THE TRANSPOSABLE ELEMENT Ds AFFECTS the PATTERN of INTRAGENIC RECOMBINATION at the BZ and R LOCI in MAIZE

THE TRANSPOSABLE ELEMENT Ds AFFECTS THE PATTERN OF INTRAGENIC RECOMBINATION AT THE BZ AND R LOCI IN MAIZE

H.K.DOONER' AND J. L. KERMICLEt "Advanced Genetic Sciences, 6701 San Pablo, Oakland, California 94608, and TDepartment of Genetics, University of Wisconsin, Madison, Wisconsin 53706 Manuscript received May 17, 1985 Revised copy accepted January 16, 1986

ABSTRACT Insertion of the transposable element Ds into either the bz or R locus affects intragenic recombination in various ways. We have examined here one aspect of this problem; namely, the distribution of flanking markers among intragenic recombinants produced by different types of heterozygotes carrying Ds insertion mutations. Heteroallelic combinations of a Ds insertion mutation and a mutation borne on a structurally normal chromosome generate a majority of intragenic recombinants of a crossover type. In contrast to this, most intragenic recombinants obtained from heterozygotes between two different Ds insertion mutations have a parental arrangement of outside markers. Therefore, the resolution of the recombination intermediate would appear to depend on the nature of the mutations in the heterozygote.

HE insertion of a transposable element into a chromosome can be ex- T pected a priori to interfere with the normal homologous pairing of that chromosome at meiosis and, consequently, to affect recombination. Various effects of transposable insertion elements on interallelic recombination have been reported in maize. NEUFFER(1 965) observed no intragenic recombination between heteroallelic a1 mutations that could be destabilized by two different transposable elements, Dt and Ac. On the other hand, MCCLINTOCK(1965) reported putative cases of intragenic recombination between transposable element a1 mutations belonging to the Spm and Dt systems. Yet, the occurrence of such cases depended on the state of activity of the Spm element in one of the alleles. NELSON(1968) found that recombination occurred between all the transposable element wx mutations tested, as well as between such alleles and most stable alleles, and was able to map the insertions within the wx locus. More recently, GREENBLATT(1 98 1) observed an unexpected enhancement of recombination by the transposable element Mp in an intergenic interval delim- ited by the P locus and an adjacent translocation breakpoint, and he postulated that the insertion of the element Mp reduced the pairing stresses inherent in a translocation heterozygote. We report here a different effect of transposable element insertions on re-

Genetics 113: 135-143 May, 1986. 136 H. K. DOONER AND J. L. KERMICLE combination. We have compared the patterns of distribution of linked markers among intragenic recombinants produced by two types of heteroallelic combinations involving Ds insertion mutations: those in which the contrasting allele is borne on a structurally normal homologue and those in which the heteroallele is a different Ds insertion mutation. We find that the heteroallelic makeup profoundly affects whether intragenic recombinants are borne on recombinant or parental chromosomes. The following constitutes a report of observations made independently by the two of us at different loci, bz and R. Because of the remarkable similarities between our observations, we have decided to report them jointly.

