Preparation and analysis of spermatocyte meiotic pachytene bivalents of pigs for gene mapping

Well-spread meiotic pachytene bivalents were obtained by using the prolonged hypotonic treatment com-bined with high chloroform Carnory‘s fixative solution from cells of the testes of domestic pigs. Comparisonin the division index and length of pachytenc bivalents with metaphase chromosomes showed that those ofthe former are 5 times higher and 3.42(1.87-5.98) times longer than those of the latter. Comparative studieson chromomere maps of bivalents and mitotic chromosomal G-bands were conducted by using the chromo-some 12 as a example. Sex vesicle and various shapes of synaptic sex chromosomes have been observed.Two-color PRimed IN Situ (PRINS) labeling has been conducted successfully on pachytene bivalents of pigs.     

Preparation and analysis of spermatocyte meiotic pachytene bivalents of pigs for gene mapping

Well-spread meiotic pachytene bivalents were obtained by using the prolonged hypotonic treatment combined with high chloroform Carnory's fixative solution from cells of the testes of domestic pigs. Comparison in the division index and length of pachytene bivalents with metaphase chromosomes showed that those of the former are 5 times higher and 3.42(1.87-5.98) times longer than those of the latter. Comparative studies on chromomere maps of bivalents and mitotic chromosomal G-bands were conducted by using the chromosome 12 as a example. Sex vesicle and various shapes of synaptic sex chromosomes have been observed. Two-color PRimed IN Situ (PRINS) labeling has been conducted successfully on pachytene bivalents of pigs.     

Mapping of pachytene bivalents of female codling moth Cydia pomonella (Linnaeus) (Lep., Tortricidae)

Spread pachytene nuclei of codling moth Cydia pomonella (Linnaeus) (Lep., Tortricidae) females of a Syrian strain (SY) were used to investigate chromomere patterns of chromosome bivalents and determine their length. The karyotype of female codling moths consists of 28 chromosome bivalents, of which seven are clearly distinguishable using chromosome length and the number and size of the chromomeres in the pachytene stage. One autosome bivalent has two nucleolar organizing regions (NORs) that are located at the opposite ends of the chromosome and appear as distinct structural landmarks. In female codling moths, the WZ sex chromosome bivalent was easily identified in pachytene oocytes according to the heterochromatic thread of the W chromosome. This study contributed to the knowledge and identification of pachytene chromosomes of female codling moths.     

Super-stretched pachytene chromosomes for fluorescence in situ hybridization mapping and immunodetection of DNA methylation

Meiotic pachytene chromosome-based fluorescence in situ hybridization (FISH) mapping is one of the most important tools in plant molecular cytogenetic research. Here we report a simple technique that allows stretching of pachytene chromosomes of maize to up to at least 20 times their original size. A modified Carnoy's II fixative (6:1:3 ethanol:chloroform:acetic acid) was used in the procedure, and proved to be key for super-stretching of pachytene chromosomes. We demonstrate that super-stretched pachytene chromosomes provide unprecedented resolution for chromosome-based FISH mapping. DNA probes separated by as little as 50 kb can be resolved on super-stretched chromosomes. A combination of FISH with immunofluorescent detection of 5-methyl cytosine on super-stretched pachytene chromosomes provides a powerful tool to reveal DNA methylation of specific chromosomal domains, especially those associated with highly repetitive DNA sequences.     

Primed in situ labelling detection of 5S rDNA sequences in normal and androgenetic rainbow trout

Sex genotypes in mature androgenetic and control rainbow trout Oncorhynchus mykiss were determined using primed in situ labelling (PRINS) detection of 5S rDNA sequences on the X chromosomes. Three sex genotypes, corresponding to phenotypic sex, were revealed: female XX, male XY and supermale YY.     

