Dominant Locus Research Articles

Autosomal dominant hereditary spastic paraplegia with axonal sensory motor polyneuropathy maps to chromosome 21q 22.3

Aim: Hereditary spastic paraplegia (HSP) are a genetically and clinically heterogeneous group of disorders. At present, 19 autosomal dominant loci for HSP have been mapped. We ascertained an American family of European descent segregating an autosomal dominant HSP associated with peripheral neuropathy. Methods: A genome wide scan was performed with 410 microsatellite repeat marker (Weber lab screening set 16) and following linkage and haplotype analysis, fine mapping was performed. Established genes or loci for HSP were excluded by direct sequencing or haplotype analysis. Results: All established loci for HSP were excluded. Fine mapping suggested a locus on chromosome 21q22.3 flanked by markers D21S1411 and D21S1446 with a maximum logarithm of odds score of 2.05 and was supported by haplotype analysis. A number of candidate genes in this region were analyzed and no disease-producing mutations were detected. Conclusion: We present the clinical and genetic analysis of an American family with autosomal dominant HSP with axonal sensory motor polyneuropathy mapping to a novel locus on chromosome 21q22.3 designated SPG56.

Abstract 35: Identification of a Mouse Strain Modifier Locus for Factor V Leiden/Tissue Factor Pathway Inhibitor Dependent Thrombosis

Factor V Leiden (FVL) is the most common genetic risk factor for venous thromboembolism (VTE). FVL is incompletely penetrant as only 10% of FVL heterozygotes develop VTE. Previously, nearly uniform perinatal lethality was observed in C57BL/6J (B6) mice hemizygous for tissue factor pathway inhibitor (Tfpi+/-) on a homozygous FVL background (FVQ/Q). However, inbred mouse strains exhibit significant genetic variation and can exert a profound influence on phenotype. We tested the lethality of FVQ/Q Tfpi+/- on the DBA/2J inbred strain background by crossing FVQ/+ Tfpi+/- to DBA/2J followed by a double heterozygous (FVQ/+ Tfpi+/-) x FVQ/Q cross. Surprisingly, FVQ/Q Tfpi+/- mice on the DBA/2J genetic background are born at expected Mendelian frequencies (N=62, nexp=15, nobs=15, N.S.) and display no overt thrombosis. This demonstrates the existence of dominant antithrombotic DBA/2J strain-specific thrombosis modifier gene(s). Analysis of progeny from a DBA/2J-B6 F1 FVQ/Q Tfpi+/- x B6 FVQ/Q cross yielded equal proportions of FVQ/Q Tfpi+/- and FVQ/Q Tfpi+/+ (N=130, nexp=43, nobs=63, p<0.001) which suggested that multiple modifier genes were contributing to the phenotype. To isolate individual dominant modifier loci, we adopted a serial backcrossing strategy in which F1 FVQ/Q Tfpi+/- mice were serially backcrossed to B6 FVQ/Q mice for 17 generations. A standard mouse positional cloning strategy was used on the 110 surviving FVQ/Q Tfpi+/- mice from these crosses which identified a chromosome 2 modifier locus (148-169 Mb) under genetic selection. Currently there are 16 genes in this locus with known annotated non-synonymous coding variants. Ongoing studies are focused on identifying the responsible genetic DBA/2J variant within this locus. Identification of this modifier gene will represent a significant advance for the genetics of thrombotic disease. Not only will it give insight into the pathways leading to thrombosis, it may also reveal previously unexplored therapeutic targets for the prevention of thrombotic events.

Twenty-five thousand years of fluctuating selection on leopard complex spotting and congenital night blindness in horses.

Leopard complex spotting is inherited by the incompletely dominant locus, LP, which also causes congenital stationary night blindness in homozygous horses. We investigated an associated single nucleotide polymorphism in the TRPM1 gene in 96 archaeological bones from 31 localities from Late Pleistocene (approx. 17 000 YBP) to medieval times. The first genetic evidence of LP spotting in Europe dates back to the Pleistocene. We tested for temporal changes in the LP associated allele frequency and estimated coefficients of selection by means of approximate Bayesian computation analyses. Our results show that at least some of the observed frequency changes are congruent with shifts in artificial selection pressure for the leopard complex spotting phenotype. In early domestic horses from Kirklareli-Kanligecit (Turkey) dating to 2700-2200 BC, a remarkably high number of leopard spotted horses (six of 10 individuals) was detected including one adult homozygote. However, LP seems to have largely disappeared during the late Bronze Age, suggesting selection against this phenotype in early domestic horses. During the Iron Age, LP reappeared, probably by reintroduction into the domestic gene pool from wild animals. This picture of alternating selective regimes might explain how genetic diversity was maintained in domestic animals despite selection for specific traits at different times.

