22 cell types to LD across billions of pairs of loci in the human genome. These computationally intensive analyses revealed that there is no concordance between LD and chromatin interactions, even at genomic distances below 25 kilobases (kb) where both tend to be high. At genomic distances where LD is approximately zero, chromatin interactions are frequent. While LD is somewhat elevated between distal regulatory elements and their promoters, LD block boundaries are depleted—not enriched—at chromatin boundaries. Finally, gene expression and ontology data suggest that chromatin contacts identify regulatory variants more reliably than do LD and genomic proximity. We conclude that the genomic architectures of genetic and physical interactions are independent, with important implications for gene regulatory evolution, interpretation of genetic association studies, and precision medicine.

Boost-HiC: computational enhancement of long-range contacts in chromosomal contact maps.

Genome-wide chromosomal contact maps are widely used to uncover the 3D organization of genomes. They rely on collecting millions of contacting pairs of genomic loci. Contacts at short range are usually well measured in experiments, while there is a lot of missing information about long-range contacts. We propose to use the sparse information contained in raw contact maps to infer high-confidence contact counts between all pairs of loci. Our algorithmic procedure, Boost-HiC, enables the detection of Hi-C patterns such as chromosomal compartments at a resolution that would be otherwise only attainable by sequencing a hundred times deeper the experimental Hi-C library. Boost-HiC can also be used to compare contact maps at an improved resolution. Boost-HiC is available at https://github.com/LeopoldC/Boost-HiC. Supplementary data are available at Bioinformatics online.

Population genetics of Indian giant river-catfish,Sperata seenghala(Sykes, 1839) using microsatellite markers

The giant river-catfishSperata seenghalais one of the commercially important freshwater catfishes of India with wide distribution in all major rivers and reservoirs. This fish has huge demand in domestic market due to high nutritional value and low number of intramuscular bones. Conversely, the culture practices for this fish have not yet been standardized and capture fisheries is the only source to meet the demand. This may lead to over exploitation of resources and subsequent population reduction. Knowledge on genetic structure of populations is prerequisite to formulate sustainable management and conservation measures. In the present study, 15 microsatellites were used to characterize population genetics ofS. seenghalacollected from river Brahmaputra, Ganga, Godavari, Mahanadi and Narmada. Locus-wise, the number of alleles varied from 8 to 19 with an average of 12 alleles per locus. The mean observed and expected heterozygosity values varied from 0.622 to 0.699 and 0.733 to 0.774, respectively. Several loci have shown deviation from Hardy–Weinberg equilibrium and no significant linkage disequilibrium between pairs of loci was detected. Pair-wiseFSTvalues between populations ranged from 0.135 (Brahmaputra–Ganga) to 0.173 (Brahmaputra–Narmada) and confirmed the moderate to high genetic differentiation among the populations. AMOVA, Structure and Principal Co-ordinate analyses showed significant genetic differentiation among the sampled populations ofS. seenghala. A total of 65 private alleles were recorded across populations. This study confirmed the distinctiveness of each population ofS. seenghalafrom five major rivers of India. These populations could be treated as distinct management units (MUs) for assessment and management purpose.

Genetic dissection of harvest index and related traits through genome-wide quantitative trait locus mapping in Brassica napus L.

The harvest index (HI) is the ratio of grain yield to the total biomass and represents the harvestable yield of crops. In Brassica napus, the HI is lower than that of other economically important crops, and limited relevant studies have been carried out regarding this issue. In this study, phenotypic analyses of 11 related traits showed the complexity of HI and the feasibility of cultivating desirable varieties with high HI. Quantitative trait loci (QTL) mapping based on a high-density genetic map identified 160 QTL, 163 epistatic loci pairs for HI and three closely related traits: seed yield (SY), biomass yield (BY) and plant height (PH), including two, five and three major QTL for HI, SY and PH, respectively. The related candidate genes underlying the QTL and epistatic loci with coding region variation were identified and investigated, including BnaA02g14010D, homologous to OsTB1, which functions as a negative regulator for lateral branching, and BnaA02g18890D, homologous to OsGW2, which controls grain width and weight. The complex correlation of HI with related traits, numerous QTL and epistatic loci and the candidate genes identified here provide new insights into the genetic architecture of HI, which might further enhance effective breeding strategies for yield improvement in rapeseed.

Forensic parameters of 15 autosomal STRs (Identifiler™ kit) in three Mayan groups and one Mestizo population from Guatemala.

