Second-division Segregation Research Articles

Amplified fragment length polymorphism analysis to assess crossover interference and homozygosity in gynogenetic diploid Pacific abalone (Haliotis discus hannai)

Recombination analysis in gynogenetic diploids is a powerful tool for assessing the degree of inbreeding, investigating crossover events and understanding chiasma interference during meiosis. To estimate the marker-centromere recombination rate, the inheritance pattern of 654 amplified fragment length polymorphism (AFLP) markers was examined in the 72-h veliger larvae of two meiogynogenetic diploid families in the Pacific abalone (Haliotis discus hannai). The second-division segregation frequency (y) of the AFLP loci ranged from 0.00 to 0.96, with 23.9% of loci showing y-values higher than 0.67, evidencing the existence of interference. The average recombination frequency across the 654 AFLP loci was 0.45, allowing estimation of the fixation index of 0.55, indicating that meiotic gynogenesis could provide an effective means of rapid inbreeding in the Pacific abalone. The AFLP loci have a small proportion (4.4%) of y-values greater than 0.90, suggesting that a relatively low or intermediate degree of chiasma interference occurred in the abalone chromosomes. The information obtained in this study will enhance our understanding of the abalone genome and will be useful for genetic studies in the species.

Efficient tools to target DNA to Podospora anserina

Because, in filamentous fungi, integration of transgenes proceeds mainly through nonhomologous recombination, transformation results in a random distribution of these DNA sequences. Moreover, they are often found as multiple copies clustered at a single locus. Therefore, for a given transgene, expression may be highly variable among independent transformants. To get rid of both multiple copies and position effects, due to the impact of the chromatin structure upon transgene expression, we constructed tools to specifically target single copies at two well-defined loci, Pa_2_3690 and Pa_4_5450 (Espagne et al., 2008). These genes have been chosen for the following reasons: (1) they have a relatively stable expression during the entire Podospora’s life cycle, (2) complete deletion of their ORF does not lead to an abnormal phenotype (Bidard et al., 2011), (3) they are located on two different chromosomes and (4) their second division segregation (SDS) frequency is 80% and 90%, respectively. Besides, a highly efficient homologous recombination ΔPaKu70 strain is available (El-Khoury et al., 2008), which makes gene replacement fairly easy. Taking advantage of this strain, we designed two plasmid tools to target genomic integration of any DNA fragment.

Microsatellite-centromere mapping in walking catfish Clarias macrocephalus (Günther, 1864) using gynogenetic diploids

Thirty new microsatellite loci were isolated from a microsatellite-enriched genomic library of walking catfish Clarias macrocephalus. The CA motif was the most abundant, although several other motifs were also isolated. Two gynogenetic diploid families, each consisting of a female and 50 offspring, were genotyped at 56 microsatellite loci, including 30 loci developed in the present study and 26 loci reported in the previous study. Overall, 33 anonymous microsatellites derived from genomic library and 11 EST-linked microsatellites were mapped to their centromeres. High levels of chiasma interference were apparent in the walking catfish genome as indicated by an average frequency of second-division segregation (y) of 0.643 ± 0.248. Twenty-six loci (59%) showed a high microsatellite–centromere recombination, with a frequency >0.67, and three loci had recombination frequencies >0.9. This study demonstrated that gene–centromere mapping provided a rapid method for the expansion of the initial linkage map of walking catfish.

Allelic recombination in the his-I locus of Neurospora crassa in isolates showing high and low frequency of intergenic recombination

From lines selected for high and low frequency of second division segregation (SDS) for asco in linkage group (LG) VI, one wild-type isolate of mating type (mt) A was picked out from the high and the low selection line, respectively. Both isolates were chosen on the basis of showing extreme SDS-frequencies, and both belonged to the last generation of selection. The two wild-type isolates were crossed with a strain carrying marker genes suitable for the analysis of allelic recombination in the his-I locus in LG V. The progeny of mt a was analysed for the influence of high and low line background on allelic recombination. The frequency of his-I prototrophs seems to be related to the high and low line background of the isolates. The two backgrounds showed no different influence on the recombination of markers flanking the his-I locus among his-I prototrophs. Variation of the frequency of his-I prototrophs not attributable to the rec-I locus is suggested. The data are interpreted according to Holliday's model of genetic recombination.

