the waxy locus in maize 111. effect of structural

THE WAXY LOCUS IN MAIZE 111. EFFECT OF STRUCTURAL HETEROZYGOSITY ON INTRAGENIC RECOMBINATION

AND FLANKING MARKER ASSORTMENT1i2

OLIVER E. NELSON

Department of of GenetL'cs, Uniuersity of Wisconsin, Madison, Wisconsin 53706

Manuscript received May 13, 1974 Revised copy received September 10,1974

ABSTRACT

The effect of heterozygosity for structural rearrangements on recombination between two wx heteroalleles (C and 90) and the pattern of flanking markers in the resultant Wx gametes has been examined. The rearrangements are Tp9, an insertional translocation in which a segment of chromosome 3 has been inserted into the short arm of chromosome 9 close to the wx locus; In9a, a long pericentric inversion with wx in the inverted segment; and Reurr 9, a complex rearrangement of chromosome 9. Heterozygosity for rearrangements decreases the frequency of W x gametes to varying degrees.- Heterozygosity for Tp9 enhances the proportion of W x gametes that are apparent convertants and allows the conclusion that such gametes do not normally arise from an exchange in the wx locus plus a second exchange distal to wx. Heterozygosity for Zn9a markedly decreases the frequency of Wx gametes that are recombinant for outside markers but does not decrease the frequency of convertants.-Heterozygosity for Rearr 9 permits a low frequency of Wx gametes, all of which are apparent convertants.-A high proportion wf the convertants have the flanking markers that entered the cross with C so recombination is polarized in normal homologs and in heterozygotes for all rearrangements.

PREVIOUS investigations of recombination between the waxy heteroalleles wx-C and wx-90 have shown that the majority of Wx gametes are also

recombinant for the flanking markers (NELSON 1962, 1968). However, the second largest class of Wx recombinants carries the flanking markers that entered the cross linked to the wx-C allele. The frequency of this class is sufficiently high to make it improbable that these Wx recombinants arose from an exchange event within the wx locus plus a second exchange between a flanking marker, the bz locus, and the wx locus, even on the assumption that an exchange in the wx locus does not interfere with an exchange in the intervals adjacent to the waxy locus. The data are thus reminiscent of those frequently found in random spore analyses of crosses between heteroalleles in various fungi. The relatively large proportion of W x recombinants with one array of parental markers may arise by the process of gene conversion (MITCHELL 1955). The inequality in

Research supported by College of Agricultural and Life Sciences, by National Science Foundation Grants, GB-1073

Laboratory of Genetics Paper No. 1726 and 151C4, and by National Institutes of Health Grant 15422

Genetics 79: 3144 January, 1975

32 OLlVER E. NELSON

numbers of W x recombinants with one parental marker array as compared to those with the other array of parental markers indicates that recombination (conversion) may be polarized ( LISSOUBA and RIZET 1960; MURRAY 1963).

The detection in random spore analyses of a sizable proportion of apparent recombinants between mutant sites within a locus that have one or both parental marker arrays is not sufficient to indicate that conversion has taken place. Con- version is revealed by tetrad analysis as a deviation from the expected 2:2 segre- gation of a given site: e.g.; a3 f ; a3 +; + 3.; 4- a2. Alternately, BALLANTYNE and CHOVNICK (1971) have demonstrated in half-tetrad analyses of the progeny from heteroallelic rosy genotypes in Drosophila melanogaster that the r y f products with parental combinations of flanking markers arose from nonrecipro- cal events, i.e., conversion of one mutant site.

By still other means, reported here, it has been possible to demonstrate that the flanking marker pattern of Wx recombinant gametes is a reflection of events localized at the wx locus and therefore, in all probability, conversion in origin. One approach has been to utilize heterozygosity for the insertional translocation Tp9. which RHOADES (1958, 1968) reported to reduce markedly recombination between markers on the short arm of chromosome 9 where the wx locus is located. NELSON (1961) found that heterozygosity for Tp9 greatly reduced the frequency of Wx gametes in a wx-9O/wx-C heteroallelic plant but not to the same extent as it reduced crossing over between the flanking markers sh and g115. These obser- vations were interpreted as evidence that intergenic and intragenic recombination might arise from different event. In subsequent investigations, it was found that crossing over in the wx-gll5 interval in Tp9 heterozygotes was nearly equal to that observed in structurally normal homozygotes. Thus the effect on recombination of heterozygosity for the translocation lapses in the vicinity of the wx locus, and this observation renders untenable the suggestion that heterozygosity for Tp9 could differentiate between intra- and intergenic recombination. The net effect of Tp9 heterozygosity on recombination between wx-C and wx-90 is to reduce the frequency of W x gametes. It also reduces drastically recombination between the wx locus and the distal marker bz, thus effectively shifting the marker from a position 28 map units distal to a point less than 2 map units distal to the wx locus. If the bz Wx U gametes that constitute a sizable proportion of the W z gametes produced by plants that are bz (C+) u/Bz ( f 9 0 ) V have arisen from an exchange between the mutant sizes in the wx locus and a second exchange between the bz and wx loci, one would expect that the proportion of W x gametes carrying the parental markers that entered the cross with WX-C would be substantially decreased or eliminated in Tp9 heterozygotes as compared to structurally normal homozygotes.

