BERKELEY, CALIFORNIA
University of California at Berkeley
GRAND FORKS, NORTH DAKOTA
University of North Dakota Novel meiotic mutants of maize identified from Mutransposon and EMS mutant screens --Golubovskaya, IN, Sheridan, WF, Harper, LC, Cande, WZ Our goal is to identify maize genes that control key events in meiosis, such as initiation of meiosis, pairing of homologous chromosomes, synapsis, meiotic recombination, and chromosome segregation. A large collection of maize meiotic mutants will allow us to study the genetic control of meiosis and thereby further our understanding of meiosis in large genome organisms.

The first maize meiotic mutants (as1, am1, dy1, dv1, el1, po1, po1-ms6, st1, and va1) were discovered in the 1930�s and have been maintained at the Maize Genetic Stock Center. In 1974, the collection of maize meiotic mutants was doubled in size by screening M3 populations treated with N-nitroso-N-methyl urea. Several new, unique mutants and new alleles of known meiotic genes were isolated from this screen, such as afd1, am1-praI, dsy1, dsy2, dsy3, dsy4, Mei025, ms28, ms43, pam1, pam2, and po1-ms4. All these mutants, except for Mei025, are recessive (Golubovskaya, Adv. Genet. 26:141-192, 1989)

New meiotic mutants. As a part of a long-term collaboration between I. N. G. and W. F. S., the collection of meiotic mutants was further increased in number by screening a maize population with active Mu transposons. The goal of using an active Mu population was to facilitate cloning of the maize meiotic genes. We screened the same F2 population that was previously used to isolate 51 maize embryo-specific mutants (Clark and Sheridan, Plant Cell 3:935-951, 1991; Sheridan and Clark, Plant J. 3:347-358). From 1991 to 1998, we discovered and characterized 18 new meiotic mutants (Table 1). In addition, in 1993, we isolated three meiotic mutants from active Ac stocks; these mutants have desynaptic phenotypes and were designated dsy9307, dsy9308, and dsy9309.

Some of the new mutants found in the Mu screen have unique phenotypes. For example, in mac1 plants, multiple archesporial cells undergo differentiation into megaspore mother cells in the ovule instead of one as in normal maize plants (Sheridan et al., Genetics 142:1009-1020, 1996; and Sheridan et al., Genetics 153:933-941, 1999). Other mutants from this screen have phenotypes similar to previously described mutants. Two mutants exhibited phenotypes similar to ameiotic1 and turned out to be new alleles of the am1 gene. These alleles were designated am1-485 and am1-489 (Golubovskaya et al. Genetics 147:1339-1350, 1997).

Another mutant, po495, had a polymitotic phenotype, distinguishable by a precocious post-meiotic mitosis that starts without a preceding S phase. However, it is not allelic to the original polymitotic1 mutation first described by Beadle (Cytologia 5:118-130, 1933). The pam495 mutant is similar in phenotype to the pam1 and pam2 mutants and is characterized by the presence of multinuclear conglomerates (coenocytes) in prophase I as well as abnormal chromosome synapsis and chromosome missegregation in anaphase I. However, pam495 is not allelic to either pam1 or pam2. The sticky485 mutant is similar to the Beadle�s sticky1 mutants. During prophase I, chromosomes in this mutant do not separate but rather condense into a tight cluster and cannot be distinguished individually under a microscope; chromosome fragmentation and missegregation occur in anaphase I. The allelic relationship between sticky485 and sticky1 has not been tested. The remainder of the new mutants have abnormal chromosome pairing or synapsis and were designated with a prefix dsy (see Table 1). One mutant, originally designated as segregationII-513 (segII-513), turned out to exhibit abnormal homologous synapsis after a more detailed cytological examination. All meiotic mutants found in this screen were completely male and female sterile.

Allelism tests showed that four new mutants with desynaptic phenotypes, dsy523, dsy9307, dsy9308, and dsy9309, were alleles of the dsy1 locus, in which two other mutations were previously identified, dsy1-1, induced by chemical mutagenesis in 1974 (Golubovskaya and Mashnenkov, Genetika (Russ.) 12:7-14, 1976) and dsy1-9101, discovered as a spontaneous mutation in 1991 (Golubovskaya et al. Devel. Genetics, 13:411-24, 1992; Golubovskaya et al. Devel. Genetics, 21:146-59, 1997). However, analysis of the pedigree of the new alleles isolated in Ac active families suggested that they likely represented the same mutant allele and they were designated as dsy1-9307. The dsy523 is the dsy1-9101 allele. Two other new desynaptic mutants, dsy498 and asy498, showed allelism in our test, but pedigree analysis again indicated that they too represented the same mutation event. The other new desynaptic mutants did not show allelism among themselves nor to any of the existing desynaptic maize mutants that we tested (Table 2).

