Unequivocal determination of sire allele origin for multiallelic microsatellites when only the sire and progeny are genotyped

The effect of a segregating economic trait locus (ETL) can be detected with the aid of a linked genetic marker, if specific alleles of each locus are in association among the individuals genotyped for the genetic marker. For dairy cattle this can be achieved by application of the ‘granddaughter design’. If only the sires and their sons are genotyped for the genetic markers, then the allele origin of sons having the same genotypes as their sires cannot be determined. Seven sires and 101 sons were genotyped for five microsatellites. The mean frequency of heterozygous sires was 77%. The mean number of alleles per locus was 8.2. Frequency of informative sons per locus ranged from 60% to 80% with a mean of 72%. With highly polymorphic microsatellites, at least 60% more grandsire families can be included in the analysis, and the number of sons assayed can be reduced by 40%, as compared to diallelic markers.     

Methylation of KvDMR1 involved in regulating the imprinting of CDKN1C gene in cattle

The CDKN1C gene encodes a cyclin‐dependent kinase inhibitor and is one of the key genes involved in the development of Beckwith–Wiedemann syndrome and cancer. In this study, using a direct sequencing approach based on a single nucleotide polymorphism (SNP) at genomic DNA and cDNA levels, we show that CDKN1C exhibits monoallelic expression in all seven studied organs (heart, liver, spleen, lung, kidney, muscle and subcutaneous fat) in cattle. To investigate how methylation regulates imprinting of CDKN1C in cattle, allele‐specific methylation patterns in two putative differential methylation regions (DMRs), the CDKN1C DMR and KvDMR1, were analyzed in three tissues (liver, spleen and lung) using bisulfite sequencing PCR. Our results show that in the CDKN1C DMR both parental alleles were unmethylated in all three analyzed tissues. In contrast, KvDMR1 was differentially methylated between the two parental alleles in the same tissues. Statistical analysis showed that there is a significant difference in the methylation level between the two parental alleles (P < 0.01), confirming that this region is the DMR of KvDMR1 and that it may be correlated with CDKN1C imprinting.     

The S11 and S13 self incompatibility alleles in Solanum chacoense Bitt. are remarkably similar

A genomic clone of the S11 allele from the self-incompatibility locus (S locus) in Solanum chacoense Bitt. has been isolated by cross-hybridization to the S. chacoense S13 allele and sequenced. The sequence of the S11 allele contains all the features expected for S genes of the Solanaceae, and S11 expression, as assessed by northern blots and RNA-PCR, was similar to that of other S. chacoense S alleles. The S11 protein sequence shares 95% identity with the phenotypically distinct S13 protein of S. chacoense and is the gametophytic S allele with the highest similarity to an existing allele so far discovered. Only 10 amino acid changes differentiate the mature proteins from these two alleles, which sets a new lower limit to the number of changes that can produce an altered S allele specificity. The amino acid substitutions are not clustered, suggesting that an accumulation of random point mutations can generate S allele diversity. The S11 intron is unusual in that it could be translated in frame with the coding sequence, thus suggesting an additional mechanism for the generation of new S alleles.     

Null alleles at two lactate dehydrogenase loci in rainbow trout are associated with decreased developmental stability

Previous studies with rainbow trout (Oncorhynchus mykiss) have shown that allozymic heterozygotes have increased developmental stability, as measured by reduced fluctuating bilateral asymmetry. In this paper, we examine the phenotypic effects of null alleles at two lactate dehydrogenase (LDH) loci. If the association between allozymic heterozygosity and developmental stability is due largely to linked chromosomal segments, then we would expect null allele heterozygotes to have increased developmental stability. In contrast, heterozygotes for LDH null alleles in three populations have reduced developmental stability. This suggests that the reduction in enzyme activity at these loci is having a deleterious effect on development that is strong enough to mask any beneficial effects that may be associated with heterozygosity for these chromosomal segments. The LDH loci examined in this study are members of two different paralogous pairs of duplicate genes produced by the polyploidization of the ancestral salmonid genome. The apparent deleterious effects of these null alleles in heterozygotes could retard the possible loss of duplicate gene expression.     

Resolving genetic relationships with microsatellite markers: a parentage testing system for the swallow Hirundo rustica

Eight polymorphic microsatellite markers from the swallow were isolated and characterized. Extraordinary variability was revealed at the HrU6 locus with 45 different alleles scored among 46 unrelated individuals. The probability that the same genotype combination would occur in two random and unrelated individuals at six selected loci was as low as 1.3 × 10^-8 and the combined exclusion probability was 0.9996. Stable Mendelian inheritance was observed in about 1000 meioses. No significant linkage was revealed and for almost all combinations of marker-pairs, linkage closer than 5 cM could be excluded. At two loci, null (nonamplifying) alleles were encountered. Thirteen (30%) extra-pair offspring were identified in 5 (56%) broods when applying the marker set on a nearly complete swallow colony. We were able to identify a single male from the other families in the colony as the most likely father for nine of the 13 extra-pair offspring.     