For W x gametes with parental combinations of flanking markers, the data cannot discriminate between gene conversion and a model involving high negative interference as the basis of their origin. However, evidence from tetrad analysis in Neurospora (STADLER and TOWE 1963) and Saccharomyces (FOGEL and HURST 1967) has shown that recombinants within a cistron that carry parental combinations of flanking markers usually result from gene conversion at the site of the

INTRAGENIC RECOMBINATION IN M A I Z E 33

mutant with that parental marker combination since one observes a 3:l segre- gation for that site. The previously cited evidence frolm Drosophila (BALLANTYNE and CHOVNICK 1971) indicates that for the rosy locus all ry+ gametes with parental combinations of markers have arisen from gene conversion. In this paper, W z gametes with parental combinations of markers originating in plants heteroallelic for wz mutations will be referred to as convertants. I t should be understood, however, that the evidence that such gametes arose from non-reciprocal events is indirect.

Additionally, the effects of heterozygosity for two structural rearrangements, Zn9a (RHOADES and PATTERSON 1957) and Rearranged chromosome 9 (MCCLIN- TOCK 1941) have been examined for the 90/C heteroallelic combination. Zn9a is a long pencentric inversion that includes the waxy locus. Rearranged (Rearr) 9 is a complex chromosome rearrangement, and since recombination in the wx region generates dicentric chromatids, it may be considered for our purposes as equivalent to a paracentric inversion. Since a single crossover within the inverted region produces inviable gametes owing to deficiencies, duplications, or dicentric chromatids, the purpose of such an examination is to ascertain whether the frequency of convertants is reduced for the inversion heterozygotes. CHOVNICK (1973) has reported for Drosophila that heterozygosity for a paracentric inversion with the rosy locus approximately at its center does not decrease the frequency of convertants in the progeny of flies heteroallelic for two rosy mutations relative to the frequency observed in structurally normal flies. At the same time, the frequency of ry+ recombinants with outside markers recombined decreases drastically. More recently, MCCARRON, GELBART and CHOVNICK ( 1974) have shown that with heterozygosity for a long pericentric inversion which includes the rosy locus and for two ry heteroalleles, the frequency of conversion of the two ry mutants is as high as when the heteroalleles are both located on normal homologs. Further, they found some crossovers between the two ry heteroalleles but not as many as with normal homologs. This study confirms the production of approximately the same frequency of W z convertants from wz-90 and wx-C in Zn9a heterozygotes as in structurally normal plants, and a pronounced decrease in W z gametes with outside markers recombined. In heterozygotes for Rearr 9 where one breakpoint is close to the waxy locus, the frequency of Wx convertants is much reduced, and only convertants are found.

The polarity which is observed when the heteroalleles are located on structurally normal chromosomes is also of interest with regard to the location of one heteroallele in a rearranged chromosome. It will be shown that no structural rearrangement investigated changes the marked tendency of W z gametes with parental markers to have those markers that entered the cross linked to wx-C.

MATERIALS A N D METHODS

Detection of non-uaxy p d e n grains and seeds: The frequency of non-waxy (Wz) pollen grains in the F, progeny of a cross between two stocks carrying different LUX alleles was estimated in gelatin mounts following staining with I,-KI solution. The method has been described in detail by NELSON (1968).

34 OLIVER E. NELSON

In the analyses where the F, cross between the two wx heteroalleles is used as the male parent on a bz wx-C u/bz wx-C U; ae/ae tester stock, detection of seeds with Wx/wx/wx endosperms is facilitated since all stocks are homozygous for the amylose-extender (ae) mutant. The bronze (bz ) locus is located 28 map units distal to the wx locus. The B z allele in the presence of the complementary color factors A,,A,,C, and R conditions the production of anthocyanin pigment in the aleurone. The bz allele in the presence of the same complementary factors results in seeds colored tan to bronze. The virescent-1 (U) locus is located 7 map units from the wx locus and is across the centromere in the long arm of chromosome 9. Plants that are U / U ap? yellowish-white as seedlings but become green rapidly with age. The amylose-extender (ae) locus is located on the long arm of Chromosome 5. Endosperms that are ae/ae/ae have a vitreous appearance, and the percentage of amylose is increased from 25% in A&/- / - endosperms to between 45% and 60%. Endosperms that are ae/ae/ae; wx/wx/wx are markedly wrinkled (NEUFFER, JONES and ZUBER 1968). Endosperms that are Wx/wx/wx; ae/ae/ae are noticeably less defective in appearance than are double mutant wx/wx/wx; ae/ae/ae endosperms. Such putative Wx/wx; ae/ae seeds stain an intense blue-black with an I,-KI solution, clearly dif- ferentiating them from wx/wx; ae/ae seeds. Having all stocks ae/ae also constitutes a check for Wx/wx seeds that are derived from contamination by Wx; Ae pollen. Plants grown from these Wx/wx seeds are crossed by the bz wx-C u/bz wx-C U; ae/ae tester to verify the allelic state at the bz and U loci. The appearance of wx/wx; Ae/ae kernels with plump endosperms, a character- istic, dull appearance, and tan color after reaction with an I,-KI stain indicates that the W X / W X seed arose from fertilization by a contaminating Wx, Ae pollen grain (NELSON 1968).