Different effects of temperature on recombination in lines of Neurospora crassa selected for high and for low recombination frequency

Wild-type isolates of Neurospora crassa, picked out from lines selected for high and for low frequency of second division segregation (SDS) at 25oC for asco in linkage group (LG) VI, were scored for temperature effects on SDS-frequency for asco and on recombination frequencies in two adjacent intervals in LG II. Results obtained at 25oC and 17oC were compared. In the high selection line, the SDS-frequency for asco in both mating types (mt:s) decreased at 17oC compared to results obtained at 25oC. In the low selection line the SDS-frequency for asco in mt A increased at 17oC compared to results obtained at 25oC, while no significant effect of temperature on SDS-frequency for asco could be established in mt a. In LG II, temperature effects on recombination frequencies could only be established in the interval spanning the centromere, in one of the crosses involving an isolate from the high selection line. The recombination frequency increased at 17oC compared to results obtained at 25oC. The results are interpreted in terms of varying access in different genotypes, to gene products promoting and obstructing meiotic recombination, respectively.

Recombination in other chromosomal regions than the interval subjected to selection, in lines of Neurospora crassa selected for high and for low recombination frequency

Recombination studies involving regions of the linkage groups (LG:s) I, II, III, IV, V and VI were performed in wild-type isolates of Neurospora crassa picked out from lines selected for high and for low frequency of second division segregation (SDS) for asco in the left arm of LG VI. Significant differences in recombination frequencies between high and low selection lines were obtained when proximal regions in the LG:s I, IV, V and the left arm of LG VI were scored, while no difference between lines could be established in the regions located in the LG:s II, III and in the right arm of LG VI. The correlation between SDS-frequencies for asco obtained with different asco tester strains was much stronger than the correlation between SDS-frequencies for asco and the recombination frequency in any of the other regions tested. This suggests that some of the genetic factors separating the recombination frequencies of the two selection lines operate mainly within the asco-centromere interval.

Variation of high and low recombination frequencies in Neurospora crassa owing to genetic and environmental conditions

From lines selected for high and low frequency of second division segregation (SDS) for asco in linkage group (LG) VI, one wild-type isolate of mating type (mt) a showing extreme SDS-frequency was picked out from the high and the low selection line, respectively. Both wild types were crossed with a strain carrying a met-8 (LG III) and a his-1 (LG V) allele. The met-8′ progeny of mt A was analysed with reference to SDS-frequency for asco. As all crosses were performed in repeats, the influence of environmental conditions could be analysed. No significant influence of the regions surrounding the his-I locus could be established, neither for high, nor for low SDS-frequencies. Environmental conditions significantly influenced the SDS-frequencies of isolates which had a high line background. The isolates with a low line background were less affected in this respect. The SDS-frequencies of isolates with a low line background showed a bimodal-like distribution. This suggests the influence of a major genetic factor. If this is located in any of the LG:s I, III and V, it would not be closely linked to the mt, the met-8 or the his-I locus.

Ascospores of large-spored Metschnikowia species are genuine meiotic products of these yeasts.

The asci of Metschnikowia species normally contain two ascospores (never more), raising the question of whether these spores are true meiotic products. We investigated this problem by crossing genetically-marked strains of the haploid, heterothallic taxa Metschnikowia hawaiiensis, Metschnikowia continentalis var. continentalis, and M. continentalis var. borealis. Asci were dissected and the segregation patterns for various phenotypes analyzed. In all cases (n = 47) both mating types (h+ and h-) were recovered in pairs of sister spores, casting further uncertainty as to whether normal meiosis takes place. However, the segregation patterns for cycloheximide resistance and several auxotrophic markers were random, suggesting that normal meiosis indeed occurs. To explain the lack of second-division segregation of mating types, we concluded that crossing-over does not occur between the mating-type locus and the centromere, and that meiosis I is tied to spore formation, which explains why the number of spores is limited to two. The latter assumption was also supported by fluorescence microscopy. The second meiotic division takes place inside the spores and is followed by the resorption of two nuclei, one in each spore.