Quantitative trait loci on chromosome 5 for susceptibility to frequency-specific effects on hearing in DBA/2J mice.

The DBA/2J strain is a model for early-onset, progressive hearing loss in humans, as confirmed in the present study. DBA/2J mice showed progression of hearing loss to low-frequency sounds from ultrasonic-frequency sounds and profound hearing loss at all frequencies before 7 months of age. It is known that the early-onset hearing loss of DBA/2J mice is caused by affects in the ahl (Cdh23ahl) and ahl8 (Fscn2ahl8) alleles of the cadherin 23 and fascin 2 genes, respectively. Although the strong contributions of the Fscn2ahl8 allele were detected in hearing loss at 8- and 16-kHz stimuli with LOD scores of 5.02 at 8 kHz and 8.84 at 16 kHz, hearing loss effects were also demonstrated for three new quantitative trait loci (QTLs) for the intervals of 50.3–54.5, 64.6–119.9, and 119.9–137.0 Mb, respectively, on chromosome 5, with significant LOD scores of 2.80–3.91 for specific high-frequency hearing loss at 16 kHz by quantitative trait loci linkage mapping using a (DBA/2J × C57BL/6J) F1 × DBA/2J backcross mice. Moreover, we showed that the contribution of Fscn2ahl8 to early-onset hearing loss with 32-kHz stimuli is extremely low and raised the possibility of effects from the Cdh23ahl allele and another dominant quantitative trait locus (loci) for hearing loss at this ultrasonic frequency. Therefore, our results suggested that frequency-specific QTLs control early-onset hearing loss in DBA/2J mice.

粳稻落粒性遗传分析

落粒性是水稻的重要生物学性状之一，对于提高产量降低损失具有重要意义，为了阐明水稻落粒性机理，本文研究了难落粒粳稻品种C418与易落粒粳稻品种春江100及其杂种后代F1、F2落粒性的遗传变异特点。结果表明：1) C418的难落粒与春江100的易落粒表现差异受一对细胞核基因控制，无细胞质效应；2) C418为难落粒显性基因携带品种，文章将该基因命名为Sh-t，春江100为易落粒隐性基因携带品种，文章将该基因命名为sh-t。 Seed shattering is one of the important biological traits in rice. To reveal the genetic character of the seed shattering in Japonic rice, a cross between C418 (japonica cv., nonshattering) and Chun-jiang100 (japonica cv., shattering) was made. Seed shattering of F1, F2 progenies derived from re-ciprocal crosses between C418 and Chunjiang100 and their parents were recorded. The results revealed that there was only one dominant gene locus involved in the control of the seed shattering in this cross. The nonshattering gene carryied by C418 was temporarily designated Sh-t, and the shattering genes carryied by Chunjiang100 was temporarily designated sh-t, respectively.

A reference genetic linkage map of apomictic Hieracium species based on expressed markers derived from developing ovule transcripts.

Apomixis in plants generates clonal progeny with a maternal genotype through asexual seed formation. Hieracium subgenus Pilosella (Asteraceae) contains polyploid, highly heterozygous apomictic and sexual species. Within apomictic Hieracium, dominant genetic loci independently regulate the qualitative developmental components of apomixis. In H. praealtum, LOSS OF APOMEIOSIS (LOA) enables formation of embryo sacs without meiosis and LOSS OF PARTHENOGENESIS (LOP) enables fertilization-independent seed formation. A locus required for fertilization-independent endosperm formation (AutE) has been identified in H. piloselloides. Additional quantitative loci appear to influence the penetrance of the qualitative loci, although the controlling genes remain unknown. This study aimed to develop the first genetic linkage maps for sexual and apomictic Hieracium species using simple sequence repeat (SSR) markers derived from expressed transcripts within the developing ovaries. RNA from microdissected Hieracium ovule cell types and ovaries was sequenced and SSRs were identified. Two different F1 mapping populations were created to overcome difficulties associated with genome complexity and asexual reproduction. SSR markers were analysed within each mapping population to generate draft linkage maps for apomictic and sexual Hieracium species. A collection of 14 684 Hieracium expressed SSR markers were developed and linkage maps were constructed for Hieracium species using a subset of the SSR markers. Both the LOA and LOP loci were successfully assigned to linkage groups; however, AutE could not be mapped using the current populations. Comparisons with lettuce (Lactuca sativa) revealed partial macrosynteny between the two Asteraceae species. A collection of SSR markers and draft linkage maps were developed for two apomictic and one sexual Hieracium species. These maps will support cloning of controlling genes at LOA and LOP loci in Hieracium and should also assist with identification of quantitative loci that affect the expressivity of apomixis. Future work will focus on mapping AutE using alternative populations.