MATERIALS AND METHODS Stocks: All the alleles used in these experiments were in the genetic background of the inbred W22. Description of bz alleles: The aleurone phenotypes conditioned in the presence of all the complementary factors required for anthocyanin pigmentation are given in pa- rentheses. Bz-McC (purple): the normal progenitor allele of all the bz insertion mutations ana- lyzed in this study; extracted from a C-I sh Bz chromosome obtained from BARBARA MCCLINTOCK. bz-ml (bronze, in the absence of Ac; spotted, in its presence): an allele arising from insertion of Ds in Bz-McC (MCCLINTOCK1951). bz-m2 (bronze-purple variegation): an allele arising from insertion of Ac in Bz-McC (MCCLINTOCK1955). bz-m2(DI) and bz-m2(DIZ) (bronze, in the absence of Ac; spotted, in its presence): the first and second derivatives from bz-m2 harboring Ds elements at the locus as a conse- quence of mutations from Ac to Ds (MCCLINTOCK1955). Bz-WZ2 (purple): the normal Bz isoallele carried in the W22 inbred. bz-E2 and bz-E5 (bronze): ethyl methanesulfonate (EMS) induced mutants from Bz- W22, which lack UDPG-flavonoid glucosyltransferase activity (UFGT-) but make cross- reacting material (CRM+) (DOONER 198 1 and unpublished results). bz-R (bronze): the stable, reference allele at the locus. Description of r alleles: Phenotypes conditioned by the R alleles used in this study have been described elsewhere (KERMICLE1970, 1984; DOONERand KERMICLE1974). The principal action, genic element constitution according to STADLER’S(1954) duplication model, and origin of these factors are as follows: R-r:standard (purple aleurone, red plant parts) = (P) (S). Collected allele. r-r:n46 and r-r:n142 (colorless aleurone, red plant parts) = (P). Derivatives from R- r:standard. r-r:n35 and r-r:nlOl (colorless aleurone, red plant parts) = (P) (s). Derivatives from Rxstandard. r-g:1557-2 (colorless aleurone, green plant parts) = (s). Carries the (s) component of r-r:n35. R-sc:124 (purple aleurone, green plant parts) = (Sc). Derivative carrying the self- colored aleurone (Sc) component from R-st (R-stippled) allele. r-ml, r-m3 and r-m9 (pale or colorless aleurone, green plant parts) = (Sc-Ds). Three independent mutations arising from insertions of Ds into R-sc:124. r-g (colorless aleurone, green plant parts). A null allele carried by the g, wx tester stock (see below). r-g:nc3-5 (nearly colorless aleurone, green plant parts). A variant of R-st that is vir- tually spotless in the absence of M-st (Modifier of R-stippled), but stippled in its presence. R-g:8 pale (pale aleurone, green plant parts). A variant of dark seeded R-g:8, an (S) derivative of Rxstandard. EFFECT OF DS ON RECOMBINATION 137 Markers: The mutations sh (shrunken endosperm) and wx (waxy endosperm) were used as flanking markers in bz heteroallelic crosses. In a W22 background these markers map, respectively, about 3 units distal and 25 units proximal to bz in 9s. The markers g (golden leaf) and M-st served to flank r mutants in the R locus experiments. These markers are located, respectively, 20 units proximal and 6 units distal to R in 1OL. Selection and analysis of purple kernel derivatives: Bz: bz heteroallelic and homoallelic combinations, homozygous for r, were crossed by a sh bz-R wx; R-r tester in an isolated detasselling plot. Putative Bz intragenic recombinants or reversions were selected as individual mottled purple kernels against a background of mottled bronze kernels. In this scheme, Bz contaminants from nearby com- mercial fields (almost exclusively r in constitution) appear as colorless kernels and are not selected. The selections were classified for flanking markers and then backcrossed to the male parent to verify heritability of the selection and recovery of the pollen markers. R-sc: r heteroallelic and homoallelic combinations were pollinated with g r-g m-st; wx and g R-g:8 pale m-st; wx in isolated detasselling plots. Purple kernel selections were evaluated for the proximal marker golden and were crossed with an r-g:nc3-5 stock carrying wx. Authentic R-sc derivatives were identified on the basis of their progeny segregation for r and wx. Presence or absence of some 6% of stippled kernels within the r-g:nc3-5 kernel class indicated, respectively, the presence or absence of M-st in the R-sc chromosome.