Pseudosynapsis and Decreased Stringency of Meiotic Repair Pathway Choice on the Hemizygous Sex Chromosome of Caenorhabditis elegans Males

During meiosis, accurate chromosome segregation relies on homology to mediate chromosome pairing, synapsis, and crossover recombination. Crossovers are dependent upon formation and repair of double-strand breaks (DSBs) by homologous recombination (HR). In males of many species, sex chromosomes are largely hemizygous, yet DSBs are induced along nonhomologous regions. Here we analyzed the genetic requirements for meiotic DSB repair on the completely hemizygous X chromosome of Caenorhabditis elegans males. Our data reveal that the kinetics of DSB formation, chromosome pairing, and synapsis are tightly linked in the male germ line. Moreover, DSB induction on the X is concomitant with a brief period of pseudosynapsis that may allow X sister chromatids to masquerade as homologs. Consistent with this, neither meiotic kleisins nor the SMC-5/6 complex are essential for DSB repair on the X. Furthermore, early processing of X DSBs is dependent on the CtIP/Sae2 homolog COM-1, suggesting that as with paired chromosomes, HR is the preferred pathway. In contrast, the X chromosome is refractory to feedback mechanisms that ensure crossover formation on autosomes. Surprisingly, neither RAD-54 nor BRC-2 are essential for DSB repair on the X, suggesting that unlike autosomes, the X is competent for repair in the absence of HR. When both RAD-54 and the structure-specific nuclease XPF-1 are abrogated, X DSBs persist, suggesting that single-strand annealing is engaged in the absence of HR. Our findings indicate that alteration in sister chromatid interactions and flexibility in DSB repair pathway choice accommodate hemizygosity on sex chromosomes.     

A preliminary pachytene analysis of two species of Arachis L.

Summary The morphology of pachytene chromosomes was studied in A. glabrata Benth. and A. pusilla Benth. belonging respectively to the sections Rhizomatosae and Triseminale. These two species can not be crossed with the cultivated groundnut A. hypogaea L. All 20 chromosomes of A. glabrata could be identified individually and further classified into 5 basic types. The features that enabled the identification of chromosomes were: total length, arm ratios, nucleolus attachment and position and extent of heterochromatin. A simple key has been proposed for classifying different chromosomes to facilitate their easy identification. The genomes of A. glabrata did not resemble those of A. hypogaea except for the presence of an A chromosome, 2 euchromosomes and 2 nucleolus organisers. A. glabrata did not appear to be an amphidiploid but rather an allopolyploid hybrid. The genome of A. pusilla contained chromosomes unlike those of any other species of section Arachis. It was concluded that both these species are quite unrelated to other species of the section Arachis.     

The porcine proteasome subunit A4 (PSMA4) gene: isolation of a partial cDNA, linkage and physical mapping

A partial cDNA clone encoding the porcine proteasome subunit A4 ( PSMA4 or proteasome subunit C9) has been isolated from a porcine muscle cDNA library and sequenced. A biallelic Taq I RFLP was identified in Large White, Landrace and Duroc breeds. Moreover, the 3'-untranslated region of the gene showed a triallelic SSCP. By linkage analysis the PSMA4 locus was assigned to pig chromosome 7 and by radioactive in situ hybridization this locus was mapped to the region 7q13–q14.     

Coevolutionary dynamics of polyandry and sex‐linked meiotic drive

Segregation distorters located on sex chromosomes are predicted to sweep to fixation and cause extinction via a shortage of one sex, but in nature they are often found at low, stable frequencies. One potential resolution to this longstanding puzzle involves female multiple mating (polyandry). Because many meiotic drivers severely reduce the sperm competitive ability of their male carriers, females are predicted to evolve more frequent polyandry and thereby promote sperm competition when a meiotic driver invades. Consequently, the driving chromosome's relative fitness should decline, halting or reversing its spread. We used formal modeling to show that this initially appealing hypothesis cannot resolve the puzzle alone: other selective pressures (e.g., low fitness of drive homozygotes) are required to establish a stable meiotic drive polymorphism. However, polyandry and meiotic drive can strongly affect one another's frequency, and polyandrous populations may be resistant to the invasion of rare drive mutants.     

