Genetic Dissection of Quantitative Trait Loci for Hemostasis and Thrombosis on Mouse Chromosomes 11 and 5 Using Congenic and Subcongenic Strains

Susceptibility to thrombosis varies in human populations as well as many inbred mouse strains. Only a small portion of this variation has been identified, suggesting that there are unknown modifier genes. The objective of this study was to narrow the quantitative trait locus (QTL) intervals previously identified for hemostasis and thrombosis on mouse distal chromosome 11 (Hmtb6) and on chromosome 5 (Hmtb4 and Hmtb5). In a tail bleeding/rebleeding assay, a reporter assay for hemostasis and thrombosis, subcongenic strain (6A-2) had longer clot stability time than did C57BL/6J (B6) mice but a similar time to the B6-Chr11^A/J consomic mice, confirming the Hmtb6 phenotype. Six congenic and subcongenic strains were constructed for chromosome 5, and the congenic strain, 2A-1, containing the shortest A/J interval (16.6 cM, 26.6 Mbp) in the Hmtb4 region, had prolonged clot stability time compared to B6 mice. In the 3A-2 and CSS-5 mice bleeding time was shorter than for B6, mice confirming the Hmtb5 QTL. An increase in bleeding time was identified in another congenic strain (3A-1) with A/J interval (24.8 cM, 32.9 Mbp) in the proximal region of chromosome 5, confirming a QTL for bleeding previously mapped to that region and designated as Hmtb10. The subcongenic strain 4A-2 with the A/J fragment in the proximal region had a long occlusion time of the carotid artery after ferric chloride injury and reduced dilation after injury to the abdominal aorta compared to B6 mice, suggesting an additional locus in the proximal region, which was designated Hmtb11 (5 cM, 21.4 Mbp). CSS-17 mice crossed with congenic strains, 3A-1 and 3A-2, modified tail bleeding. Using congenic and subcongenic analysis, candidate genes previously identified and novel genes were identified as modifiers of hemostasis and thrombosis in each of the loci Hmtb6, Hmtb4, Hmtb10, and Hmtb11.     

Genetic Mapping of Linked Antibiotic Resistance Loci in Neisseria gonorrhoeae

Loci for resistance to several antibiotics in laboratory-derived strains of Neisseria gonorrhoeae were mapped by genetic transformation. Genes for high-level resistance to streptomycin (str) and spectinomycin (spc) and for low-level resistance to tetracycline (tet) and chloramphenicol (chl) were linked. Also, a locus for high-level resistance to rifampin (rif) was linked to str and tet. The apparent order was rif... str... tet... chl... spc. Loci for resistance to other antibiotics (penicillin, erythromycin) were transferred independently of each other and were not linked to the cluster around str. Similar linkage relationships were found with str, tet, chl, and spc loci obtained from naturally occurring (clinical) isolates of N. gonorrhoeae.     

Genetic Mapping of Novel Loci Affecting Canine Blood Phenotypes

Since the publication of the dog genome and the construction of high-quality genome-wide SNP arrays, thousands of dogs have been genotyped for disease studies. For many of these dogs, additional clinical phenotypes are available, such as hematological and clinical chemistry results collected during routine veterinary care. Little is known about the genetic basis of variation in blood phenotypes, but this variation may play an important role in the etiology and progression of many diseases. From a cohort of dogs that had been previously genotyped on a semi-custom Illumina CanineHD array for various genome-wide association studies (GWAS) at Cornell University Hospital for Animals, we chose 353 clinically healthy, adult dogs for our analysis of clinical pathologic test results (14 hematological tests and 25 clinical chemistry tests). After correcting for age, body weight and sex, genetic associations were identified for amylase, segmented neutrophils, urea nitrogen, glucose, and mean corpuscular hemoglobin. Additionally, a strong genetic association (P = 8.1×10⁻¹³) was evident between a region of canine chromosome 13 (CFA13) and alanine aminotransferase (ALT), explaining 23% of the variation in ALT levels. This region of CFA13 encompasses the GPT gene that encodes the transferase. Dogs homozygous for the derived allele exhibit lower ALT activity, making increased ALT activity a less useful marker of hepatic injury in these individuals. Overall, these associations provide a roadmap for identifying causal variants that could improve interpretation of clinical blood tests and understanding of genetic risk factors associated with diseases such as canine diabetes and anemia, and demonstrate the utility of holistic phenotyping of dogs genotyped for disease mapping studies.     