Ascospores of large-spored Metschnikowia species are genuine meiotic products of these yeasts

The asci of Metschnikowia species normally contain two ascospores (never more), raising the question of whether these spores are true meiotic products. We investigated this problem by crossing genetically-marked strains of the haploid, heterothallic taxa Metschnikowia hawaiiensis, Metschnikowia continentalis var. continentalis, and M. continentalis var. borealis. Asci were dissected and the segregation patterns for various phenotypes analyzed. In all cases (n=47) both mating types (h+ and h−) were recovered in pairs of sister spores, casting further uncertainty as to whether normal meiosis takes place. However, the segregation patterns for cycloheximide resistance and several auxotrophic markers were random, suggesting that normal meiosis indeed occurs. To explain the lack of second-division segregation of mating types, we concluded that crossing-over does not occur between the mating-type locus and the centromere, and that meiosis I is tied to spore formation, which explains why the number of spores is limited to two. The latter assumption was also supported by fluorescence microscopy. The second meiotic division takes place inside the spores and is followed by the resorption of two nuclei, one in each spore.

Suppressed recombination and a pairing anomaly on the mating-type chromosome of Neurospora tetrasperma.

Neurospora crassa and related heterothallic ascomycetes produce eight homokaryotic self-sterile ascospores per ascus. In contrast, asci of N. tetrasperma contain four self-fertile ascospores each with nuclei of both mating types (matA and mata). The self-fertile ascospores of N. tetrasperma result from first-division segregation of mating type and nuclear spindle overlap at the second meiotic division and at a subsequent mitotic division. Recently, Merino et al. presented population-genetic evidence that crossing over is suppressed on the mating-type chromosome of N. tetrasperma, thereby preventing second-division segregation of mating type and the formation of self-sterile ascospores. The present study experimentally confirmed suppressed crossing over for a large segment of the mating-type chromosome by examining segregation of markers in crosses of wild strains. Surprisingly, our study also revealed a region on the far left arm where recombination is obligatory. In cytological studies, we demonstrated that suppressed recombination correlates with an extensive unpaired region at pachytene. Taken together, these results suggest an unpaired region adjacent to one or more paired regions, analogous to the nonpairing and pseudoautosomal regions of animal sex chromosomes. The observed pairing and obligate crossover likely reflect mechanisms to ensure chromosome disjunction.

The fungal genetic system: A historical overview

J. Goner., Vol. 75, Number 3, December 1996, pp. 245 - 253. O Indian Academy of Sciences The fungai genetic system: a historical overview ROWLAND H. DAVIS Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697-3900, USA The early years The genetics of filamentous fungi was initiated through the efforts of rather few people, whose work led to the explosive development of the genetics and biochemistry of Neurospora crassa and Aspergitlus nidulans in the 1940s and 1950s. Among the major contributors is David Perkins, whom this issue of Journal of Genetics honours, and who continues to this day to be an international resource of new knowledge about N. crassa. This article is biased towards Neurospora, in keeping with its intent to honour David and with the focus of many of the articles to follow. I have chosen a historical theme, leaving to others the task of illuminating the present. Neurospora became part of a continuous line of organisms underlying the twentieth- century revolution in biology. Many of the interests and traditions of geneticists working with Drosophila, mouse and corn were carried over to N. crassa, together with a new ambition to understand the relationship between genes and enzymes. It is easy for today's student to forget that work on Neurospora genetics and biochemistry preceded the modern development of these areas in Escherichia coli and yeast. In fact, E. coli genetics originated in the laboratory of E. L. Tatum, already a pioneer in Neurospora. The clarification of the sexual life histories of N. crassa, N. sirophila and N. tetrasperma by B. O. Dodge (Shear and Dodge 1927) and his advocacy of the organism to others sparked the interest of T. H. Morgan. Morgan accepted the idea that Neurospora might be a useful experimental organism for genetic work. Morgan urged Neurospora upon Lindegren, who did the first detailed genetic studies on N. crassa, confirmed the observations by Dodge on second-division segregation, and demonstrated linkage (Perkins 1992). He thus began to realize the potential of this organism for other studies in which genetics might play a role. In a brief letter to Dodge in 1941, Beadle asked for N. crassa cultures, and with astonishing speed, he and Tatum published their first paper on the biochemical genetics of N. crassa later that year (Beadle and Tatum 1941). Work on the genetics, biology and bio- chemistry of N. crassa was soon extended to A. nidulans, with its parasexual mode of recombination. This mechanism was then quite novel, and it broadened the defini- tion of sexuality to a point that it could include E. coli and bacteriophage as well. However, before and during the time of Dodge's studies with Neurospora, another tradition was developing elsewhere. Early studies on basidiomycetes by Kniep, Buller and others had revealed a multifactorial system of mating types, some of the properties of which were analysed correctly through tetrad analysis (Raper 1966). The two ascomycetes and several basidiomycetes dominated development of the field of fungal genetics.