Stocks: The two wx heteroalleles, C and 90 were selected for this investigation since in the absence of a structural heterozygosity a high frequency (72 to 131 x 10-5) of Wx pollen grains has been observed in heteroallelic plants (NELSON (1958, 1962, 1968; BRICGS and SMITH 1965; YU and PETERSON 1973). In genotypes these alleles will be written as ( C + ) and (+go) to illustrate the relation of the mutant sites. The wx-C allele is the reference waxy allele. It has been shown that the mutant site in wx-C is distal to that in wx-90 (NELSON 1962).

Tp9: The Tp9 stock, which was isolated by FRANCES CLARK BEARD, carries an insertional translocation in which a large segment of chromosome 3 is inserted in chromosome 9 between the bz and wx loci (RHOADES 1958, 1968). The stock was received from M. M. RHOADES as the heterozygous translation stock, Sh B z Tp9 Wx/sh bz N9 wx. A homozygous translocation stock of the constitution Sh Bz Tp9 (C+) gZl5/Sh B z Tp9 (C+) gZ15; Ae,’Ae was isolated. The shrunken-l (sh) locus is located 30 map units distal to the wx locus and 2 units distal to the bz locus. The sh alleles condition the production of seeds in which the endomsperm has collapsed during maturation to give a deeply indented crown which contrasts with the smooth or slightly indented crowns of seeds that carry Sh. The glossy-15 (gZ15) locus is 10 units from wx and is on the long arm of chromosome 9. Seedlings that are gZlS/gZ15 develop a glossy appearance (and water sprayed on the leaves remains in prominent drops) at about the time that the fourth leaf appears. This g€ossy appearance is lost later in development (NEUFFER, JONES and ZUBER 1968). This translocation stock was crossed by a Sh bz (C+) u/Sh bz (C+) U ; ae/ae line. The resulting F, was crossed by a sh bz ( f 9 0 ) u/sh bz (+so) U ; ae/ae line to obtain (in the absence of recombination) 4 classes of kernels-sh B z Tp9 (C+) V gl15/sh bz N9 (+90) U GZ15 and either ae/ae or Ae/Ae as well as Sh bz N9 (C+) U Gl15/sh bz N9 f+90) U G115 ae/ae or Ae/ae kernels. The ae/ae kernels constituted the material with which to measure the effect of heterozygosity for Tp9 on the flanking marker pattern in Wx recombinants. The Ae/ae kernels were usead to verify that the F, plant was indeed heterozygous for the translocation since recombination between bz and U was found to be 6.8% (206 plants). The standard map distance between these loci is 31 units (NEUFFER, JONES and ZUBER 1968). RHOADES (1968) has reported pronounced reduction in recombination between loci on the short arm of chromosome 9 in plants heterozygous for Tp9.

Znuersion 9a: This inversion in chromosome 9 is a long pericentric inversion with breakpoints at 9S.7 and 9L.9 as determined by C. H. LI (RHOADES and PATTERSON 1957). The waxy locus lies within the inverted segment, but the bz locus is in the uninverted segment of the

INTRAGENIC RECOMBINATION IN MAIZE 35

short arm 9. The inversion stock as received from the Maize Genetics Cooperative carried the standard wx allele (C) in the inverted segment. From this accession, Bz Zn9a [V (+e)/, a stock was derived that was also homozygous for the ae mutation.

Rearr. 9: This is a complex rearrangement of chromosome 9 which MCCLINTOCK (1941) has shown to result from three breaks in chromosome 9. The terminal knob on the short arm of the chromosome has been broken into two unequal parts and the smaller piece together with three- quarters of the short arm is inserted into the long arm of chromosome 9. A segment of the chromosome containing the remainder of the short arm of chromosome 9 and the proximal one- third of the long arm is inverted and attached to the remainder of the terminal knob. A diagrammatic representation of this rearrangement is shown in Figure 1. This stock which carries the reference wx allele (C) was received from MCCLINTOCK in 1960 as the heterozygous stock, Rearr 9 S h Bz (C+)/N9 sh bz IC+). A line that was homozygous for the rearrangement and for ae was then derived.

RESULTS

Heterozygosity for Tp9: Since the heterozygotes for Tp9 were derived from a cross, Sh Bz Tp9 (C+) V/Sh Bz N9 (C+) U ; Ae/ae X sh bz N9 (4-90) v/sh bz N9 (4-90) U ; ue/ue, there were in addition to the Sh Bz Tp9 (e+) V/sh bz N9 (4-90) U ; ae/ue seeds which were heterozygous for the wx hetemalleles C and 90 and for Tp9, seeds which were heterozygous for the wx heteroalleles but nomt for the translocation. These seeds, detected by their bronze (bz) phenotype, serve as a control group to estimate the recombination between wx-C and wz-90 in this particular genetic background in the absence of the translocation. However, there is a marker ( S h vs sh) only on the distal side of the waxy locus.