RESULTS AND DlSCUSSION Intragenic recombination between an insertion mutation and a mutation borne on a structurally normal homologue In order to map the sites of Ds insertion mutations in bz and R relative to other sites in these loci, we adopted a conventional mapping strategy that consisted in testcrossing heteroallelic combinations of an insertion mutation and a contrasting allele not borne on an insertion chromosome. That is, one of the parents (the female parent in both cases) was structurally heterozygous for an insertion. The purple kernel selections obtained from these testcrosses represent putative intragenic recombinants. Their linked marker distribution confirmed their origin as recombinants and allowed us to place the mutations relative to each other in the chromosome. In the bz locus experiment the Ds insertion mutants were bz-ml, bz-m2(DI) and bz-m2(DZI). These have been characterized molecularly as being insertions of 1.1, 3.5 and 3.9 kb, respectively, within the bz locus (DOONERet al. 1985). They were isolated by BARBARAMCCLINTOCK in a strain carrying a Bz isoallele that we now designate Bz-McC. The contrasting alleles were the EMS-induced mutants bz-E2 and bz-E5. Both mutants are UFGT-, CRM+, i.e., they make normal amounts of an inactive glucosyltransferase enzyme and represent, therefore, missense mutations in the bz coding sequences. These mutants were isolated by EMS mutagenesis of a stock carrying the Bz-W22 isoallele, which shares considerable restriction homology with Bz-McC (H. K. DOONER,unpublished observations). The results obtained from heteroallelic combinations of the type bz-m/bz-E are presented in Table 1. An examination of the linked marker composition of the Bz selections reveals the following: (1) Most Bz derivatives are borne on recombinant chromosomes. Their origin from one heteroallelic combination is 138 H. K. DOONER AND J. L. KERMICLE TABLE 1 Bz selections obtained from heteroallelic combinations of Ds insertion mutants with EMS- induced mutants

Er selections

Linked marker composition - Kernel Frequency Genotype population (X 1o-~) -

sh br-ml wx --- 114,653 6.1 0 0 7 0 Sh b~-E2WX sh bz-ml wx 123,632 10.5 1 2 10 0 Sh bz-E5 Wx

sh bz-m2(DI) wx -~-217,823 2.8 0 0 0 6 Sh bz-E2 WX

sh bz-m2(DI) wx 194,462 2.6 0 0 0 5 Sh b.~-E5 WX

FIGURE 1 .-Origin of recombinant Bz and R-sc exceptions from representative heteroallelic combinations of an insertion .mutation and a mutation borne on a structurally normal homologue. illustrated in Figure 1A. (2) In all cases there is a single recombinant class; therefore, it is possible to unequivocally order the mutants in a genetic map. The order from telomere to centromere is m2(DI) E2/E5 ml. (3) Few Bz chromosomes are marked parentally-too few to identify a clear majority class. In the R locus experiment, the Ds insertion mutants used (r-ml, r-m3 and r- m9) derive from a stock carrying the solidly pigmenting allele R-sc;124. Unlike other R alleles that are complex, R-sc:124 consists of a single genic element (Sc) specifying seed color. The contrasting alleles descend from the R duplication chromosome R-mtandard. This duplication embodies a tandem arrangement of two anthocyanin genes that retain extensive duplication homology but confer different tissue specificities, so that the proximal member (P) controls plant pigmentation, whereas the distal member (S) controls seed pigmentation. Three of the alleles, r-r:n46, r-rm142 and r-g:1557-2 have lost one member of the duplication, as well as the ability to pigment seed; r-g:1557-2 has also EFFECT OF DS ON RECOMBINATION 139 TABLE 2 R-se derivatives from heterozygotes between r-nt insertion mutants and either r-r or r-g heteroallelic mutations