Multiple Trait Analysis of Genetic Mapping for Quantitative Trait Loci

We present in this paper models and statistical methods for performing multiple trait analysis on mapping quantitative trait loci (QTL) based on the composite interval mapping method. By taking into account the correlated structure of multiple traits, this joint analysis has several advantages, compared with separate analyses, for mapping QTL, including the expected improvement on the statistical power of the test for QTL and on the precision of parameter estimation. Also this joint analysis provides formal procedures to test a number of biologically interesting hypotheses concerning the nature of genetic correlations between different traits. Among the testing procedures considered are those for joint mapping, pleiotropy, QTL by environment interaction, and pleiotropy vs. close linkage. The test of pleiotropy (one pleiotropic QTL at a genome position) vs. close linkage (multiple nearby nonpleiotropic QTL) can have important implications for our understanding of the nature of genetic correlations between different traits in certain regions of a genome and also for practical applications in animal and plant breeding because one of the major goals in breeding is to break unfavorable linkage. Results of extensive simulation studies are presented to illustrate various properties of the analyses.     

A Single Genetic Determinant that Prevents Sex Reversal in C57BL-YPOS Congenic Mice

Sex determination in the mammalian embryo begins with the activation of a gene on the Y chromosome which triggers a cascade of events that lead to male development. The mechanism by which this gene, designated SRY in humans and Sry in mice (sex determining region of the Y chromosome), is activated remains unknown. Likewise, the downstream target genes for Sry remain unidentified at present. C57BL mice carrying a Y chromosome from Mus musculus musculus or molossinus develop normally as males. In contrast, C57BL/6 mice with the Y chromosome from M. m. domesticus often show sex reversal, i.e., develop as XY females. It has been documented that C57BL mice with the Y chromosome from Poschiavinus (Y^POS), a domesticus subtype, always develop as females or hermaphrodites. This suggests that a C57BL gene either up- or downstream of Sry is ineffective in interacting with Sry, which then compromises the processes that lead to normal male sex development. Nonetheless, by selective breeding, we have been able to generate a sex reversal-resistant C57BL/6-congenic strain of mice in which the XY^POS individuals consistently develop as normal males with bilateral testes. Because the resistance to sex reversal was transferred from strain 129S1/Sv (nonalbino) by simple selection over 13 backcross generations, it is inferred that a single autosomal gene or chromosomal region confers resistance to the sex reversal that would otherwise result. XY^POS normal males generated in these crosses were compared to XY^POS abnormal individuals and to C57BL/6 controls for sexual phenotype, gonadal weight, serum testosterone, and major urinary protein (MUP) level. A clear correlation was found among phenotypic sex, MUP level, and testis weight in the males and in the incompletely masculinized XY^POS mice. The fully masculinized males of the congenic strain resemble C57BL/6 males in the tested parameters. DNA analysis confirmed that these males, in fact, carry the Y^POSSry gene.     

Genetic Study of Cationic ATPase Activities and Audiogenic Seizure Susceptibility in Recombinant Inbred and Congenic Strains of Mice

Total, Mg2+, and Na+, K+ ATPase activities were studied in fresh brain homogenates of the audiogenic seizure (AGS)-resistant C57BL/6J (B6) and AGS-susceptible DBA/2J (D2) inbred strains and in 13 B6 X D2 (BXD) recombinant inbred (RI) strains. These activities were also studied in the D2.B6-Iasb congenic mice, that are similar genetically to D2 mice, except for the Iasb gene which inhibits the spread of AGS activity. The total and Mg2+ ATPase activities of the brainstem were significantly lower in the D2 than in the B6 mice at 21 days of age. No differences were found between these strains for Na+,K+ ATPase activity. The total, Mg2+, and Na+,K+ ATPase activities in the B6 brainstem did not change noticeably from 21 to 80 days of age. In the D2 brainstem, however, the Mg2+ activity increased with age, and the Na+,K+ ATPase activity decreased from 30 to 80 days of age. No genetic associations could be found between AGS susceptibility and total or Mg2+ ATPase activities in the D2.B6-Iasb mice or among the 13 BXD RI strains. Hence, differences in genetic background, rather than differences in AGS susceptibility, can account for the lower ATPase activities in 21-day-old D2 mice. Further, the Mg2+ and Na+,K+ ATPase activities appear to be regulated by more than one gene. This study emphasizes the utility of RI and congenic strains for testing the biochemical basis of AGS susceptibility in mice.     