The Vat locus encodes for a CC-NBS-LRR protein that confers resistance to Aphis gossypii infestation and A.gossypii-mediated virus resistance.

Aphis gossypii is a polyphagous sucking aphid and a vector for many viruses. In Cucumis melo, a dominant locus, Vat, confers a high level of resistance to Aphis gossypii infestation and to viruses transmitted by this vector. To investigate the mechanism underlying this double resistance, we first genetically dissected the Vat locus. We delimited the double resistance to a single gene that encodes for a coiled-coil-nucleotide-binding-site-leucine-rich repeat (CC-NBS-LRR) protein type. To validate the genetic data, transgenic lines expressing the Vat gene were generated and assessed for the double resistance. In this analysis, Vat-transgenic plants were resistant to A.gossypii infestation as well as A.gossypii-mediated virus transmission. When the plants were infected mechanically, virus infection occurred on both transgenic and non-transgenic control plants. These results confirmed that the cloned CC-NBS-LRR gene mediates both resistance to aphid infestation and virus infection using A.gossypii as a vector. This resistance also invokes a separate recognition and response phases in which the recognition phase involves the interaction of an elicitor molecule from the aphid and Vat from the plant. The response phase is not specific and blocks both aphid infestation and virus infection. Sequence analysis of Vat alleles suggests a major role of an unusual conserved LRR repeat in the recognition of A.gossypii.

Sequence analysis of the Ras-MAPK pathway genes SOS1, EGFR & GRB2 in silver foxes (Vulpes vulpes): candidate genes for hereditary hyperplastic gingivitis.

Hereditary hyperplastic gingivitis (HHG) is an autosomal recessive disease that presents with progressive gingival proliferation in farmed silver foxes. Hereditary gingival fibromatosis (HGF) is an analogous condition in humans that is genetically heterogeneous with several known autosomal dominant loci. For one locus the causative mutation is in the Son of sevenless homologue 1 (SOS1) gene. For the remaining loci, the molecular mechanisms are unknown but Ras pathway involvement is suspected. Here we compare sequences for the SOS1 gene, and two adjacent genes in the Ras pathway, growth receptor bound protein 2 (GRB2) and epidermal growth factor receptor (EGFR), between HHG-affected and unaffected foxes. We conclude that the known HGF causative mutation does not cause HHG in foxes, nor do the coding regions or intron-exon boundaries of these three genes contain any candidate mutations for fox gum disease. Patterns of molecular evolution among foxes and other mammals reflect high conservation and strong functional constraints for SOS1 and GRB2 but reveal a lineage-specific pattern of variability in EGFR consistent with mutational rate differences, relaxed functional constraints, and possibly positive selection.

Fluorescent in situ hybridization shows DIPLOSPOROUS located on one of the NOR chromosomes in apomictic dandelions (Taraxacum) in the absence of a large hemizygous chromosomal region.

Apomixis in dandelions (Taraxacum: Asteraceae) is encoded by two unlinked dominant loci and a third yet undefined genetic factor: diplosporous omission of meiosis (DIPLOSPOROUS, DIP), parthenogenetic embryo development (PARTHENOGENESIS, PAR), and autonomous endosperm formation, respectively. In this study, we determined the chromosomal position of the DIP locus in Taraxacum by using fluorescent in situ hybridization (FISH) with bacterial artificial chromosomes (BACs) that genetically map within 1.2-0.2 cM of DIP. The BACs showed dispersed fluorescent signals, except for S4-BAC 83 that displayed strong unique signals as well. Under stringent blocking of repeats by C0t-DNA fragments, only a few fluorescent foci restricted to defined chromosome regions remained, including one on the nucleolus organizer region (NOR) chromosomes that contains the 45S rDNAs. FISH with S4-BAC 83 alone and optimal blocking showed discrete foci in the middle of the long arm of one of the NOR chromosomes only in triploid and tetraploid diplosporous dandelions, while signals in sexual diploids were lacking. This agrees with the genetic model of a single dose, dominant DIP allele, absent in sexuals. The length of the DIP region is estimated to cover a region of 1-10 Mb. FISH in various accessions of Taraxacum and the apomictic sister species Chondrilla juncea, confirmed the chromosomal position of DIP within Taraxacum but not outside the genus. Our results endorse that, compared to other model apomictic species, expressing either diplospory or apospory, the genome of Taraxacum shows a more similar and less diverged chromosome structure at the DIP locus. The different levels of allele sequence divergence at apomeiosis loci may reflect different terms of asexual reproduction. The association of apomeiosis loci with repetitiveness, dispersed repeats, and retrotransposons commonly observed in apomictic species may imply a functional role of these shared features in apomictic reproduction, as is discussed.