Amplification, analysis and chromosome mapping of novel homeobox-containing and homeobox-flanking sequences in rice

Homeobox genes, widely distributed among animal and plant kingdoms, play an important role in developmental process. Several homeobox conserved fragments were amplified by PCR and the flanking regions were also obtained by an LM-PCR procedure. Sequencing and Southern analysis showed that they belong to a homeobox gene family of rice. Six homeobox-containing fragments were mapped on the molecular linkage map of rice. They were located on chromosomes 3, 4 and 7 respectively. It is noteworthy that there are 4 homeobox fragments located on rice chromosome 3 and the result is also consistent with the comparative genomics between rice and maize.     

Effects of sex and dietary lysine on performances and serum and meat traits in finisher pigs

A total of 160 Duroc×(Landrace×Large White) pigs, 50% barrows and 50% gilts, of 28.3±4.52 kg of BW were used to study the effect of lysine (Lys) restriction in the finisher period, on growth performances and serum and carcass and meat quality traits. The grower diet (from 30 to 90 kg BW) was slightly Lys-restricted (7.8 g standardised ileal digestible (SID) Lys/kg) in accordance with results from a previous trial. During the finisher period (90 to 130 kg BW), four experimental diets with decreasing SID Lys contents (6.3, 5.6, 4.2 and 3.2 g/kg) were tested. Each of the eight treatments (two sexes×four levels of Lys) was replicated five times. Each replicate was a pen with four pigs allocated together. When animals achieved 129±2.59 kg were slaughtered and carcass and meat characteristics were evaluated. No significant interaction sex×diet was found. During the finisher period, barrows grew faster (P<0.001) and ate more feed (P<0.001) but tended to be less efficient (P=0.055) than gilts. The Lys restriction affected linearly (P<0.001) all productive performance traits; daily BW gain and feed intake decreased and feed conversion ratio increased. Also, the concentration of serum urea at slaughter tended to be higher in barrows than in gilts (P=0.065) and was reduced quadratically by the restriction of Lys in feed (P<0.001). Carcasses from barrows had higher backfat thickness (P<0.01) and lower weight of main trimmed lean cuts (ham+shoulder+loin; P<0.05) than those from gilts. The Lys restriction during the finisher period decreased carcass yield (quadratic; P<0.001) and the weight of major cuts (linear; P<0.001). Sex and diet had limited effect on meat characteristics; the Lys restriction decreased quadratically the proportion of protein (P<0.01) and increased linearly the intramuscular fat (IMF) content (P<0.001). We can conclude that dietary Lys restriction during finisher period in pigs impaired growth performances and was not successful to increase the carcass fat deposition, although it could have positive effects on IMF proportion of pork.     

The porcine fat mass and obesity associated (FTO) gene is associated with fat deposition in Italian Duroc pigs

In humans, common variants in the fat mass and obesity associated ( FTO ) gene are associated with body mass index and obesity. Here we sequenced exon 4, parts of introns 3 and 4 and two portions of the 3'-untranslated region of the porcine FTO gene in a panel of nine pigs of different breeds and identified three SNPs. Allele frequencies of the g.276T>G ( AM931150 ) mutation were studied in seven pig breeds. This mutation was used to linkage-map FTO to SSC6. Association analyses between the g.276T>G polymorphism and several traits [pH of semimembranosus muscle and estimated breeding values (EBV) for average daily gain, back fat thickness, lean cuts, ham weight and feed:gain ratio] were carried out in 257 sib-tested Italian Large White pigs. Only feed:gain ratio showed P  <   0.05. A selective genotyping approach was applied, analysing two extreme and divergent groups of Italian Large White pigs selected on the basis of back fat thickness EBV (50 with most positive and 50 with most negative values). Fisher's exact test (two-tailed) was not significant when comparing the allele frequencies of these two groups. The same approach was used in the Italian Duroc breed for which two extreme and divergent groups of animals were selected according to visible intermuscular fat EBV. Differences of allele frequencies between these two groups were highly significant ( P  <   0.00001, P  <   0.001 and P  <   0.0001, considering all animals or only two- or three-generation unrelated animals respectively), indicating association between the analysed FTO marker and intermuscular fat deposition.     

