Neurospora heterokaryons with complementary duplications and deficiencies in their constituent nuclei provide an approach to identify nucleus-limited genes

Abstract

Introgression is the transfer of genes or genomic regions from one species into another via hybridization and back-crosses. We have introgressed four translocations (EB4, IBj5, UK14-1, and B362i) from Neurospora crassa into N. tetrasperma. This enabled us to construct two general types of heterokaryons with mat-A and mat-a nuclei of different genotypes: one type is [T + N] (with one translocation nucleus and one normal sequence nucleus), and the other is [Dp + Df] (with one nucleus carrying a duplication of the translocation region and the other being deleted for the translocation region). Self-crossing these heterokaryons again produced [T + N] and [Dp + Df] progeny. From conidia (vegetative spores) produced by the heterokaryotic mycelia, we obtained self-fertile (heterokaryotic) and self-sterile (homokaryotic) derivative strains. [T + N] heterokaryons produced homokaryotic conidial derivatives of both mating types, but [Dp + Df] heterokaryons produced viable conidial homokaryons of only the mating type of the Dp nucleus. All four [T + N] heterokaryons and three [Dp + Df] heterokaryons produced both self-sterile and self-fertile conidial derivatives, but the [Dp(B362i) + Df(B362i)] heterokaryons produced only self-sterile ones. Conceivably, the Df(B362i) nuclei may be deleted for a nucleus-limited gene required for efficient mitosis or nuclear division, and whose deficit is not complemented by the neighboring Dp(B362i) nuclei. A cross involving Dp(EB4) showed repeat-induced point mutation (RIP). Because RIP can occur in self-crosses of [Dp + Df] but not [T + N] heterokaryons, RIP alteration of a translocated segment would depend on the relative numbers of [Dp + Df] vs. [T + N] ancestors.

TheNeurospora crassastandard Oak Ridge background exhibits an atypically efficient meiotic silencing by unpaired DNA

“…The results of crosses with the ::r1 OR and ::r1 B/S1 strains are presented in Table S2, and those with ::r3 OR and ::r3 B/S1 are presented in Table 1. tetrasperma to construct the strains T(EB4) Nt and T(IBj5) Nt , and from the T Nt x 85 crosses we obtained self-fertile dikaryotic [T Nt + N] and [Dp Nt + Df Nt ] strains (Giri et al, 2015). From the dikaryons we obtained homokaryotic conidial derivatives of genotype T(EB4) Nt a, T(IBj5) Nt a, Dp(EB4) Nt A and Dp(IBj5) Nt a and used them to make the crosses whose results are presented in Table 3.…”

“…We have now introgressed an ::r transgene from N. crassa OR into N. tetrasperma 85, and the resulting ::r Nt testers were used to make ::r Nt x 85 crosses. Additionally, by introgressing the N. crassa T(EB4) and T(IBj5) translocations into N. tetrasperma we had previously obtained T(EB4) Nt , T(IBj5) Nt , Dp(EB4) Nt and Dp(IBj5) Nt strains in which nominally only the rearrangement breakpoints were derived from N. crassa while the rest of the genome was derived from N. tetrasperma strain 85 (Giri et al, 2015). These strains enabled us to ask whether Dp-heterozygous crosses in N. tetrasperma are barren like their N. crassa counterparts.…”

A novel meiotic drive in Neurospora crosses heterozygous for hybrid translocation strains disfavors homokaryotic progeny derived from alternate, but not adjacent-1 segregation

“…Recently, we introgressed four insertional translocations (IT; EB4, IBj5, UK14-1, and B362i) from Neurospora crassa into N. tetrasperma, to make heterokaryons with complementary duplications and deficiencies in their constituent nuclei ([Dp + Df]; Giri et al, 2015). Construction of the [Dp + Df] heterokaryon relied on (1) the difference in N. crassa and N. tetrasperma ascogenesis, and (2) adjacent-1 segregation in IT-heterozygous crosses.…”

“…In contrast, isosequential crosses (i.e., N x N or T x T) produce mostly 8B:0W asci (Perkins 1997). In RT x N, all eight ascospores from ADJ have complementary Dp/Df (i.e., Dp2/Df1 and Dp1/Df2), and the asci are 0B:8W, thus 8B:0W = 0B:8W is characteristic of RT x N. ALT and ADJ in IT (and QT) x N crosses in N. tetrasperma, generate 4B:0W asci with, respectively, [T + N] and [Dp + Df] ascospores (Giri et al, 2015). The two genotypes can interconvert via self-crosses.…”

“…The RIP mutational process, targeting duplicated DNA (Selker 1990), can occur in self-crosses of [Dp + Df] but not [T + N]. Crosses of T Nt strains with N. tetrasperma strains 85 A or 85 a produced mostly four-spored asci, but the rare eight-spored asci included 8B:0W, 4B:4W, and 6B:2W types (Giri et al, 2015). More eight-spored asci were produced in crosses of T Nt with E. The crosses of T(EB4) Nt and T(UK14-1) Nt with E produced comparable numbers of 8B:0W and 4B:4W asci, and also 6B:2W.…”