Identification and Molecular Mapping of the RsDmR Locus Conferring Resistance to Downy Mildew at Seedling Stage in Radish (Raphanus sativus L.)

Downy mildew (DM), caused by the fungus Peronospora parasitica, is a destructive disease of radish (Raphanus sativus L.) worldwide. Host resistance has been considered as an attractive and environmentally friendly approach to control the disease. However, the genetic mechanisms of resistance in radish to the pathogen remain unknown. To determine the inheritance of resistance to DM, F1, F2 and BC1F1 populations derived from reciprocal crosses between a resistant line NAU-dhp08 and a susceptible line NAU-qtbjq-06 were evaluated for their responses to DM at seedling stage. All F1 hybrid plants showed high resistance to DM and maternal effect was not detected. The segregation for resistant to susceptible individuals statistically fitted a 3:1 ratio in two F2 populations (F2(SR) and F2(RS)), and 1:1 ratio in two BC1F1 populations, indicating that resistance to DM at seedling stage in radish was controlled by a single dominant locus designated as RsDmR. A total of 1972 primer pairs (1036 SRAP, 628 RAPD, 126 RGA, 110 EST-SSR and 72 ISSR) were screened, and 36 were polymorphic between the resistant and susceptible bulks, and consequently used for genotyping individuals in the F2 population. Three markers (Em9/ga24370, NAUISSR826700 and Me7/em10400) linked to the RsDmR locus within a 10.0 cM distance were identified using bulked segregant analysis (BSA). The SRAP marker Em9/ga24370 was the most tightly linked one with a distance of 2.3 cM to RsDmR. These markers tightly linked to the RsDmR locus would facilitate marker-assisted selection and resistance gene pyramiding in radish breeding programs.

Endogenous retrovirus insertion in the KIT oncogene determines white and white spotting in domestic cats.

The Dominant White locus (W) in the domestic cat demonstrates pleiotropic effects exhibiting complete penetrance for absence of coat pigmentation and incomplete penetrance for deafness and iris hypopigmentation. We performed linkage analysis using a pedigree segregating White to identify KIT (Chr. B1) as the feline W locus. Segregation and sequence analysis of the KIT gene in two pedigrees (P1 and P2) revealed the remarkable retrotransposition and evolution of a feline endogenous retrovirus (FERV1) as responsible for two distinct phenotypes of the W locus, Dominant White, and white spotting. A full-length (7125 bp) FERV1 element is associated with white spotting, whereas a FERV1 long terminal repeat (LTR) is associated with all Dominant White individuals. For purposes of statistical analysis, the alternatives of wild-type sequence, FERV1 element, and LTR-only define a triallelic marker. Taking into account pedigree relationships, deafness is genetically linked and associated with this marker; estimated P values for association are in the range of 0.007 to 0.10. The retrotransposition interrupts a DNAase I hypersensitive site in KIT intron 1 that is highly conserved across mammals and was previously demonstrated to regulate temporal and tissue-specific expression of KIT in murine hematopoietic and melanocytic cells. A large-population genetic survey of cats (n = 270), representing 30 cat breeds, supports our findings and demonstrates statistical significance of the FERV1 LTR and full-length element with Dominant White/blue iris (P < 0.0001) and white spotting (P < 0.0001), respectively.

A Dominant Locus, qBSC-1, Controls β Subunit Content of Seed Storage Protein in Soybean (Glycine max (L.) Merri.)