R-sc derivatives

Linked marker composition

Frequency Genotype Kernel population (XlO-') G r-r:n46 + 39,246 6.62 1 2 23 0 g r-ml M-st g r-r:n142 M-st 26,697 4.49 0 0 12 0 G r-m3 + * M-St -g r-r 6,370 6.28 0 0 4 0 G r-m9 + G r-g:1557-2 M-st - - 69,413 6.77 2 1 44 0 g r-ml + * Pooled data from r-r:n35 and r-r:nl02. lost its plant pigmenting ability. The remaining two, r-r:n35 and r-r:nlOl, represent mutations of the seed pigmenting component of R-r. Because they retain the duplication, the latter were considered less than ideal for present purposes. Data involving them provide a link, however, with a previous study that mapped Ds in the three insertions distal to the (Sc) component of R-sc:124 (KERMICLE1980). Table 2 presents the results from r heteroallelic combinations. Clearly, the distribution of linked markers reveals the same features as the br data. Most R-sc derivatives fall in one recombinant class, and very few are borne on parentally marked chromosomes. The recombinant linked marker composition of the R-sc selections confirms that the site of Ds insertion lies distal to the R locus component governing tissue-specific expression @.e., seed vs. plant color) in all three r-m mutants (Figure 1B). These data, obtained largely from heterozygotes that utilized a nonduplication derivative of R-mtundurd against r- ml and r-m3, do not differ appreciably from those reported earlier (KERMICLE 1980) for heterozygotes between these Ds insertion mutations and r-r alleles carrying the duplication. Intragenic recombination between two different Ds insertion mutations The results discussed in the previous section stand in sharp contrast to those obtained from heterozygotes carrying two different insertion mutations. An examination of the linked marker distribution among Bz selections (Table 3) and R-sc selections (Table 4) from heteroallelic combinations of two Ds insertion mutants reveals a preponderance of parentally marked chromosomes and a dearth of recombinant types. In fact, it would be difficult to map the insertions relative to each other from these data. There are scarcely more recom- 140 H. K. DOONER AND J. L. KERMICLE

TABLE 3 Bz selections obtained from heteroallelic combinations of different bz-m insertion mutants

~ ~ ~~~~ Bz selections

Linked marker composition - Kernel Frequency Genotype population (XI 0-5) - sh br-ml Wx ____- 210,914 17.5 23 7 5 2 Sh br-m2(DI) wx sh bz-ml Wx - 224,020 13.8 16 3 10 2 sh bz-mZ(DII) wx sh bz-m2(DI) wx 295,461 0 0 0 0 0 sh bz-m2(DII) ~ ~~ ~ ~

TABLE 4 R-sc derivatives obtained from heteroallelic combinations of r-mZ, r-m3 and r-m9

R-sc derivatives

Linked marker composition - Frequency Genotype Kernel population (XIO-') - G r-m3 + ___- 70,531 10.78 38 12 4 22 g r-ml M-st G r-m9 + 60,890 1.81 2 8 1 0 g r-ml M -st G r-m9 + 32,584 7.67 4 15 3 3 E r-m3 M-st binants, based on the number of parentally marked cases, than one would expect due to coincident reversion and recombination in a flanking region. Results from the homoallelic combinations show that all the insertion mutations are extremely stable. No Bz reversions were detected when populations greater than 250,000 gametes were screened for each bz-m mutant. Likewise, no rev- ertants to R-sc were obtained from any of the r-m mutants tested in somewhat smaller populations (KERMICLE1980). Therefore, the Bz and R-sc selections obtained from heterozygotes between two different Ds insertion mutations must arise from some type of recombinational event; however, the resolution of that event favors heavily the formation of parentally marked chromosomes. The origin of the Bz and R-sc majority types from the heterozygotes corre- sponding to the first entries in Tables 3 and 4, respectively, is diagramed in Figure 2 (A and B). EFFECT OF DS ON RECOMBINATION 141

