Wild-isolated Neurospora crassa strains that increase fertility of crosses with segmental aneuploids used to establish that a large duplication suppresses RIP in a smaller duplication

Crosses involving strains bearing large segmental duplications are characteristically barren and produce only very few viable ascospores. We report here that the productivity of duplication x euploid crosses can be influenced by the euploid parent. The yield of ascospores was exceptionally low in crosses with the wild-isolated strains Golikro (FGSC# 4830) and Costa Rica (FGSC# 852) and exceptionally high in crosses with Lahore-1 (FGSC #1824), Dagguluru-1 (FGSC #3360), Okeechobee (FGSC # 3968), and Tiassale (FGSC # 4825). The four strains that increased productivity were used in crosses with strains bearing the duplication Dp(IBj5) together with a small (1.3 kb) duplication of the erg-3 gene. This made it possible to obtain sufficient numbers of progeny to establish that presence of Dp(IBj5) suppresses RIP in erg-3 .

Wild-isolated Neurospora crassa strains that increase fertility of crosses with segmental aneuploids used to establish that a large duplication suppresses RIP in a smaller duplication Mario Fehmer, Ashwin Bhat, Felicite K. Noubissi, and Durgadas P. Kasbekar.Centre for Cellular and Molecular Biology, Hyderabad 500 007, India.
Crosses involving strains bearing large segmental duplications are characteristically barren and produce only very few viable ascospores.We report here that the productivity of duplication x euploid crosses can be influenced by the euploid parent.The yield of ascospores was exceptionally low in crosses with the wild-isolated strains Golikro (FGSC# 4830) and Costa Rica (FGSC# 852) and exceptionally high in crosses with Lahore-1 (FGSC #1824), Dagguluru-1 (FGSC #3360), Okeechobee (FGSC # 3968), and Tiassale (FGSC # 4825).The four strains that increased productivity were used in crosses with strains bearing the duplication Dp(IBj5) together with a small (1.3 kb) duplication of the erg-3 gene.This made it possible to obtain sufficient numbers of progeny to establish that presence of Dp(IBj5) suppresses RIP in erg-3 .

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Neurospora strains bearing large (e.g., >100 kb) chromosomal segment duplications (segmental aneuploids) can be obtained as segregants from crosses heterozygous for some insertional or quasiterminal translocations (reviewed in Perkins 1997 Adv. Genet. 36: 239-398).Sexual crosses made with segmental aneuploid strains display a characteristic "barren" phenotype; they form perithecia but only a few viable ascospores.Although the barren phenotype is useful in signalling the presence of segmental aneuploidy, it makes subsequent genetic analyses very tedious because of the difficulty in obtaining sufficient numbers of progeny.Therefore a need was felt to identify euploid strains that might increase ascospore yields in crosses with segmental aneuploid strains.
A recent study involving crosses between Dp(AR17) duplication strains and eight wild-isolated strains revealed that crosses with two wild strains, Golikro (FGSC# 4830) and Costa Rica (FGSC# 852), produced significantly (approx 100x) fewer ascospores (Noubissi et al. 2000 Fungal Genet. Biol. 31: 91-97).Results summarized in Table 1 show this is true even in crosses with two other duplication strains, Dp(OY329) and Dp(S1229) and suggested that the productivity of barren crosses can be influenced by the euploid parent.This motivated us to examine wild-isolated strains for any that increased ascospore production in otherwise barren crosses.

Dp(IBj5) suppresses RIP in Dp1.3 ec hph:
We wanted to test whether Dp(IBj5) also suppresses RIP in a small duplication.Dp( IBj5) is an insertional duplication on IR of a VIL segment (from cpc-1 through ylo-1) and strains bearing this duplication can be obtained as segregants from crosses of the translocation strain T(VIL > IR) IBj5 cpc-1 mat A (FGSC #443) with normal sequence strains.The small duplication was represented by Dp 1.3 ec hph, which is comprised of a 1.3 kb fragment of erg-3 that is marked with the hph gene for hygromycinresistance, and targets RIP to the erg-3 locus on LGIIIR (Prakash et al., 1999Microbiology 145: 1443-1451).Colonies generated from erg-3 mutant ascospores have a characteristic morphology on Vogel's-sorbose agar medium and are easily scorable under a dissection microscope (Noubissi et al. 2000 Fungal Genet. Biol. 31: 91-97).The IBj5 translocation strain was crossed with a Dp 1.3 ec hph strain to generate Dp 1.3 ec hph ; Dp(IBj5) segregants.Crosses of these segregants with the standard laboratory wild type strains 74-OR23-1 mat A or OR8-1 mat a were so severely barren that despite repeated attempts we could not obtain sufficient numbers of progeny to test whether Dp(IBj5) suppresses RIP in Dp 1.3 ec hph.Therefore we resorted to crossing a Dp 1.3 ec hph ; Dp(IBj5) mat a segregant with the Lahore-1, Dagguluru-1, Okeechobee and Tiassale strains.In a single attempt we recovered, respectively, 182, >2500, 81 and 34 progeny from these crosses.None of these progeny were mutant in erg-3.Combining the results for the crosses with the Lahore-1, Okeechobee and Tiassale strains, the erg-3 mutation frequency is estimated to be less than 1/297 (i.e., < 0.3%).We examined 1003 progeny from the cross with Dagguluru-1, thus the erg-3 mutation frequency in this cross was less than 1/1003 (i.e., < 0.1%).These values are much lower than the erg-3 mutation frequencies in crosses of these four wild-isolates with a Dp 1.3 ec hph mat a strain (Noubissi et al. 2000 Fungal Genet. Biol. 31: 91-97) and therefore allow us to conclude that the IBj5 duplication, like the AR17, OY329 and S1229 duplications, suppresses RIP in the smaller duplication Dp 1.3 ec hph.
AB was supported by a CSIR-UGC Junior Research Fellowship, FKN by the Third World Organization of Women in Science.
Published by New Prairie Press, 2017