Soybean seed storage protein is one of the most important plant vegetable proteins, and β subunit is of great significance to enhance soybean protein quality and processing property. F2 segregated population and residual heterozygous lines (RHL) derived from the cross between Yangyandou (low level of β subunit) and Zhonghuang 13 (normal level of β subunit) were used for mapping of β subunit content. Our results showed that β subunit content was controlled by a single dominant locus, qBSC-1 (β subunit content), which was mapped to a region of 11.9 cM on chromosome 20 in F2 population of 85 individuals. This region was narrowed down to 2.5 cM between BARCSOYSSR_20_0997 and BARCSOYSSR_20_0910 in RHL with a larger population size of 246 individuals. There were 48 predicted genes within qBSC-1 region based on the reference genome (Glyma 1.0, Williams 82), including the two copies of β subunit coding gene CG4. An InDel marker developed from a thymine (TT) insertion in one copy of CG4 promoter region in Yangyandou cosegregrated with BARCSOYSSR_20_0975 within qBSC-1 region, suggesting that this InDel marker maybe useful for marker-assisted selection (MAS).

Detecting molecular signatures of natural selection in Morus alba populations from trans‐Himalaya

AbstractGenome scan was done with 439 dominant marker loci to identify outlier loci in three populations of Morus alba from the trans‐Himalayan region. Employing finite and hierarchical island population genetic models, we detected 30 (6.85%) loci under selection, of which 3.64% were under directional selection and 3.19% under balancing selection. The hierarchical island model was efficient in detecting signatures of natural selection with low occurrence of false positives. Data showed significant spatial genetic structure (SGS), especially in population from Nubra valley with significantly high SGS. Bayesian approach revealed high genetic differentiation and inbreeding in Nubra population. Indus and Suru valleys populations showed comparatively low SGS, genetic differentiation, and inbreeding. The study showed that M. alba falls in the range of mixed mating and outcrossing species. However, the population of Nubra valley showed selfing with high SGS. Significant SGS strengthens the presence of natural selection in M. alba natural populations.

A single locus confers tolerance to continuous light and allows substantial yield increase in tomato.

An important constraint for plant biomass production is the natural day length. Artificial light allows for longer photoperiods, but tomato plants develop a detrimental leaf injury when grown under continuous light--a still poorly understood phenomenon discovered in the 1920s. Here, we report a dominant locus on chromosome 7 of wild tomato species that confers continuous light tolerance. Genetic evidence, RNAseq data, silencing experiments and sequence analysis all point to the type III light harvesting chlorophyll a/b binding protein 13 (CAB-13) gene as a major factor responsible for the tolerance. In Arabidopsis thaliana, this protein is thought to have a regulatory role balancing light harvesting by photosystems I and II. Introgressing the tolerance into modern tomato hybrid lines, results in up to 20% yield increase, showing that limitations for crop productivity, caused by the adaptation of plants to the terrestrial 24-h day/night cycle, can be overcome.

Evolution of apomixis loci in Pilosella and Hieracium (Asteraceae) inferred from the conservation of apomixis-linked markers in natural and experimental populations.

The Hieracium and Pilosella (Lactuceae, Asteraceae) genera of closely related hawkweeds contain species with two different modes of gametophytic apomixis (asexual seed formation). Both genera contain polyploid species, and in wild populations, sexual and apomictic species co-exist. Apomixis is known to co-exist with sexuality in apomictic Pilosella individuals, however, apomictic Hieracium have been regarded as obligate apomicts. Here, a developmental analysis of apomixis within 16 Hieracium species revealed meiosis and megaspore tetrad formation in 1 to 7% of ovules, for the first time indicating residual sexuality in this genus. Molecular markers linked to the two independent, dominant loci LOSS OF APOMEIOSIS (LOA) and LOSS OF PARTHENOGENESIS (LOP) controlling apomixis in Pilosella piloselloides subsp. praealta were screened across 20 phenotyped Hieracium individuals from natural populations, and 65 phenotyped Pilosella individuals from natural and experimental cross populations, to examine their conservation, inheritance and association with reproductive modes. All of the tested LOA and LOP-linked markers were absent in the 20 Hieracium samples irrespective of their reproductive mode. Within Pilosella, LOA and LOP-linked markers were essentially absent within the sexual plants, although they were not conserved in all apomictic individuals. Both loci appeared to be inherited independently, and evidence for additional genetic factors influencing quantitative expression of LOA and LOP was obtained. Collectively, these data suggest independent evolution of apomixis in Hieracium and Pilosella and are discussed with respect to current knowledge of the evolution of apomixis.

