New multicent linkage testers for centromere-linked genes and rearrangements in Neurospora

A tester described in 1972 contained readily-scored markers near the centromeres of all seven linkage groups (Perkins, Neurospora Newsl. 19:33). That tester, now called multicent-1, was somewhat more laborious to score than alcoy (Perkins et al. 1969 Genetica 40:247-278) or its successor alcoy;csp-2 (Perkins and Björkman 1979 Neurospora Newsl. 26:9-10). multicent-1 had the advantage over alcoy of requiring no follow-up cross to distinguish alternatives, and it was somewhat more effective in detecting linkage of left-arm markers. Because alcoy itself contains three translocations, multicent-1 was more likely to identify the chromosomes involved in new centromere-linked translocations (Perkins and Barry 1977 Adv. Genet. 19:133-285). Creative Commons License This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. This regular paper is available in Fungal Genetics Reports: http://newprairiepress.org/fgr/vol37/iss1/19 New multicent linkage testers for centromere-linked genes and rearrangements in Neurospora D.D. Perkins Department of Biological Sciences, Stanford University, Stanford CA 94305-5020 A tester described in 1972 contained readily-scored markers near the centromeres of all seven linkage groups (Perkins, Neurospora Newsl. 19:33). That tester, now called multicent-1, was somewhat more laborious to score than alcoy (Perkins et al. 1969 Genetica 40:247-278) or its successor alcoy;csp-2 (Perkins and Björkman 1979 Neurospora Newsl. 26:9-10). multicent-1 had the advantage over alcoy of requiring no follow-up cross to distinguish alternatives, and it was somewhat more effective in detecting linkage of left-arm markers. Because alcoy itself contains three translocations, multicent-1 was more likely to identify the chromosomes involved in new centromere-linked translocations (Perkins and Barry 1977 Adv. Genet. 19:133-285). One disadvantage with multicent-1 was the marker balloon, whose restricted colonial growth meant that most of the other markers could be scored readily only in the bal+ half of the progeny. bal was also inconvenient for fertilization and stock preservation. We have therefore developed three new multicent testers. These are designated multicent-3 to -5 (Table I). Independently, Metzenberg et al. (1984 Neurospora Newsl. 31:35-39) devised a strain designated multicent-2, with six centromeres marked. This has been used for mapping cloned DNA fragments. Table I. Constitution of multicent testers 1 through 5 Strain Designation I II III IV V VI VII multicent-1 A or a bal acr-2 pdx at ylo-1 wc-1 multicent-2 un-2 a arg-5 thi-4 pyr-1 lys-1 ars-1 inl nic-3 multicent-3 In(IL;IR)OY323 arg-5 acr-2 pdx at ylo-1 wc-1 A or a multicent-4 A or a arg-5 acr-2 psi at ylo-1 wc-1 multicent-5 In(IL;IR)OY323 arg-5 acr-2 psi at ylo-1 wc-1 A or a FGSC Numbers helper* A a A a multicent-1 2014 2015 multicent-2 4488 multicent-3 6824 6825 6826 6827 multicent-4 6828 6829 6830 6831 multicent-5 6832 6833 6834 6835 See FGSC stock list for allele numbers of markers. Published by New Prairie Press, 2017 * In heterokaryon with inactive mating helper strain am1 ad-3B cyh-1, FGSC 4564 (Perkins 1984 Neurospora Newsl. 31:41-42. The phenotypically wild type heterokaryon can be used as femal parent on minimal medium, but the am1 ad-3B cyh-1 component does not participate in the cross The multicent-3 tester incorporates two changes. arg-5 is substituted for bal in linkage group II, and a long inversion, OY323, has been inserted as a crossover-suppressor in linkage group I. Progeny are isolated either to complete medium or to minimal supplemented with arginine and pyridoxine. As a result of the heterozygous inversion, mating type shows little or no recombination with markers throughout two-thirds of I, which is the longest linkage group. In multicent-4 also, arg-5 is substituted for bal. In addition, psi replaces pdx as a marker for IV. psi (protein-synthesis-inhibited) is a readily-scored temperature-sensitive conditional mutant that grows normally on minimal medium at 25°C but does not grow at 34°. Ascospores must be germinated at 25° because psi progeny do not survive at the restrictive temperature. Replacing pdx with psi means that only two media are required. arg-5 is scored by transferring progeny to minimal medium at 25°, psi by transfer to arginine-supplemented minimal at 34°. multicent-5 differs from multicent-4 only in having the OY323 inversion present in linkage group I. multicent-5 is preferred for mapping mutants that are scorable by vegetative phenotype. As with the other testers, unmapped translocations can be recognized by linkage between markers that are normally independent. If it should be desired to determine marker-breakpoint linkage by scoring for aborted ascospores in progeny tests, it is possible to score the new rearrangement only in the noninversion half of the progeny; these are recognized as noninversion because they are of the same mating type as the noninversion parent. Scoring for the other markers is as follows: Tests for mating type are most readily accomplished on fluffy lawns in petri dishes (Perkins et al. 1989 Fungal Genet. Newsl. 36:64-66). at (attenuated morphology) is readily scorable on minimal (with or without supplements) at two or three days (34°C). Growth is flat on the surface, with scattered specks of conidiation. wc-1 (white collar) is expressed most clearly at 25°C or higher. Carotenoids are absent in mycelia, though not in conidia. Germinants are incubated until conidia become orange, preferably under illumination. Scoring of ylo-1 (yellow) improves with age, and is likely to be unreliable with young cultures, especially in combination with wc. Carotenoids in ylo-1 look orange at first, then become yellow. ylo-1 scoring at three or four days should be considered preliminary, and should be checked later. acr-2 is scored clearly by resistance to 50 μg acriflavine/ml on solid medium. With any of the testers, progeny are scored for markers sequentially beginning with the visible markers at, wc-1, and ylo-1. If linkage is apparent, the remaining markers need not be tested. If the unmapped mutant is unlinked to a visible marker, the markers that require transfer are then tested serially until linkage is seen. With translocations, the normally independent multicent markers are examined for linkages to one another. The new multicent testers are heterokaryon-compatible with strains of OR background (het-c het-d; het-e). All three are available as heterokaryons in combination with the inactive-matingtype "helper" strain am1 ad-3B cyh-1 (Table I). The heterokaryons are phenotypically wild-type http://newprairiepress.org/fgr/vol37/iss1/19 DOI: 10.4148/1941-4765.1484 and highly fertile. Although the homokaryotic testers can be used satisfactorily for crossing, using a heterokaryon as female parent saves labor by making it unnecessary to supplement the crossing medium. Fertility may also be improved when the heterokaryon is used. Published by New Prairie Press, 2017