B

FIGURE 1 .-A) The breakpoints that occurred in a normal chromosome 9 prior to formation of Rearr. 9. B) Rearr. 9. C) The synaptic configuration at meiotic prophase permitting maximum synapsis of homologous segments in a plant heterozygous for Rearr. 90/N9. The figure is adapted from MCCLINTOCK (1941).

36 OLIVER E. NELSON

TABLE 1

The frequency of Wx pollen grains in Sh Bz Tp9 (C+) V/sh bz N9 (fW) v heterozygotes (41192) and in Sh bz N9 (C+) v/sh bz N9 (+go) v heterozygotes (41193)-1971

Estimated no. Estimated no. Plant no. pollen grains scored W x X Plant no.' pollen grains scored W r X loe5

41192-1 43,188 -2 46,500 -3 71,876 -3.5 50,875 -4 44,813 -5 48,125 -6 42,063 -7 41,750 -8 36,875 -9 32,188

-1 1 33,000 -1 2 41,313 -13 101,938

Z = 681,504

-10 47,000

12 13 8 2

16 21 12 29 22 12 9

18 12 8

t = 12.7

41 193-2 -3 -4 -5 -6 -7 -8 -10 -1 1 -12 -13

52,000 38,313 55,625

36,938 92,626 35,062 43,313 43,750 55,562 42,438

44,375

77 47 70 65 79 62 57 58 39 36 35

2: = 539,903 t = 56.8

* Plants -1 and -9 were not considered in the totals since each had few (<20) W z gametes X 10-5 and were probably crossovers between bz and Tp9 in the previous generation.

In 1971, the two classes of seeds were planted in adjacent rows. A section of the main spike of each plant was collected for pollen analysis, and numerous pollinations were made from each plant on a bz (C+) u/bz (C+) U ; ae/ae tester. The results of the pollen analyses are given in Table 1 and the kernel analyses in Table 2. As has been previously reported (NELSON 1961 ) on the basis of pollen analyses alone, heterozygosity for Tp9 markedly depresses the frequency of recombination between the heteroalleles wx-90 and wx-C. With a concomitant conventional analysis here, it is possible to compare the pattern of flanking

TABLE 2

The frequency of wx/wx/Wx* seeds found when Sh Bz Tp9 (C+) V/sh bz N9 (+W) v; ae/ae heterozygotes (4192) and Sh bz NS (C+) v/sh bz N9 (+90) v; ae/ae heterozygotes (4193)

were used as male parents on Sh bz (C+) v/Sh bz (C+) v; ae/ae tester-1971

4192 4193 Total Number Freq. (X lF) Total Number Freq. (X

kernels wx/wx/Wr wx/wx/Wx kemels wx/wx/Wr wx/wr/Wz

156,015 24 14.7 65,495 38 58.0 Bz I/ E?: U bz V bz U + Sh sht 10 2 10 2 8 30

*Only those kernels are enumerated that have given plants on which test croeses on a

t This distribution corrected as described in the text for the weak competitive ability of Tp9-

$ Since the tester stock was Sh/Sh, it was necessary to self-pollinate the plants from non-waxy

biz (C+) v; ae tester enable the exclusion of contamination as an origin.

carrying gametes.

seeds to ascertain if the W z gamete was Sh o r sh.

INTRAGENIC RECOMBINATION IN MAIZE 37

markers in the W z recombinants to the pattern when no structural rearrangement is involved.

The Wx recombinants formed in the Sh Bz Tp9 (CY-) V/sh bz N9 (+90) U

heterozygotes had the following flanking marker pattern: 5 Bz V, 1 Bz U , 15 bz V and 3 bz U . However, these data must be corrected to compensate for the relatively poor competitive effectiveness of pollen grains that are Tp9; N3 and hence have the piece of chromosome 3 involved in the insertional translocation dupli- cated, as compared to pollen grains that are N9; N3. This was first observed by RHOADES (1958). NELSON (1964) reported that when plants of the genotype Sh Tp9 ( f 9 0 ) G115/sh N9 (C+) g115 were tested as male parents on an sh N9 (C+) g215/sh N9 (C+) gZ15 tester the percentages of fertilization effected by the Tp9 gametes were 25.4, 29.8, 26.5 and 28.9 for four different males. These values are similar to that observed in this experiment where in a total of 7261 kernels from 24 ears (2 each from 12 of the 13 heterozygotes used as male parents), 1748 or 24.1% had the bz marker that came into the cross linked to Tp9. Assuming no recombination between Bz and Tp9, this figure (24.1) indicates the percentage of megaspores fertilized by Tp9; N3 gametes competing with N9; N3 gametes. Thus the flanking marker pattern given above is distorted by fertilization by N9 gametes in 76% of the events. If fertilization had occurred in accordance with the gametic frequencies of 0.5 Tp9; 0.5 N9, one should observe (on correction to reflect the flanking marker pattern) a shift to 10 Bz V , 2 Bz U , 10 bz V a n d 2 bz U .