Double trouble: combined action of meiotic drive and Wolbachia feminization in Eurema butterflies

Arthropod sex ratios can be manipulated by a diverse range of selfish genetic elements, including maternally inherited Wolbachia bacteria. Feminization by Wolbachia is rare but has been described for Eurema mandarina butterflies. In this species, some phenotypic and functional females, thought to be ZZ genetic males, are infected with a feminizing Wolbachia strain, wFem. Meanwhile, heterogametic WZ females are not infected with wFem. Here, we establish a quantitative PCR assay allowing reliable sexing in three Eurema species. Against expectation, all E. mandarina females, including wFem females, had only one Z chromosome that was paternally inherited. Observation of somatic interphase nuclei confirmed that W chromatin was absent in wFem females, but present in females without wFem. We conclude that the sex bias in wFem lines is due to meiotic drive (MD) that excludes the maternal Z and thus prevents formation of ZZ males. Furthermore, wFem lines may have lost the W chromosome or harbour a dysfunctional version, yet rely on wFem for female development; removal of wFem results in all-male offspring. This is the first study that demonstrates an interaction between MD and Wolbachia feminization, and it highlights endosymbionts as potentially confounding factors in MD of sex chromosomes.     

Methylation differences of the neuronatin gene promoter region in liver between normal and cloned pigs

Abnormal phenotypes in cloned pigs can be partly due to changes in epigenetic modifications such as methylation levels of promoter CpG islands. Neuronatin is an imprinted gene, conserved in human, pig, cattle and mouse, which is expressed exclusively from the paternal allele. Three CpG islands located in the promoter region of the porcine neuronatin gene have the potential to regulate the gene expression by cytosine methylation. To illustrate whether neuronatin was differentially expressed among nuclear transfer macroglossia–positive and nuclear transfer macroglossia–negative pigs and in vitro‐fertilized pigs, we detected its expression level by qRT‐PCR and further quantified methylation levels by pyrosequencing DNA from the liver. The results showed that neuronatin was expressed at a significantly higher level in livers of nuclear transfer macroglossia‐positive pigs compared with normal cloned and in vitro‐fertilized pigs. Livers of nuclear transfer macroglossia‐positive pigs also had a significantly lower methylation level at CpG island 2 and CpG island 3 in the promoter region.     

The analysis of telomere length and telomerase activity in cloned pigs and cows

Inefficiency in the production of cloned animals is most likely due to epigenetic reprogramming errors after somatic cell nuclear transfer (SCNT). In order to investigate whether nuclear reprogramming restores cellular age of donor cells after SCNT, we measured telomere length and telomerase activity in cloned pigs and cattle. In normal pigs and cattle, the mean telomere length was decreased with biological aging. In cloned or transgenic cloned piglets, the mean telomere length was elongated compared to nuclear donor fetal fibroblasts and age-matched normal piglets. In cloned cattle, no increases in mean telomere length were observed compared to nuclear donor adult fibroblasts. In terms of telomerase activity, significant activity was observed in nuclear donor cells and normal tissues from adult or new-born pigs and cattle, with relatively higher activity in the porcine tissues compared to the bovine tissues. Cloned calves and piglets showed the same level of telomerase activity as their respective donor cells. In addition, no difference in telomerase activity was observed between normal and transgenic cloned piglets. However, increased telomerase activity was observed in porcine SCNT blastocysts compared to nuclear donor cells and in vitro fertilization (IVF)-derived blastocysts, suggesting that the elongation of telomere lengths observed in cloned piglets could be due to the presence of higher telomerase activity in SCNT blastocysts. In conclusion, gathering from the comparative studies with cattle, we were able to demonstrate that telomere length in cloned piglets was rebuilt or elongated with the use of cultured donor fetal fibroblasts.     