Segregation analyses and gene-centromere distances in zebrafish.

The gol-1, gol-2, alb-1 and spa-1 mutations affect pigment pattern in the zebrafish. We show here that these loci are unlinked to each other. In addition, gene-centromere distances were determined for these loci by analysis of half-tetrads obtained by the inhibition of the second meiotic division. The fractions of tetratype (second-division segregation) tetrads range from 0.24 (spa-1) to 0.89 (gol-1). The observation of greater than 0.67 second-division segregation indicates that the zebrafish has high chiasma interference.

Dominant enhancer effect of the meiotic mei4 mutant on recombination frequencies restricted to linkage group VI in Podospora anserina

A mutant which increases second division segregation (SDS) frequency of locus 110 (linkage group VI) was isolated. It was called mei4 because of its meiotic deficiency. The present paper deals with its effect on meiotic recombination when heterozygous. mei4 then only acts on linkage group VI. The SDS frequencies were increased for all markers used, except locus 5 located very close to the centromere. This quasi general enhancement results exclusively in an enlargement of map distance on linkage group VI's proximal part. Crosses involving three mutant genes allowed to check that the distances on the distal part were constant. This is due to a real lack of crossover frequency modification in this region and not to a change ofchiasma interference. Among the seven linkage groups of Podospora anserina, group VI exhibits several other particularities concerning meiotic recombination, especially a lower positive chiasma interference and a more regular crossover distribution, suggesting a particular recombination regulation.

Crossing-over across the centromere in the mating-type chromosome of Neurospora crassa.

The frequency of crossing-over in the two short regions on opposite arms, adjacent to the centromere of the mating-type chromosome ofNeurospora crassa is controlled, independently in each arm, by at least two genes with equal and additive effect. These genes segregate on inbreeding and cause great variability in both the frequency of recombination and the frequency of second-division segregation of loci situated within these regions. Recombination values between loci situated beyond these sensitive regions is not affected; the relative increase or decrease in their centromere distances may be attributed to change in the recombination frequency about the centromere only.

Unbiased second-division segregation in Neurospora.

Results of crosses of a large number of Neurosporas of different origin, including several distinct strains ofN. sitophila, were utilized to re-examine the question of whether certain wild-type Neurosporas other thanN. crassashow biases in the two types of second-division segregation. Segregations for alleles of the mating type andpeakloci on a wide variety of genetic backgrounds gave little evidence for excess either of symmetrical or asymmetrical ‘post-reduction’ asci.

Circular genetic maps.

The widespread occurrence of genetic circularity suggests a selective advantage to map circularity per se. Circularity permits gene clustering relations not possible in linear maps; that is, every gene in a circular map can have two nearest map neighbors, two next nearest, etc. It seems possible that map circularity is a consequence of the selective forces responsible for the clustering of genes of related function. Certain features of the pattern of crossing-over in various fungi suggest that circularity of linkage maps is there to be found. A concurrence of high frequencies of second-division segregation and negative chromatid interference across the centromere, both of which phenomena have been reported, is a feature of a simple hypothetical crossover pattern that generates circular linkage maps.