The control plants in this experiment, Sh bz N9 (C+) u/sh bz N9 (4-90) U ,

had recombinant W z gametes marked only distally ( S h us. sh) . Since the tester stock used was Sh/Sh, the plants from non-waxy seeds were self-pollinated to ascertain whether the W z gametes had been Sh or sh. Of the 38 Wx gametes recovered, 30 or 79% carried the distal marker linked with 90. The data presented by NELSON (1968) for flanking markers in W z gametes from the same heteroallelic cross show that 73% of the Wx gametes carried the distal marker ( B z ) linked to 90. Considering that the sh locus is more distal (2 crossover units) to the w x locus than is bz, the percentages of Wx gametes carrying the distal marker linked to 90 are in reasonable agreement for the two experiments.

It is interesting to compare the pattern of flanking markers from the experiment involving normal chromosome 9’s (NELSON 1968) to that observed here where one chromosome 9 has the insertional translocation. In the test with normal homologs where the genotype was Bz (4-90) V/bz (C+) U , there were 37 authenticated Wx gametes of which 1 was Bz V , 26 Bz U , 1 bz V and 9 bz U .

This observation confirms the conclusion that the site of wx-C is distal to that of wx-90 (NELSON 1962). Although the frequency of Wx gametes produced by (C+)/(+90) plants heterozygous for Tp9 was much reduced, the predominant class recombinant for flanking markers carried the distal marker originally linked with wx-90 and the proximal marker originally linked to wx-C, as when wx-90 and wx-C were both on structurally normal chromosomes. The larger class of W x gametes with a parental array of markers has the markers linked originally with wx-C, as was the case when both chromosome 9’s were normal. With

38 OLIVER E. ISELSON

TABLE 3

The frequency of W x pollen grains in Rearr. 9 (C+) V/N bz ($90) v heterozygotes-1967

Estimated no. Estimated no. Plant no. pollen grains scored W z X Plant no. pollen grains scored W z X

39276-1 139,813 16 39277-1 113,563 16 -2 -3 -4 -5 -6 -7 -8 -9 -10 -1 1 -12

161,313 112,688 97,500 90,3 13

117,938 133,468 119,188 140,0163 115,251 147,563 133,813

17 8

22 20 11 19 12 9

15 18 13

-2 128,563 -3 110,438 -4 122,688 -5 152,126 -6 130,688 -7 87,313 -8 184,875 -9 11 1,5oO -10 113,750 -1 1 139,48 -13 153,875 -14 139,500 -1 5 140,500

z = 3,337,698

7 13 11 18 12 5 8 9 4

19 14 14 10

Z=13 -

heterozygosity for Tp9, however, a much higher proportion of the W x gametes had the flanking markers associated with wx-C.

Heterozygosity for Rearr. 9: Tables 3 and 4 summarize the pollen and kernel data, respectively, for plants that were heterozygous Rearr. 9 Bz (C+) V / N 9 bz (+90) U. The frequency of W x pollen grains is reduced to approximately the same level as in heterozygotes for Tp9. Not as many w x / w x seeds were detected as the frequency of W x pollen grains would have predicted (1 7) for a population of this size. The W x gametes recovered have only the parental combinations of flanking markers. As found in the tests of normal chromosomes or the other two rearrangements, when the parental combinations are considered, the markers that entered the cross linked to wx-C are in the majority. A subsequent test indicated that the 4 Bz W x V gametes carried Rearr. 9 while the 2 bz W x U gametes did not.

Heterozygosity for In9a: The heterozygous plants from which male gametes were sampled were Bz Zn9a [V (+C) J/bz N9 ( f 9 0 ) U ; ae/ue. There were no

TABLE 4

The frequency of wx/wx/Wx* seeds detected when Rearr. 9 Bz (C+) V/bz N9 (+90) v; ae/ae heterozygotes were used as male parents on a bz (C+) v/bz (C+) v; ae/ae tester-1967

No. Xreq. (X 10-5) Total kernels wz/wx/wx wx/wx/Wx

131,863 6 4.6 Bz V Bz U bz V bz U

4 0 0 2

* Only those kernels are enumerated that have given plants on which test crosses enabled the exclusion osf contamination as an origin.

I N T R A G E N I C R E C O M B I N A T I O N I N M A I Z E 39

TABLE 5

The frequency of Wx pollen grains in Bz In9a [V(+C)]/bz N9 ($90) v heterozygotes-1966

Estimated no. wx x 10-5 Plant no. pollen grains scored

3M5-1 -2 -3 -4 -5 -6 -7 -8 -10 -1 1 -12 -1 3

42,0010

43,187 28,937 78,312 M,625 76,562 27,875 33,437 31,250 22,062 64,000

W,WO

Z = 532,,247

52 38 60 55 33 56 33 47 33 64 46 4a

t=46

normal plants testing the recombination between wx-90 and wx-C in this particular background, and comparisons must be between the values for plants heterozygous for In9a and the values observed for recombination between wx-90 and wx-C in other backgrounds. The pollen analysis data are given in Table 5 and the data from the analyses of conventional crosses in Table 6.