------. Sh wx bz-m2(DI)7'------

FIGURE2.-Origin of the Bz and R-sc majority types from representative heterozygotes between two different Ds insertion mutations. No Bz selections were recovered from bz-m2(DZ)/bz-m2(DZZ) heterozygotes, although a population of almost 300,000 kernels was screened (Table 3). This result is readily explainable. Both of these mutations carry Ds insertions which arose by deletion of an internal segment of the Ac element present in the bz- m2 parent allele. The site of the insertion has been mapped molecularly and found to be the same in both mutants (H. DOONER, E. WECKand J. ENGLISH, unpublished results). Thus, the heterozygotes bz-ml/bz-m2(DZ) and bz-ml/bz- m2(DZZ) are similar in structure and yield similar frequencies of Bz derivatives. In both cases there is a majority parental class carrying the markers flanking the proximal heteroallele. Similar observations have been interpreted in fungi (MURRAY1963) to indicate polarity in recombination. Yet, we do not see this effect when only one member of the heteroallelic pair is an insertion mutation (Table 1). Alternatively, the observed differential recovery of the two parentally marked classes could be related to the different size of the insertion in the two mutations, because the Ds element in the proximal heteroallele, bz-ml, is more than 2-kb smaller than the one present in bz-m2(DZ) or bz m2(DZZ). The heterozygote bz-m2(DZ)/bz-m2(DZZ)also failed to yield any spotted kernels. These would be expected from reconstitution of a functional Ac element by recombination within the Ds elements present in bz-m2(DZ) and bz-m2(DZZ). The failure to recover spotted kernel exceptions among a large population of kernels (Table 3) is understandable because the segments deleted in the two Ds elements overlap (H. DOONER, E. WECK and J. ENGLISH,unpublished result s). One feature of the data in Table 4, for which at present we do not have an adequate explanation, is the variation in total frequency of R-sc derivatives from the three heteroallelic combinations examined. The differences could be a property of the individual r-m alleles, with m3 > ml > m9, or they could be a function of the relative distances between the Ds insertion sites. We have presented data in this report showing that the type of intragenic recombinant generated in heterozygotes involving transposable element insertion mutations depends on the nature of the heterozygote. Heteroallelic combinations of an insertion mutation and a mutation borne on a structurally normal homologue generate a majority of intragenic recombinants of a crossover type, i.e., having a recombinant arrangement of outside markers. In con- 142 H. K. DOONER AND J. L. KERMICLE trast to this, intragenic recombination between two different insertion mutations yields a majority of recombinants with a parental arrangement of outside markers. This result is unexpected. In yeast, where intragenic recombination has been studied most extensively, conversion of deletions and insertions has been reported (FINK and STYLES1974; RADDINC 1979; FOCELet al. 1979), but, to our knowledge, the effect of insertions on the pattern of distribution of flanking markers among interallelic recombinants has not been examined. Our data would indicate that the resolution of the recombination intermediate or Hol- liday structure postulated in most recombination models (HOLLIDAY1964; ME- SELSON and RADDING 1975; POTTER and DRESSLER1976) depends on the nature of the mutations in the heterozygote, with heteroallelic insertions in- teracting to bring about a parental type of resolution. Other cases of resolution of recombination intermediates without recombination of flanking markers have been reported in yeast; however, the yeast studies have dealt with gene conversion between nonallelic sequences. KLAR and STRATHERN(1 984) found no evidence that crossing over ever accompanied gene conversion between the nonallelic HMR and MAT genes during mating-type switching. KLEIN (1984), on the other hand, examined interactions between the members of a nontan- dem inverted duplication and found that intrachromosomal gene conversion was not associated with reciprocal exchange in flanking sequences. Both reports discuss the possibility that sequences around the genes being converted may affect the isomerization of the crossed-strand Holliday structure. We can only speculate as to the basis for our observation. If in the recombination intermediate the heteroduplex region is long enough to include both insertions and if the insertions can base-pair with each other-even if not perfectly throughout their entire length-a unique figure eight would be formed in each strand, one that may promote the parental resolution of the recombination intermediate. It would be informative to study intragenic recombination in heterozygotes carrying unrelated insertions, such as Ds and the receptor element of Spm, on opposite chromosomes. If base pairing of the heteroallelic insertions is important, one would not expect preferential recovery of parental types. On the other hand, the effect described in this report may be general to insertion heteroallelism per se.

This research was supported partly by grants from the National Science Foundation (PCM 81- 09634) and Advanced Genetic Sciences to H.K.D. and from the Department of Energy (AC02- 76EVO1300) to J.L.K. This is paper no. 5-2 from AGS.

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