The differential maturation of asci and its relevance to recombination studies ofNeurospora, Sordariaand similar Ascomycetes

Four ascospore-pigmentation mutants were crossed with their wild-types by methods giving a considerable degree of synchrony in perithecial development; segregation patterns were scored from perithecia at different stages of maturity. The observed second-division segregation frequencies forascoinN. crassawere at a maximum when little or no ascal dehiscence had occurred but decreased markedly as dehiscence proceeded. No significant change in this frequency with maturity occurred fortan sporeinNeurosporabut inS. fimicolathe observed second-division segregation frequencies forgrayandhyalinerose with increasing perithecial maturity. As similar changes in this frequency were observed when asci were mounted in water instead of the 2msucrose solution normally used, it was concluded that changes in this frequency with maturity generally resulted from a differential maturation and bursting of asci with different spore arrangements, rather than from changes in crossover frequency in successive meioses. Some evidence of the latter phenomenon was found in two of theSordariacrosses.In a cross ofasco×rib-1 inNeurospora, the observed frequency of recombination between the two loci was significantly higher in dispersed spores collected from young perithecia than in those from more mature perithecia: this effect was probably an artifact resulting from the differential maturation and bursting of asci with different divisions of segregation forasco. In a cross ofcrisp× + inNeurospora, an excess ofcrispprogeny was obtained from dispersed spores and an excess of +-progeny from spores from harvested perithecia. These deviations from the expected 1:1 ratio forcr: + were thought to result from a differential maturation of asci with different segregation patterns forcrispand the failure of many dehiscing asci to expel all eight spores.Hypotheses to explain the various phenomena observed were discussed. Suggestions were made concerning the avoidance of bias from the differential maturation and bursting of asci in experimental procedures used in the study of recombination and gene conversion. This bias may be responsible for some phenomena previously attributed to events at meiosis.

Mapping chromosome centromeres from tetratype frequencies

1. If data on tetratype frequencies refer to unlinked pairs of loci, even though the loci are not necessarily all unlinked, then centromeres can be mapped if the data include an odd-numbered closed set (or cycle) of frequencies (e.g. frequencies known fora andb,b andc, c anda, wherea,b andc are mutant genes). Formulae are given for calculating second-division segregation frequencies and their standard errors from such odd-numbered cycles. 2. If unlinked tetratype data include only an even-numbered cycle (e.g. data fora andb,b andc,c andd,d anda, whered is a fourth gene), then second-division segregation frequencies cannot be calculated, but a relationship exists between the tetratype frequencies. It can be used to test the homogeneity of such data. 3. If one of the tetratype frequencies refers to a linked pair of loci, centromeres can be mapped provided the frequencies form a cycle of at least three values, a.nd provided at least one of the linked genes is situated near to the centromere of its chromosome. Formulae are given for calculating second-division segregation frequencies from cycles of three and four loci, when one pair of loci show linkage.

Use of Tetratype Frequencies for estimating Spindle Overlapping at the Second Division of Meiosis in ‘Ordered’ Tetrads

WITH ordered tetrads, such as occur in the asci of Neurospora crassa, the second-division meiotic spindles do not overlap (Fig. 1a) and the subsequent mitotic spindles are also well separated, with the result that analysis of the sequence of spores within the ascus allows a distinction to be made between first- and second-division segregation for a particular pair of alleles. With unordered tetrads, second-division segregation frequencies can be calculated from the frequencies of tetratype tetrads (for example, AB, ab, Ab, aB) for three independent loci, A/a, B/b and C/c, considered in pairs (A and B, B and C, C and A)1. When there is uncertainty whether the tetrads of a particular organism are ordered or not, use can be made of tetratype frequencies to estimate the frequencies of partial and complete spindle overlapping at the second division of meiosis. It is important to recognize such overlapping, since it may lead to inaccurate mapping of the centromeres, and, moreover, partial spindle overlap may mimic the effects of two-strand double cross-overs across the centromere. Thus the excess of such cross-overs found by Lindegren and Lindegren2 in N. crassa has been attributed3 to spindle overlapping, although there is good genetic evidence that such overlapping is not responsible4.

Map Construction in Neurospora Crassa

Publisher Summary In this chapter linkage and centromere data from neurospora crassa have been compiled from all published material, as well as unpublished sources. Tetrad data from gene–centromere and gene–gene intervals have been placed on a uniform basis for mapping by computing map lengths from second-division segregation frequencies and tetratype segregation frequencies, respectively. Maps of the seven linkage groups have been constructed from tetrad data. Seventy-five loci are shown. Confidence limits are indicated for the position of each locus. Two sets of maps are presented, the first is completely uncorrected for multiple crossovers and the second is corrected by means of the mapping function. A few additional genes have been assigned to specific linkage groups on the basis of random isolates. The use of random isolates for mapping is also discussed.