The population of Guatemala includes Mestizos (admixed) and different Mayan groups (Native Americans), which have been poorly studied in regards to short tandem repeat (STR) loci used for human identification (HID) purposes. Therefore, 483 unrelated Guatemalan volunteers from one Mestizo and three Mayan populations (Poqomchi, Ixil, and Achi) were analyzed with an AmpFlSTR Identifiler™ kit. Allele frequencies and forensic parameters were obtained for 15 autosomal STRs in these populations. Hardy-Weinberg equilibrium by locus and equilibrium linkage between pair of loci were demonstrated by exact tests in all the studied populations. Larger genetic differentiation probably due to genetic drift effects was observed among the studied Guatemalan Mayan groups than the neighboring Mexican Mayas. In brief, our results validate to use the Identifiler™ kit for HID in three non-previously studied Mayan groups, and one Mestizo population from Guatemala.

Polymorphic Microsatellite Markers Selection by Cross-Species Amplification in Black Francolin (Francolinus francolinus asiae)

The cross-species amplification is an effective method in obtaining microsatellite loci for genetic studies of closely related species. In this study, the cross-species amplified markers were characterized and selected for black francolin (Francolinus francolinus asiae). It is a ground dwelling game bird. In this study total eight markers were cross-species amplified. These eight markers were selected from chicken. The number of alleles per locus ranged from 4 to 21. The observed heterozygosity ranged from 0.022 to 0.98 and the expected heterozygosity from 0.23 to 0.79. There was no evidence of linkage disequilibrium among the pair of loci. These loci were polymorphic and offered valuable tool for genetic diversity and population genetic studies of black francolin.

Isolation and characterization of 21 polymorphic microsatellite loci for the rockpool shrimp Palaemon elegans using Illumina MiSeq sequencing

The rockpool shrimp Palaemon elegans is considered an important crustacean species within the European coastline fauna. This species is experiencing an ongoing geographical expansion beyond its native distribution range due to unintentional human introductions. A better knowledge of the genetic diversity, geographic structure and connectivity of its populations is necessary. In the present study, microsatellite loci were isolated using the Illumina MiSeq platform. The microsatellite-enriched library sequencing produced 3.9 million raw reads. Reads were processed and primer pairs were designed for microsatellite sequences amplification. Ninety-six microsatellite loci were preliminary screened in individuals from Atlantic and Mediterranean localities. From them, 21 loci exhibited reliable polymorphism and were thoroughly characterized in 30 individuals from a Cantabrian locality (Spain). No linkage disequilibrium between pairs of loci was detected. Number of alleles per locus ranged from 2 to 12. Observed and expected heterozygosities ranged from 0.033 to 0.833 and from 0.033 to 0.869 respectively. No significant departure from the Hardy-Weinberg equilibrium was detected in most of loci. This is the first time that microsatellite markers have been developed for P. elegans. This characterized microsatellite suite provides new suitable tools for further analyses, facilitating the understanding of population genetics both in natural and introduced populations.

Mapping of QTLs controlling seed weight and seed-shape traits in Brassica napus L. using a high-density SNP map

Thousand seed weight (TSW) is an important trait in oilseed rape. Seed shape and seed size, which directly affect seed weight, can be characterized by seed length, seed width, length–width ratio, seed cross-section area, seed circumference, seed diameter and seed roundness. In the present study, a high-density SNP map containing 2812 bins (involving 11,458 SNPs) was used for the QTL analysis of the eight traits. As a result, 177 identified QTLs were detected in five environments, 25 for TSW and 152 for seven seed-shape traits. These QTLs were integrated into 149 consensus QTLs by a meta-analysis, including one (TSW, length–width ratio, seed length and seed width), two (seed circumference and seed diameter) and three (seed roundness) QTLs accounted for more than 10.0% of the phenotypic variation (PV). The further integration of these consensus QTLs resulted in 63 unique QTLs, of which 34 controlled at least two different traits. Notably, the unique QTL uqC2-1 affected all eight traits and explained 6.60–7.57% of the PV. Furthermore, 19 pairs of epistatic loci were detected and explained 2.39–19.17% of the PV. These results provide useful information to increase the understanding of the genetic basis of TSW and seed-shape traits.

Are TADs supercoiled?

Topologically associating domains (TADs) are megabase-sized building blocks of interphase chromosomes in higher eukaryotes. TADs are chromosomal regions with increased frequency of internal interactions. On average a pair of loci separated by a given genomic distance contact each other 2–3 times more frequently when they are in the same TAD as compared to a pair of loci located in two neighbouring TADs. TADs are also functional blocks of chromosomes as enhancers and their cognate promoters are normally located in the same TAD, even if their genomic distance from each other can be as large as a megabase. The internal structure of TADs, causing their increased frequency of internal interactions, is not established yet. We survey here experimental studies investigating presence of supercoiling in interphase chromosomes. We also review numerical simulation studies testing whether transcription-induced supercoiling of chromatin fibres can explain how TADs are formed and how they can assure very efficient interactions between enhancers and their cognate promoters located in the same TAD.