The data indicate that recombination within the wx locus (as measured by frequency of W x pollen grains) is affected less by heterozygosity for Zn9a than for the other two structural rearrangements under consideration. There is, however, a marked shift in the pattern of flanking markers. The majority of Wx gametes carry the parental combination of markers that were linked to the wx-C allele (Table 6 ) . The second most numerous class is that with the distal marker originally linked to the wx-90 allele and the proximal linked to wx-C. This arrangement of markers is the most numerous class when wz-90 and wx-C are located on normal chromosomes.

DISCUSSION

The data presented in Tables 2 ,4 and 6 in conjunction with previously pub-

TABLE 6

The frequency of wx/wx/Wx* seeds detected when Bz InQa [V(+C)]/bz N9 (+901) v; ae/ae heterozygo'tes were used as male parents on a bz (C+) v/bz (C+) v; ae/ae tester-I966

No. Freq. (X lod) Total kernels wx/wx/wx wx/wx/wx

82,311 31 38 Bz V Bz U bz V bz U 19 0 11 1

* Only these kernels are enumerated that have given plants on which test crosses enabled the exclusion of contamination as an o'rigin.

40 OLIVER E. NELSON

lished data support the conclusion that the proportion of Wx gametes that are convertants increases with heterozygosity for each of the structural rearrangements utilized in this study. In the drastic effect associated with heterozygosity for Rearr. 9, all the Wx gametes found are apparent convertants.

In order to evaluate the data in a comparative sense, it is necessary to utilize the data derived from previous experiments (NELSON 1962, 1968) as a normal standard since the most appropriate normal controls do not exist for the structural heterozygosity experiments. Secondly, although in the experiments involving Zn9a and Tp9, W x kernels were recovered at approximately the same frequency as observed in the pollen assays, this was not true of the 1968 normal experiment or the Rearr. 9 experiment. On the reasonable assumption that this failure stemmed from experimental error for the normal experiment, normalized distributions are calculated for this situation, utilizing the W x frequency in the pollen as the best estimate and then calculating the expected frequency of each flanking marker pattern from the observed proportion of each class. For plants heterozygous for Rearr. 9, there is another possible reason why the frequency of Wx gametes estimated from the conventional genetic test did not equal the frequency of Wx gametes estimated by pollen assays. As may be seen in Figure 1, a crossover between the sites of wx-C and wx-90 that includes the normal sequence at both sites is located on an acentric fragment. This fragment included in a microspore would presumably support the formation of amylose in the pollen grain, and such a pollen grain would be scored as Wx. If this ty-pe of pollen grain were to effect fertilization, the endosperm would not be Wx in phenotype since the acentric fragment would be lost early in development even if included in the nucleus that fertilized the polar bodies. For this reason, no correction is made for the Rearr. 9 data. If this is the correct explanation for the lower frequency of seeds fertilized by a Wx gamete as contrasted to the frequency of Wx pollen grains, it may be calculated from the data in Table 3 and 4 that about 35% of the events generating Wx gametes in plants heterozygous for Rearr. 9 carrying wx-C and a normal chromosome 9 carrying wx-90 result from gene conversion and the remainder, whether originating in a conversion event or not, include a single crossover between the sites of wx-90 and wx-C. This is not greatly different from that observed when wx-90 and wx-C are both located on normal chromosome 9’s. From Table 7, it can be calculated that the gene convertants (those Wx gametes with parental markers) are 31 % of the total Wx gametes. It is not clear that this comparison is meaningful, but it suggests that the same relative proportion of convertant gametes are produced under the severe restrictions imposed on recombination by heterozygosity for Rearr. 9 as in recombination between normal homologs. The summary in Table 7 presents frequencies of each marker class for the Wx gametes produced in the various experiments.

With heterozygosity for Tp9, there is a pronounced reduction in recombination within the wx locus as well as between markers distal to the wz locus (RHOADES 1958 and NELSON 1961). In this study, the proportion of Wx gametes that are the majority convertant class increases to numerical equality with the majority recombinant class which is the most numerous class in structurally normal

INTRAGENIC RECOMBINATION IN M A I Z E 41

TABLE 7

The frequency (x 1 0 - 5 ) and percentage of outside marker classes for Wx gametes produced by (C+/(+90) plants which. were either structurally normal or structural heterozygotes

as shown in the left-hand column+

Non-recombinant+ Recombinant+ I I1 I11 Iv

bz- (ct-). 22.3 2.5 2.5 44.6

Bz Tp9 IC+) V 6.4 1.2 1.2 6.4

Bz (+90) V (31%) (3.5%) (3.5%) (62%)

bz N9 ( f 9 0 ) U (42%) (8%) (8%) (42%)

bz N9 (+SO) U (62%) (3%) (3.5%)

Rearr.9 Bz (C+) V 3.0 1.5 0 0 N9 bz ($90) U (67%) (33%)