New multicent linkage testers for centromere-linked genes and rearrangements in Neurospora Stanford University, A tester described in 1972 contained readily-scored markers near the centromeres of all seven linkage groups (Perkins,Neurospora Newsl. 19:33).That tester, now called multicent-1, was somewhat more laborious to score than alcoy (Perkins et al. 1969 Genetica 40:247-278) or its successor alcoy; csp-2 (Perkins and Björkman 1979 Neurospora Newsl. 26:9-10).multicent-1 had the advantage over alcoy of requiring no follow-up cross to distinguish alternatives, and it was somewhat more effective in detecting linkage of left-arm markers.Because alcoy itself contains three translocations, multicent-1 was more likely to identify the chromosomes involved in new centromere-linked translocations (Perkins and Barry 1977 Adv. Genet. 19:133-285).
One disadvantage with multicent-1 was the marker balloon, whose restricted colonial growth meant that most of the other markers could be scored readily only in the bal+ half of the progeny.bal was also inconvenient for fertilization and stock preservation.We have therefore developed three new multicent testers.These are designated multicent-3 to -5 (Table I).Independently, Metzenberg et al. (1984 Neurospora Newsl. 31:35-39) devised a strain designated multicent-2, with six centromeres marked.This has been used for mapping cloned DNA fragments.The multicent-3 tester incorporates two changes.arg-5 is substituted for bal in linkage group II, and a long inversion, OY323, has been inserted as a crossover-suppressor in linkage group I. Progeny are isolated either to complete medium or to minimal supplemented with arginine and pyridoxine.As a result of the heterozygous inversion, mating type shows little or no recombination with markers throughout two-thirds of I, which is the longest linkage group.

Table I. Constitution of multicent testers 1 through 5
In multicent-4 also, arg-5 is substituted for bal.In addition, psi replaces pdx as a marker for IV.psi (protein-synthesis-inhibited) is a readily-scored temperature-sensitive conditional mutant that grows normally on minimal medium at 25°C but does not grow at 34°.Ascospores must be germinated at 25° because psi progeny do not survive at the restrictive temperature.Replacing pdx with psi means that only two media are required.arg-5 is scored by transferring progeny to minimal medium at 25°, psi by transfer to arginine-supplemented minimal at 34°.
multicent-5 differs from multicent-4 only in having the OY323 inversion present in linkage group I. multicent-5 is preferred for mapping mutants that are scorable by vegetative phenotype.
As with the other testers, unmapped translocations can be recognized by linkage between markers that are normally independent.If it should be desired to determine marker-breakpoint linkage by scoring for aborted ascospores in progeny tests, it is possible to score the new rearrangement only in the noninversion half of the progeny; these are recognized as noninversion because they are of the same mating type as the noninversion parent.
Scoring for the other markers is as follows: Tests for mating type are most readily accomplished on fluffy lawns in petri dishes (Perkins et al. 1989 Fungal Genet. Newsl. 36:64-66).at (attenuated morphology) is readily scorable on minimal (with or without supplements) at two or three days (34°C).Growth is flat on the surface, with scattered specks of conidiation.wc-1 (white collar) is expressed most clearly at 25°C or higher.Carotenoids are absent in mycelia, though not in conidia.Germinants are incubated until conidia become orange, preferably under illumination.Scoring of ylo-1 (yellow) improves with age, and is likely to be unreliable with young cultures, especially in combination with wc.Carotenoids in ylo-1 look orange at first, then become yellow.ylo-1 scoring at three or four days should be considered preliminary, and should be checked later.acr-2 is scored clearly by resistance to 50 µg acriflavine/ml on solid medium.
With any of the testers, progeny are scored for markers sequentially beginning with the visible markers at, wc-1, and ylo-1.If linkage is apparent, the remaining markers need not be tested.If the unmapped mutant is unlinked to a visible marker, the markers that require transfer are then tested serially until linkage is seen.With translocations, the normally independent multicent markers are examined for linkages to one another.
The new multicent testers are heterokaryon-compatible with strains of OR background (het-c het-d; het-e).All three are available as heterokaryons in combination with the inactive-matingtype "helper" strain am1 ad-3B cyh-1 (Table I).The heterokaryons are phenotypically wild-type and highly fertile.Although the homokaryotic testers can be used satisfactorily for crossing, using a heterokaryon as female parent saves labor by making it unnecessary to supplement the crossing medium.Fertility may also be improved when the heterokaryon is used.
In heterokaryon with inactive mating helper strain am1 ad-3B cyh-1, FGSC 4564(Perkins  1984 Neurospora Newsl.31:41-42.The phenotypically wild type heterokaryon can be used as femal parent on minimal medium, but the am1 ad-3B cyh-1 component does not participate in the cross See FGSC stock list for allele numbers of markers.Published by New Prairie Press, 2017 *