With the new additions during the period of 1991-1998, the collection of meiotic mutants in maize contained 37 mutants. Allelism testing indicated that they represented at least 19 meiotic genes. Most of these meiotic genes (16) were represented by single mutant alleles. Two genes (dsy1 and po1) had three alleles each and one gene (am1) was represented by five different alleles.

In 1999, by screening 1260 Mu-active families of MTM growouts at the University of California at Berkeley for male and female sterility, four new meiotic mutants were added to the collection. These mutants were designated with a prefix mtm99 (maize targeted mutagenesis). Preliminary cytological characterization showed that mtm99-14, mtm99-25, and mtm99-30 are defective in homologous synapsis. The fourth mutant, mtm99-31, exhibited normal synapsis but was defective in sister chromatid cohesion, causing the centromeres to separate precociously before metaphase II (Table 3).

In the same year, nine meiotic mutants defective in chromosome synapsis were isolated from an EMS treated population at UC Berkeley. In this mutant screen, mature pollen was mutagenized with EMS to induce mutations (Harper et al., MNL 69:22, 1995). All nine EMS mutants exhibited desynaptic phenotypes and were named dsy9901 through dsy9906 (Table 3). Preliminary results of allelism testing with the new Mu- and EMS-induced mutants indicated that they all represent new meiotic genes (Table 4). This study is in progress.

Mapping meiotic genes. Since 1999, we have mapped five new and old meiotic genes using either the T-waxy series, or molecular markers. pam1, Mei025, and afd1 were mapped to chromosomes 1, 5S, and to the distal region of chromosome 6L, respectively. dsy2 and dsy498 (renamed phs1) were mapped to centromeric regions of chromosomes 5 (Franklin et al., in preparation) and 9 (Pawlowski et al., in preparation) respectively.

Cytology. The majority of the meiotic mutants in our collection display abnormal pairing and synapsis of homologous chromosomes. To classify these mutants, we quantitatively described the patterns of chromosome pairing at diakinesis and metaphase I (Table 5). We used an average number of univalents and bivalents as a criterion to order thirteen mutants from severely desynaptic (e.g., dsyCS, dsy9906b, phs1, and segII-513) to mildly desynaptic (e.g., mtm99-30 and dsy9305). mtm99-31 showed normal chromosome pairing and synapsis in meiosis I, consistent with our previous assessment that this mutant is defective in the second meiotic division. A detailed cytological analysis of dsy498 revealed that it is defective in homologous synapsis.

We are now working toward a comprehensive characterization of the previously known and new mutants using fluorescence in situ hybridization (FISH) coupled with 3-D microscopy to study bouquet formation, pairing of the 5S rDNA loci and the behavior of telomeres and centromeres during chromosome pairing. The pam1 gene is the first example of successful efforts in this direction (Golubovskaya et al, 2002, Genetics, in press). It appears to be a bouquet mutant. Inhibition of telomere clustering is the earliest lesion detected in pam1. The failure to complete the bouquet formation leads to defects in pairing and synapsis.

Table 1. Meiotic mutants discovered in screening of Mu active stocks at UND during 1991-1998.
 

Year
Total number of screened Mu active families
  

Number of families segregated for male sterility*
Symbols of meiotic mutations
   
Total*
Independent
Meiotic mutants
  
1991
836
80
30
7
 am1-485, dsy483**, dsy485**, dsy523=dsy1-9101, mac1=lar487, po495, sticky485
1992
584
73
15
5
 am1-489, dsy498=phs1, asy498, segII-513, pam3-495**
1993
738
36
15
6
 dsy9301#, dsy9302#, dsy9303, dsy9304#, dsy9305, dsy9306
Designations: *Total number of families isolated from a total of 836 Mu screening families. If the same mutant phenotype segregated in several (3 �10) self-pollinated siblings we count them as the same mutation event (see Independent column).
**mutants that were lost during propagation;
#these mutants have not been studied since found.
Numbers after gene symbols in years 1991-1992 designate numbers of Robertson�s Mu1 sources. Numbers after gene symbols in 1993 designate year and numerical order of isolated mutants

Abreviations: am1 � ameiotic1, asy1 � asynaptic, dsy - desynaptic, lar � leptotene arrest, mac1 - multiple archesporial cells, pam3- plural abnormality of meiosis, phs1- poor homology synapsis, po � polymitotic, segII � segregation in II meiotic division.