An Immunity-Triggering Effector from the Barley Smut Fungus Ustilago hordei Resides in an Ustilaginaceae-Specific Cluster Bearing Signs of Transposable Element-Assisted Evolution

The basidiomycete smut fungus Ustilago hordei was previously shown to comprise isolates that are avirulent on various barley host cultivars. Through genetic crosses we had revealed that a dominant avirulence locus UhAvr1 which triggers immunity in barley cultivar Hannchen harboring resistance gene Ruh1, resided within an 80-kb region. DNA sequence analysis of this genetically delimited region uncovered the presence of 7 candidate secreted effector proteins. Sequence comparison of their coding sequences among virulent and avirulent parental and field isolates could not distinguish UhAvr1 candidates. Systematic deletion and complementation analyses revealed that UhAvr1 is UHOR_10022 which codes for a small effector protein of 171 amino acids with a predicted 19 amino acid signal peptide. Virulence in the parental isolate is caused by the insertion of a fragment of 5.5 kb with similarity to a common U. hordei transposable element (TE), interrupting the promoter of UhAvr1 and thereby changing expression and hence recognition of UhAVR1p. This rearrangement is likely caused by activities of TEs and variation is seen among isolates. Using GFP-chimeric constructs we show that UhAvr1 is induced only in mated dikaryotic hyphae upon sensing and infecting barley coleoptile cells. When infecting Hannchen, UhAVR1p causes local callose deposition and the production of reactive oxygen species and necrosis indicative of the immune response. UhAvr1 does not contribute significantly to overall virulence. UhAvr1 is located in a cluster of ten effectors with several paralogs and over 50% of TEs. This cluster is syntenous with clusters in closely-related U. maydis and Sporisorium reilianum. In these corn-infecting species, these clusters harbor however more and further diversified homologous effector families but very few TEs. This increased variability may have resulted from past selection pressure by resistance genes since U. maydis is not known to trigger immunity in its corn host.

Inheritance and genetic mapping of a gene for seedling resistance to powdery mildew in wheat line X3986-2

Identification and mapping new powdery mildew resistance (Pm) genes is important for resistance breeding in wheat. Common wheat (Triticum aestivum L.) line X3986-2 was tested against 27 isolates of Blumeria graminis f. sp. tritici. To identify the Pm gene(s) in X3986-2, an F2 population and its derived F2:3 lines were developed from a cross between X3986-2 and susceptible line Mingxian169. Segregation ratios indicated the presence of a single dominant Pm locus, tentatively designated PmX3986-2. Bulked segregant analysis was applied to screen for molecular markers linked to PmX3986-2. Two sequence characterized amplified region (SCAR) markers SCAR112 and SCAR203, and five simple sequence repeat markers CFD40, CFD78, CFD81, GWM293 and WMC443 on chromosome 5D were linked to PmX3986-2, with CFD81 and SCAR112 flanking PmX3986-2 at 0.6 and 1.5 cM, respectively. This suggests that PmX3986-2 may be a novel allele of loci Pm2, Pm46 and PmLX66 on chromosome arm 5DS. PmX3986-2 with its tightly linked DNA markers should be useful for broadening the genetic basis of Pm and rapidly transferring the resistance gene to susceptible cultivars or for us in gene pyramiding for resistance breeding.

The KIT gene is associated with the english spotting coat color locus and congenital megacolon in Checkered Giant rabbits (Oryctolagus cuniculus).

The English spotting coat color locus in rabbits, also known as Dominant white spotting locus, is determined by an incompletely dominant allele (En). Rabbits homozygous for the recessive wild-type allele (en/en) are self-colored, heterozygous En/en rabbits are normally spotted, and homozygous En/En animals are almost completely white. Compared to vital en/en and En/en rabbits, En/En animals are subvital because of a dilated (“mega”) cecum and ascending colon. In this study, we investigated the role of the KIT gene as a candidate for the English spotting locus in Checkered Giant rabbits and characterized the abnormalities affecting enteric neurons and c-kit positive interstitial cells of Cajal (ICC) in the megacolon of En/En rabbits. Twenty-one litters were obtained by crossing three Checkered Giant bucks (En/en) with nine Checkered Giant (En/en) and two en/en does, producing a total of 138 F1 and backcrossed rabbits. Resequencing all coding exons and portions of non-coding regions of the KIT gene in 28 rabbits of different breeds identified 98 polymorphisms. A single nucleotide polymorphism genotyped in all F1 families showed complete cosegregation with the English spotting coat color phenotype (θ = 0.00 LOD = 75.56). KIT gene expression in cecum and colon specimens of En/En (pathological) rabbits was 5–10% of that of en/en (control) rabbits. En/En rabbits showed reduced and altered c-kit immunolabelled ICC compared to en/en controls. Morphometric data on whole mounts of the ascending colon showed a significant decrease of HuC/D (P<0.05) and substance P (P<0.01) immunoreactive neurons in En/En vs. en/en. Electron microscopy analysis showed neuronal and ICC abnormalities in En/En tissues. The En/En rabbit model shows neuro-ICC changes reminiscent of the human non-aganglionic megacolon. This rabbit model may provide a better understanding of the molecular abnormalities underlying conditions associated with non-aganglionic megacolon.