棉铃虫剂量补偿相关基因Hamsl1的鉴定与功能分析

【目的】棉铃虫Helicoverpa armigera的剂量补偿(dosage compensation, DC)分子机制尚不清楚。本研究旨在通过克隆棉铃虫雄性特异性致死(male specific lethal, msl) 基因Hamsl1,利用RNA干扰技术明确其是否参与调控棉铃虫剂量补偿。【方法】利用RT-PCR同源克隆棉铃虫Hamsl1基因全长cDNA; 利用qPCR技术研究Hamsl1基因在棉铃虫不同发育时期的表达谱;通过显微注射Hamsl1 siRNA到棉铃虫3龄幼虫中对Hamsl1基因进行RNA干扰后,利用qPCR技术检测15个Z染色体基因的表达情况,分析Hamsl1是否调控Z染色体基因剂量。【结果】成功克隆了棉铃虫Hamsl1基因的cDNA序列,鉴定出Hamsl1基因mRNA存在2种剪接体,分别命名为Hamsl1a(GenBank登录号: MK564008)和Hamsl1b(GenBank登录号: MK564009)。功能域分析发现HaMSL1含有典型的PEHE和coiled-coil功能域,具有MSL1蛋白的特征。qPCR分析表明,Hamsl1基因位于棉铃虫Z染色体上;棉铃虫Hamsl1a与Hamsl1b基因表达均具有发育时期特异性,在成虫期表达量最高,且雌雄化蛹后基因表达量差异显著,具有性别特异性。通过同源比对和qPCR分析,在DNA水平鉴定了15个Z染色体候选基因。显微注射Hamsl1 siRNA于3龄幼虫体内72 h,干扰效率为36.01%~64.27%,并未发生雄性致死现象;与对照组相比,Hamsl1 RNAi处理组中棉铃虫15个Z染色体基因在雄性个体中整体呈现表达量上调趋势,而在雌性个体中平均表达水平差异不显著。【结论】本研究初步探明Hamsl1基因位于棉铃虫Z染色体上,且该基因可能通过抑制雄性棉铃虫Z染色体基因表达,调控棉铃虫Z染色体剂量补偿。本研究为深入研究棉铃虫剂量补偿分子机制和绿色防控棉铃虫提供了理论基础。     

An unusual sex-determination system in South American field mice (Genus Akodon): the role of mutation, selection, and meiotic drive in maintaining XY females

The mechanism of sex determination in mammals appears highly conserved: the presence of a Y chromosome triggers the male developmental pathway, whereas the absence of a Y chromosome results in a default female phenotype. However, if the Y chromosome fails to initiate the male pathway (referred to as Y*), XY* females can result, as is the case in several species of South American field mice (genus Akodon). The breeding genetics in this system inherently select against the Y* chromosome such that the frequency of XY* females should decrease rapidly to very low frequencies. However, in natural populations of Akodon, XY* females persist at substantial frequencies; for example, 10% of females are XY* in A. azarae and 30% in A. boliviensis. We develop a mathematical model that considers the potential roles of three evolutionary forces in maintaining XY* females: Y-to-Y* chromosome transitions (mutation), chromosome segregation distortion (meiotic drive), and differential fecundity (selection). We then test the predictions of our model using data from breeding colonies of A. azarae. We conclude that any single force is inadequate to maintain XY* females. However, a combination of segregation bias of the male and female Y chromosomes during spermatogenesis/oogenesis and increased fecundity in XY* females could account for the observed frequencies of XY* females.