Bz In9a [V (+C) ] 27.5 1.3 0 15.8 ~ _ _ _ _

~~

~ ~ ~ ~ ~ ~

* The Tp9 data have beem corrected to compensate for the relative ineffectiveness of gametes carrying Tp9 to effect fertilization when competing with normal gametes. The data for the structurally normal chromosomes have been normalized to the Wx gamete frequency as explained in the text.

t I Carried the markers that entered the cross with wx-C. I1 Carried the markers that entered the cross with wx-90. I11 Carried the distal marker ( B z or bz) that entered the cross with wx-C and the proximal

IV Carried the distal marker (Bz o r bz) that entered the cross with wx-90 and the proximal marker from wx-90.

marker from wx-C.

homozygotes. In this structural heterozygote, there is not the complication of dicentric chromatids o r deficient/duplicate gametes following an exchange event. The reduction in recombination presumably results from an interference with pairing owing to the inserted segment, thus effectively resulting in reduced genetic distance. Thus, the bz locus is tightly linked to the wx locus in Tp9 heterozygotes. If the origin of the Wx gametes with the flanking markers that entered the cross linked to wx-C were an exchange between the heteroallelic sites and a second exchange between the wx and bz loci, the expectation is that the proportion of Bz Wx V gametes should be much reduced. The finding that the proportion of such gametes is enhanced, not reduced, indicates strongly that Bz W x V gametes do not arise via two separate exchanges but are a consequence of the event that produces the Wx gametes. The higher proportion of Bz Wx V gametes in the Wx gametes produced by plants heterozygous for Tp9 may be explained in terms of the phenomenon first noted by STADLER (1959) in crosses between cysteine-requiring mutants in Neurospora. STADLER found that gene conversion did not interfere with crossing over in adjacent regions. Assuming this to be valid for maize also, some of the convertants at the wx-C site would be found to be Bz W x U in crosses of Bz ( t 9 0 ) V/bz (e+) U on structurally normal chromosomes owing to crossing over between the bz and wx loci. Between 16% and 20% recombination has been found here in various tests. In the Tp9 heterozygotes, probably no convertants at the wx-C site will be recombinant in the region between bz and wx owing to the severe restrictions on recombination

42 OLIVER E. NELSON

distal to the w z locus in Tp9 heterozygotes. Thus all the convertants at the wx-C site would be preserved as such, enhancing the proportion of this class and attenuating the proportion of those W z gametes recombinant for flanking markers.

The flanking marker pattern observed in W x gametes from plants heterozygous for the long pericentric inversion Zn9a is interesting. The frequency of convertants with the parental markers that entered the cross linked to wz-C was as high as found in structurally normal plants, but the frequency of the majority recombinant class bz V has dropped drastically. The U locus is also in the inverted segment and apparently far removed from the breakpoint in the inversion. The marked decrease in the frequency of W z gametes with the proximal marker from wz-90 and the distal marker from wz-C reinforces the conclusion drawn from the results with Tp9 that the pattern of flanking markers in W x gametes from wz-C/wx-90 heteroallelic plants with structurally normal chromosomes is a reflection of the event(s) that produced the W z recombinant with relatively slight modification owing to proximal and distal exchange events. CHOVNICK ( 1973) has reported that in Drosophila melanogaster when interallelic recombination between rosy heteroalleles with one heteroallele placed in the center of a paracentric inversion was examined, non-rosy convertants were found at approximately the same frequency as in standard chromosome studies. However, the recovery of classical crossovers was markedly depressed. MCCARRON, GELBART and CHOVNICK (1 974) have reported the recovery from flies heterozygous for a long pericentric inversion and the heteroalleles ry41 and ry5 of ry4l and ry5 convertants in the same frequencies as from structurally normal flies. They found a partial restoration in the frequency of crossovers between the ry mutants resulting from a crossover between the mutant sites and a second crossover between the ry locus and one of the inversion breakpoints. This is essentially the same result observed with heterozygosity for Zn9a in maize (Table 7).

The results in the heterozygotes for Rearr. 9 contrast with those for Zn9u. As has already been indicated, a single exchange event in the wz region places the reconstituted Wx locus on an acentric fragment. Further, the wx locus is located close to one breakpoint in the rearrangement. The data of MCCLINTOCK (1941) allow the calculation that in the gametes produced by a male parent heterozygous for Rearr. 9, recombination between the wx locus and the breakpoint is only 0.15%. Recombination between sh and wx in the same cross is only 0.54%, as contrasted to the 20% observed in structurally normal homologs. Both calcula- tions include the crossovers resulting in dicentric chromatids. Thus, there may well be mechanical stresses that make pairing od homologous regions difficult in the vicinity of the wx locus. Such an effect could account for the difference in the frequency of convertants observed in heterozygotes for Zn9u as compared to Rearr. 9. As the results in Table 3 indicate, heterozygosity for Reurr. 9 drastically reduces the frequency with which W x gametes occur in (C+)/(+90) heteroallelic plants. Also, the W x gametes all possess a parental arrangement of outside markers, with the arrangement that entered with wx-C being more numerous although not to the extent observed with the other two rearrangements or with structurally normal homozygotes.