Table 2 Allelism test of desynaptic mutants isolated at UND (1991-1998).
 
dsy1                              
dsy523 A                            
dsy9101 A A                          
dsy9307 A A A                        
dsy9308 A A A A                      
dsy9309 A  A  A A A                    
as1 No No No No  No No                  
dsy2 No No No No No No No                
afd1 No No No  No No No No No              
pam1 No No No No No No No No No            
dsy498 No No No No No No No No No No          
segII No No No No No No No No No No No        
dsy9303 No No No No No No No No No No No No      
dsy9305 No No No No No No No No No No No No? No    
dsy9506 No No No No No No No No No No No No? No No  
  dsy1 dsy523 dsy9101 dsy9307 dsy9308 dsy9309 as1 dsy2 afd1 pam1 dsy498 segII dsy9303 dsy9305 dsy9306
Designation: A � Allelic, No � nonallelic,
Note: dsy498, dsy9303, dsy9305 and dsy9306 have been isolated from families with active Mu dsy9307, dsy9308 and dsy9309 have been isolated from families with active Ac-Ds

Table 3. Meiotic mutants discovered in screening of EMS and MTM* experiments (1999-2000 UC-Berkeley).
 
Experiment Total number of families screened Number of families segregating for male sterility Number of meiotic mutants Symbols of meiotic mutations
EMS 1999
(L. Harper)		 250 14 3 dsy9901, dsy9902, dsy9903
EMS1999
(J. Hollick)		 400 11 6 dsy9904a,dsy9904b, dsy9905a, dsy9905b, dsy9906a, dsy9906b
MTM1999
(M. Freeling)		 1260 20 4 mtm99-14, mtm99-25, mtm99-30, mtm99-31
MTM2000
(M. Freeling)		 1670 15 In progress  
Abreviations: MTM - Maize Targeted Mutagenesis project; dsy � desynaptic; mtm � maize targeted mutagenesis

Table 4. Allelism test of desynaptic mutants.
 
as                                    
dsy1 No                                  
dsy2 No No                                
pam1 No No No                              
dsy498 No No No No                            
segII-513 No No No No No                          
mms25 No No No No No No                        
mtm14 ? No ? ? No ? ?                      
mtm30 No No No ? No No ? ?                    
mtm31 No No No No ? No ? ? ?                  
dsy9904a No ? No ? No No No ? No ?                
dsy9904b ? ? ? ? ? ? ? ? ? No No              
dsy9906a No No No No ? No No ? ? No ? No            
dsy9906b No No No ? No ? No No No No No ? No          
dsy9303 No No No No No No No ? ? ? No ? ? No        
dsy9305 No No No No No No ? ? ? ? No ? No ? No      
dsy9506 No No No No No No ? ? ? No No ? No ? No No    
afd1 No No No No No No No ? ? No No ? No ? No ? No  
  as1 dsy1 dsy2 pam1 dsy498 segII513 mms25 mtm14 mtm30 mtm31 dsy9904a dsy9904b dsy9906a dsy9906b dsy9303 dsy9305 dsy9306 afd1
Designation: A � Allelic, No � nonallelic, ? � unknown
Note: dsy498, segII-513, mms25, mtm13, mtm14, mtm30, mtm31, dsy9303, dsy9305, and dsy9306 have been isolated from families with active Mu.
dsy9904a, dsy9904b, dsy9906a, and dsy9906b have been isolated from families after treatment pollen grains with EMS.

Table 5. Pattern of homologous synapsis in new desynaptic mutants of maize.
 
 
METAPHASE I
  
GENOTYPE
Pattern of homologous synapsis (number of univalents and bivalents )
TOTAL
 
20 I
1 II
2II
3II
4 II
5 II
6II
7 II
8 II
9 II
10 II
  
A344 STD ( CONTROL)                
1
3
141
145
MUTANTS                        
mtm99-31                  
2
98
100
mtm99-30            
1
10
18
25
36
90
dsy9305  
2
0
2
2
2
1
1
1
6
40
57
dsy9901      
1
2
4
11
6
9
8
7
48
dsy9902
3
5
10
8
11
10
4
3
3
0
4
61
dsy9906a
31
3
6
12
21
18
17
6
2
1
2
119
dsy2
28
25
50
46
31
21
5
0
0
1
  
207
dsy9905a
107
15
7
8
10
4
4
7
5
1
0
168
mtm99-14
42
3
7
5
2
1
          
60
mtm99-25
151
20
8
8
6
            
193
dsy498=phs1
170
7
6
1
              
184
dsy9906b
88
4
2
                
94
dsyCS = mms25
141
15
2
                
158
segII-513
150
17
4
2
              
174

 
 
 
 
 

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