The Color Loci of Mice – A Genetic Century

Color loci in mammals are those genetic loci in which mutations can affect pigmentation of the hair, skin, and/or eyes. In the mouse, over 800 phenotypic alleles are now known, at 127 identified color loci. As the number of color loci passed 100 only recently, we celebrate this ‘century’ with an overview of these loci, especially the 59 that have been cloned and sequenced. These fall into a number of functional groups representing melanocyte development and differentiation, melanosomal components, organelle biogenesis, organelle transport, control of pigment‐type switching, and some systemic effects. A human ortholog has been identified in all cases, and the majority of these human genes are found to be loci for human disorders, often affecting other body systems as well as pigmentation. We expect that a significant number of color loci remain to be identified. Nonetheless, the large number known already provide a treasury of resources for reconstruction of the mechanisms, at the subcellular, cellular and tissue levels, that produce a functional pigmentary system and contribute to the normal development and functioning of many other organ systems. The mutant mice also provide valuable models for the study of human disease.     

Identification of Genetic Loci Associated with Quality Traits in Almond via Association Mapping

To design an appropriate association study, we need to understand population structure and the structure of linkage disequilibrium within and among populations as well as in different regions of the genome in an organism. In this study, we have used a total of 98 almond accessions, from five continents located and maintained at the Centro de Investigación y Tecnología Agroalimentaria de Aragón (CITA; Spain), and 40 microsatellite markers. Population structure analysis performed in ‘Structure’ grouped the accessions into two principal groups; the Mediterranean (Western-Europe) and the non-Mediterranean, with K = 3, being the best fit for our data. There was a strong subpopulation structure with linkage disequilibrium decaying with increasing genetic distance resulting in lower levels of linkage disequilibrium between more distant markers. A significant impact of population structure on linkage disequilibrium in the almond cultivar groups was observed. The mean r² value for all intra-chromosomal loci pairs was 0.040, whereas, the r² for the inter-chromosomal loci pairs was 0.036. For analysis of association between the markers and phenotypic traits, five models comprising both general linear models and mixed linear models were selected to test the marker trait associations. The mixed linear model (MLM) approach using co-ancestry values from population structure and kinship estimates (K model) as covariates identified a maximum of 16 significant associations for chemical traits and 12 for physical traits. This study reports for the first time the use of association mapping for determining marker-locus trait associations in a world-wide almond germplasm collection. It is likely that association mapping will have the most immediate and largest impact on the tier of crops such as almond with the greatest economic value.     

百脉根BIO和豌豆突变位点ELE2的比较基因组定位

豆科两侧对称花的花瓣具有背腹(DV)的分化以及可变的器官内部(IN)非对称性,在大小与形状上显示出不同的发育特征;因而花瓣的发育为克隆决定植物器官的形状与大小的关键基因提供了很好的实验系统。本研究对百脉根中BIO基因进行研究。百脉根bio突变体具有多效性,既影响花器官内部的对称性也影响器官的大小和育性,豌豆ele突变体的表型与bio相似。定位结果表明BIO和ELE2位于豆科基因组的共线性区段,提示BIO和ELE2可能是同源基因突变所致。本研究利用比较基因组定位方法,将BIO和ELE2候选基因锚定在豆科模式植物百脉根和蒺藜苜蓿基因组含有11个同源基因的BAC重叠群上。BIO和ELE2基因的克隆将有助于揭示豆科花瓣形态和大小调控的分子机理,进而为豆科作物遗传改良提供分子理论基础。     

Genetic Mapping and Non-Mendelian Segregation of Mating Type Loci in the Oomycete, Phytophthora Infestans

DNA markers linked to the determinants of mating type in the oomycete, Phytophthora infestans, were identified and used to address the genetic basis of heterothallism in this normally diploid fungus. Thirteen loci linked to the A1 and A2 mating types were initially identified by bulked segregant analysis using random amplified polymorphic DNA markers (RAPDs) and subsequently scored in three crosses as RAPD markers, restriction fragment length polymorphisms (RFLPs), single-strand conformational polymorphisms (SSCP), cleaved amplified polymorphisms (CAPS), or allele-specific polymerase chain reaction markers (AS-PCR). All DNA markers mapped to a single region, consistent with a single locus determining both mating types. Long-range restriction mapping also demonstrated the linkage of the markers to one region and delimited the mating type locus to a 100-kb region. The interval containing the mating type locus displayed non-Mendelian segregation as only two of the four expected genotypes were detected in progeny. This is consistent with a system of balanced lethal loci near the mating type locus. A model for mating type determination is presented in which the balanced lethals exclude from progeny those with potentially conflicting combinations of mating type alleles, such as those simultaneously expressing A1 and A2 functions.     