Linkage studies on an esterase locus in the mosquito Aedes togoi

An electrophoretic survey of esterases in 7 wild-type and 10 mutant strains of the mosquito Aedes (Finlaya) togoi was undertaken using thin-layer agar gels. Three esterases (designated the Est-1, Est-2, and Est-3 loci in decreasing order of electrophoretic mobility) could be detected from fourth-instar larvae, pupae, and 2- to 5-day-old adults. Homogenates of the larvae gave the most intensely stained bands in the gels, especially for Est-3. The three esterases were designated carboxylesterases based on their response to the two esterase inhibitors, eserine and paraoxon (diethyl-p-nitrophenyl phosphate). The Est-3 locus was found to have five alleles including at least one null. The linkage results of six backcrosses suggest that Est-3 is located only 5–8 map units from the sex allele (m) and the gene arrangement is Est-3-m-s (straw-colored larva) in linkage group I.This work was supported by National Institutes of Health Grant AI 16983-01.     

Analysis of a case of somatic instability in a strain of maize from India

A case of somatic instability affecting aleurone colour in a strain of maize from India with flint background was analysed. The somatic instability is localized to theC ¹ (Inhibitor) allele ofC locus on the short arm of chromosome 9. Molecular tests indicated thatAc is not present in the Indian stock and the evidence is consistent with the involvement of theEn (Spm) transposable element in the instability. The presence of theEn (Spm)-like element in the stock would suggest that these elements have been present in the maize genome for a long time. A new allele ofshrunken (sh1) gene with a somewhat unorthodox breeding behaviour is also described.     

Bi-FoRe:an efficient bidirectional knockin strategy to generate pairwise conditional alleles with fluorescent indicators

Gene expression labeling and conditional manipulation of gene function are important for elaborate dissection of gene function.However,contemporary generation of pairwise dual-function knockin alleles to achieve both conditional and geno-tagging effects with a single donor has not been reported.Here we first developed a strategy based on a flipping donor named FoRe to generate conditional knockout alleles coupled with fluorescent allele-labeling through NHEJ-mediated unidirectional targeted insertion in zebrafish facilitated by the CRISPR/Cas system.We demonstrated the feasibility of this strategy at sox10 and isl1 loci,and successfully achieved Cre-induced conditional knockout of target gene function and simultaneous switch of the fluorescent reporter,allowing generation of genetic mosaics for lineage tracing.We then improved the donor design enabling efficient one-step bidirectional knockin to generate paired positive and negative conditional alleles,both tagged with two different fluorescent reporters.By introducing Cre recombinase,these alleles could be used to achieve both conditional knockout and conditional gene restoration in parallel;furthermore,differential fluorescent labeling of the positive and negative alleles enables simple,early and efficient realtime discrimination of individual live embryos bearing different genotypes prior to the emergence of morphologically visible phenotypes.We named our improved donor as Bi-FoRe and demonstrated its feasibility at the sox10 locus.Furthermore,we eliminated the undesirable bacterial backbone in the donor using minicircle DNA technology.Our system could easily be expanded for other applications or to other organisms,and coupling fluorescent labeling of gene expression and conditional manipulation of gene function will provide unique opportunities to fully reveal the power of emerging single-cell sequencing technologies.     

Distribution of HLA class Ⅰ and class Ⅱ haplotypes in Chinese Han population based on family segregation

HLA haplotype analysis has important application value in human population genetics, anthropological research and HLA matching transplantation. Based on HLA-A, -B, -C, -DRB1 and -DQB1 genotyping data from 663 families including 663 leukemia patients and 991 related donors, the allele frequency (AF) and haplotype frequency (HF) of two-, three- and five-locus haplotype distribution patterns in the Chinese Han population were determined by family segregation. A total of 38 alleles at A locus, 75 alleles at B locus, 35 alleles at C locus, 53 alleles at DRB1 locus and 22 alleles at DQB1 locus were discovered in this population. The frequencies of these alleles were basically consistent with those of previous reports except for some tiny differences. The study found 11 A-C, 15 C-B, 4 B-DRB1 and 11 DRB1-DQB1 two-locus haplotypes with a frequency over 2%. The number of A-C-B and A-B-DRB1 three-locus haplotype with a frequency over 1% were 11 and 3 respectively. The most common HLA-A-C-B-DRB1-DQB1 haplotype (HF>1%) were A*3001-C*0602-B*1302-DR*0701-DQ*0202 (4.30%), A*0207-C*0102-B*4601-DR*0901-DQ*0303 (3.07%), A*3303-C*0302-B*5801-DR*0301-DQ*0201 (1.49%) and A*1101-C*0102-B*4601-DR*0901-DQ*0303 (1.01%). The results are helpful for finding matching donors for hematopoietic stem cell transplant patients and also contribute to transplant immunology, HLA-related diseases, research of human genetics and other fields.