Agrobacterium mediated genetic transformation of commercial jute cultivar Corchorus capsularis cv. JRC 321 using shoot tip explants

We have developed a reproducible method of Agrobacterium tumefaciens mediated stable genetic transformation of white jute (Corchorus capsularis cv. JRC 321) utilizing the shoot organogenesis potential of the shoot tip apical meristem. A. tumefaciens strain LBA4404 harboring the binary vector pCAMBIA 1301 was used in the transformation experiments. The explants were subjected to varying durations of preculture and cocultivation with A. tumefaciens in the presence of acetosyringone in order to optimize the conditions conducive for the highest expression of transgene. A schedule of 1 day preculture of shoot tips followed by 3 days cocultivation was optimized for Agrobacterium mediated stable genetic transformation of C. capsularis cv. JRC 321. The optimized lethal doses of the antibiotic hygromycin B for shoot tips (12 mg/L) and for 5 days old seedlings (14 mg/L) were employed in efficient selection of the transformed tissues. This method of transformation resulted in a mean transformation efficiency of 4.09 %. Stable expression of the intron harbored gusA transgene was observed in mature organs of the transformed plants and their progenies. Genomic integration and inheritance of the hpt transgene was further confirmed by Southern blot analysis. The transformed plants exhibited normal morphology and most of them produced viable progenies, many of which segregated in a 3:1 ratio following Mendelian inheritance for a single dominant locus. However, strong P value support for 3:1 segregation ratio was obtained in case of two lines of independent transformants. Nevertheless, the method of transformation mentioned in this protocol could be effectively implemented in genetic transformation of many other cultivars of jute due to the genotype independent regeneration potential of the shoot tip explants.

Estimation of U.S. Yorkshire breed composition using genomic data 1

The Yorkshire breed uses white coat color as a selection requirement to indicate breed purity. In this study, genomic information from chromosomal regions, as well as the whole genome, was applied to estimate breed composition of purebred Yorkshire animals. Genotypes for approximately 60,000 SNP from the Illumina PorcineSNP60 BeadChip (60K) were available for reference animals for which the genetic background was known, study animals that included Yorkshire sires (Tes_York, n = 889), and known crossbred animals that had Yorkshire heritage (Tes_U, n = 12). Haplotypes of SNP flanking the KIT (Dominant white locus) and MC1R (Melanocortin receptor 1) genes were developed for reference animals for the Duroc, Hampshire, Landrace, Yorkshire, and Pietrain breeds. For the KIT region, haplotypes observed in Yorkshire reference animals were also found in 84 and 7% of the haplotypes in Landrace and Pietrain reference animals, respectively. They were not found in Duroc or Hampshire reference animals. The sensitivity and specificity of haplotype analysis was 0.93 and 0.75, respectively. In addition, whole genome SNP information was used in regression analyses to further differentiate breed composition. Using 60K, 90% of regression coefficients for Yorkshire, indicating relative Yorkshire composition, ranged from 0.791 to 1.073 and 0.524 to 1.06 in Tes_York and Tes_U, respectively. Regression coefficients for 90% of Hampshire ranged from -0.029 to 0.052 and -0.005 to 0.379 in Tes_York and Tes_U, respectively. Animals in Tes_U were likely of Yorkshire and Hampshire breed origin. The sensitivity and specificity of regression analysis was 0.96 and 0.58, respectively. Combining haplotype and regression analyses, 810 Tes_York animals were accepted as purebred Yorkshire. Genomic information can be used as a tool to describe an animal's breed composition and reduce the need for progeny testing for white coat color verification.