Identifying Loci Contributing to Natural Variation in Xenobiotic Resistance in Drosophila

Natural populations exhibit a great deal of interindividual genetic variation in the response to toxins, exemplified by the variable clinical efficacy of pharmaceutical drugs in humans, and the evolution of pesticide resistant insects. Such variation can result from several phenomena, including variable metabolic detoxification of the xenobiotic, and differential sensitivity of the molecular target of the toxin. Our goal is to genetically dissect variation in the response to xenobiotics, and characterize naturally-segregating polymorphisms that modulate toxicity. Here, we use the Drosophila Synthetic Population Resource (DSPR), a multiparent advanced intercross panel of recombinant inbred lines, to identify QTL (Quantitative Trait Loci) underlying xenobiotic resistance, and employ caffeine as a model toxic compound. Phenotyping over 1,700 genotypes led to the identification of ten QTL, each explaining 4.5–14.4% of the broad-sense heritability for caffeine resistance. Four QTL harbor members of the cytochrome P450 family of detoxification enzymes, which represent strong a priori candidate genes. The case is especially strong for Cyp12d1, with multiple lines of evidence indicating the gene causally impacts caffeine resistance. Cyp12d1 is implicated by QTL mapped in both panels of DSPR RILs, is significantly upregulated in the presence of caffeine, and RNAi knockdown robustly decreases caffeine tolerance. Furthermore, copy number variation at Cyp12d1 is strongly associated with phenotype in the DSPR, with a trend in the same direction observed in the DGRP (Drosophila Genetic Reference Panel). No additional plausible causative polymorphisms were observed in a full genomewide association study in the DGRP, or in analyses restricted to QTL regions mapped in the DSPR. Just as in human populations, replicating modest-effect, naturally-segregating causative variants in an association study framework in flies will likely require very large sample sizes.     

10个近交系小鼠的微卫星位点分析及其在遗传监测中应用的探讨（一）

选用不同染色体上的８个微卫星位点,应用ＰＣＲ技术对１０个品系的近交系小鼠进行遗传分析,研究结果表明：在无任何亲缘关系的近交系小鼠品系之间,该８个微卫星位点均具有不同的等位基因。含有不同等位基目的微卫星位点所占的百分比从ＭＳＭ／ＭＳ与Ｃ３Ｈ／ＨｅＪ的１００％到（ＣｘＳ）Ｆ与Ｃ５７ＢＬ／６Ｊ间的２５％,平均５６．７２％,在重组近交系间及重组近交系与亲本之间,此比率从（ＣｘＳ）Ｄ或（ＣｘＳ）Ｍ与ＢＡＬＢ／ＣＨｅＡ间的５０％到（ＣｘＳ）Ｄ或（ＣｘＳ）Ｍ与ＳＴＳ／Ａ,（ＣｘＳ）Ｄ与（ＣｘＳ）Ｍ间的２５％,此８个微卫星位点有可能做为在基因水平上进行近交系小鼠遗传监测的标记。     

Mapping and Genetic Characterization of Loci Controlling the Restoration of Male Fertility in Ogura CMS Radish

Three Raphanus populations (BC1, F2 and R8) each segregating for the restoration of Ogura CMS were used tomap restorer loci. The three restorer loci, Rf1, Rf2 and Rf3, each exhibited dominant restoring alleles and wereeach mutually epistatic. Rf1 was mapped to the upper region of Rs1 using data from each population. Rf2 wasmapped to the middle of Rs2 using both the F2 and R8 populations. Rf3 was mapped to the upper region of Rs7using the R8 population. The marker analysis and linkage mapping of the BC1 and F2 populations were describedpreviously (Bett and Lydiate, 2003). Scoring at 114 marker loci in R8 population allowed a new map ofthe Raphanus genome to be integrated with the consensus map. The complex genetic control of the restoration ofOgura CMS in Raphanus is compared with the more simple genetic control of this trait previously described inB. napus. Markers linked to each of the three restorer loci will allow the routine generation and verification ofdefined restorer and maintainer lines for various combinations of defined restorer loci. Although the restorationof Ogura CMS in Raphanus probably involves additional loci, the identification of three loci and diagnosticmarkers for each provides a solid foundation for the development of a holistic model for the genetic control ofthis trait through mapping in additional populations.     

Genetic Dissection of Behavioural and Autonomic Effects of Δ9-Tetrahydrocannabinol in Mice

Marijuana and its main psychotropic ingredient Δ⁹-tetrahydrocannabinol (THC) exert a plethora of psychoactive effects through the activation of the neuronal cannabinoid receptor type 1 (CB1), which is expressed by different neuronal subpopulations in the central nervous system. The exact neuroanatomical substrates underlying each effect of THC are, however, not known. We tested locomotor, hypothermic, analgesic, and cataleptic effects of THC in conditional knockout mouse lines, which lack the expression of CB1 in different neuronal subpopulations, including principal brain neurons, GABAergic neurons (those that release γ aminobutyric acid), cortical glutamatergic neurons, and neurons expressing the dopamine receptor D1, respectively. Surprisingly, mice lacking CB1 in GABAergic neurons responded to THC similarly as wild-type littermates did, whereas deletion of the receptor in all principal neurons abolished or strongly reduced the behavioural and autonomic responses to the drug. Moreover, locomotor and hypothermic effects of THC depend on cortical glutamatergic neurons, whereas the deletion of CB1 from the majority of striatal neurons and a subpopulation of cortical glutamatergic neurons blocked the cataleptic effect of the drug. These data show that several important pharmacological actions of THC do not depend on functional expression of CB1 on GABAergic interneurons, but on other neuronal populations, and pave the way to a refined interpretation of the pharmacological effects of cannabinoids on neuronal functions.     

The Nxsm Recombinant Inbred Strains of Mice: Genetic Profile for 58 Loci Including the Mtv Proviral Loci

We report the construction of 17 recombinant inbred (RI) strains of mice derived from the progenitor strains NZB/BINRe and SM/J and the typing of this RI strain set, designated NXSM, for 58 loci distributed on 16 autosomes and the X chromosome. Two backcrosses involving NZB/BINJ and SM/J were constructed to confirm chromosomal assignments and determine gene orders suggested from NXSM RI strain data. From these results we recommend that chromosomal assignments and gene orders suggested from analyses of RI strain sets be confirmed using data obtained by other means. We also typed NZB/BINJ and SM/J for mammary tumor proviral (Mtv) loci. Both strains share three previously described Mtv loci: Mtv-7, Mtv-14 and Mtv-17. In addition, NZB/BINJ contains the previously described Mtv-3 and Mtv-9 loci and two new Mtv proviral loci: Mtv-27 located on chromosome (Chr) 1 and Mtv-28 located on the X chromosome. SM/J contains the previously described loci Mtv-6 and Mtv-8. Four LTR, mink cell focus-forming murine leukemia viral loci were identified and mapped: Ltrm-1 on Chr 12, Ltrm-2 on Chr 16, Ltrm-3 on Chr 5, and Ltrm-4 on Chr 13. The Tgn locus was positioned proximal to the Ly-6 locus on Chr 15.     

小鼠15号染色体上脊髓重数量性状基因座的精细定位

目的以前的研究结果表明,控制小鼠脊髓重的一个数量性状基因座(QTL)位于15号染色体D15Mit158附近,跨度约30cM。为分离和确认脊髓重相关基因,本文对该QTL区域进行了精细定位。方法以高级互交系小鼠A/J×C57BL/6J(F4)为研究对象,选择脊髓重偏向两极的个体,在D15Mit158位点附近作高密度局部基因组扫描,用Map Manager QTX19软件对脊髓重与基因型进行连锁不平衡分析。结果在15号染色体D15Mit107附近出现了一个很强的连锁峰,LRS值为17.3(P=1.8×10-4),变异解释率为27%,LOD值达到3.75,可以认定为一主效QTL。该QTL跨度范围为3.2cM。另一个提示可能具有连锁关系的QTL位点在D15Mit28附近,LRS值为7.6(P=0.02),变异解释率为13%,跨度范围为5.0cM。结论控制小鼠脊髓重的D15Mit158区域实际上含有两个QTL,其中一个主效QTL位于15号染色体上宽约3.2cM的D15Mit107位点附近;另一个可能的QTL位于宽约5.0cM的D15Mit28附近。