INTRAGENIC RECOMBINATION I N MAIZE 43 A constant feature of recombination data from wx-C/wx-90 heterozygotes is

the polarity observed-the parental arrangement of markers that entered the cross linked to wx-C is always in excess of the opposite arrangement. This tendency persists in all rearrangements, including Reurr. 9 where the order of mutant sites in the wz locus relative to the centromere is reversed. This agrees with the observation of MURRAY (1968) in Neurospora that direction of polarity is independent of orientation of a chromosome segment relative to the centromere. However, the reports of LEBLON (1972a,b) suggest that in Ascobolus the nature of the mutation, frameshift resulting from a base deletion, or base sub- stitution, may be the crucial determinant in crosses to wild type as to which strand will be converted preferentially. If the same considerations apply to maize, it would be expected that if there were excess conversion of wz-C in wx-C/wx-90 heterozygotes with structurally normal homologs, there would be excess conversion of wx-C in all structural heterozygotes.

I am indebted to HARLINGA KISDARJONO, AGNES TAN, MARGARET LIEN and MINC Tu CHANG for expert technical assistance.

LITERATURE CITED

BALLANTYNE, G. H. and A. CHOVNICK, 1971 Gene conversion in higher organisms: Non- reciprocal recombination events at the rosy cistron in Drosophila melanogaster. Genet. Res. 17: 139-149.

BRIGGS, R. W. and H. H. SMITH, 1965 Effects of X-radiation on intracistron recombination at the waxy locus in maize. J. Heredity 56: 157-162.

CHOVNICK, A., 1973 Gene conversion and transfer of genetic information within the inverted region of inversion heterozygotes. Genetics 75: 123-131.

FOGEL, S. and D. D. HURST, 1967 Meiotic gene conversion in yeast tetrads and the theory of recombination. Genetics 57: 455-481.

LEBLON, G., 1972a Mechanism of gene conversion in Ascobolus immersus. I. Existence of a correlation between the origin of mutants induced by different mutagens and their conversion spectrum. Mol. Gen. Genet. 115: 36-48. -, 1972b Mechanism of gene conversion in Ascobolus immersus. 11. The relationships between the genetic alterations in bl or b2 mutants and their conversion spectrum. Mol. Gen. Genet. 116: 322-335.

LISSOUBA, P. and G. RIZET, 1960 Sur 1' existence d'une unit6 gkn6tique polarisbe ne subissant que des kchanges non reciproques. Compt. Rend. Acad. Sci. D 250: 3408-3410.

MCCARRON, M., W. GELBART and A. CHOVNICK, 1974 Intracistronic mapping of electrophoretic sites in Drosophila melanogaster : Fidelity of information transfer by gene conversion. Genetics 76: 289-299.

MCCLINTOCK, B., 1941 26: 234-282.

The stability of broken ends of chromosomes in Zea mays. Genetics

MITCHELL, M. B., 1955 Aberrant recombination of pyridoxine mutants of Neurospora. Proc. Natl. Acad. Sci. U.S. 41: 215-220.

MURRAY, N. E., 1963 Polarized recombination and fine structure within the me-2 gene of Neurospora. Genetics 48: 1163-1183. - , 1968 Polarized intragenic recombination in chromosome rearrangements of Neurospora. Genetics 58: 181-191.

44 OLIVER E. NELSON

NELSON, 0. E., 1958 Intracistron recombination in the W d w x region of maize. Science 130: 794-795. -, 1961 The effect of heterozygosity for cytological aberrations on recombination within the waxy locus of maize. Genetics 46: 887 (Abstr.). -- , 1962 The waxy locus in maize. I. Intralocus recombination frequency estimates by pollen and by conventional analyses. Genetics 47: 737-742. -, 1964 Differential crossing Over in male and female gametes of plants heterozygous for Dp9. Maize Genet. Coop. News Letter 38: 124-126. -, 1968 The waxy locus in maize. 11. The location of the controlling element alleles. Genetics 60: 507-524.

The mutants of maize. Crop Sci. Soc., Amer., Madison, WI. pp. 74.

A genetic analysis of a duplication and a deficiency involving chromosomes 9 and 3. Maize Genet. Coop News Letter 32: 66-70. 1968 Studies on the cytological basis of crossing over. pp. 228-241. In: Replication and Recombination of Genetic Material. Edited by W. J. PEACOCK and R. D. BROCK. Aust. Acad. Sci., Canberra.

RHOADES, M. M. and E. B. PATTERSON, 1957 Fuither studies on the Li pericentric inversion in chromosome 9. Maize Genet. Coop. News Letter 31: 7677.

STADLER, D. R., 1959 The relation of gene conversion to crossing over in Neurospora. Proc. Natl. Acad. Sci. U.S. 45: 1625-1629.

STADLER, D. R. and A. M. TOWE, 1963 Recombination of allelic cysteine mutants in Neuro- spora. Genetics 48: 1323-1344.

Yu, M. H. and P. A. PETERSON, 1973 Influence of chromosomal gene position on intragenic

Corresponding editor: A. CHOVNICIC

NEUFFFZ, M. G., L. JONES and M. S. ZUBER, 1968

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recombination in maize. Theoret. App. Genet. 43: 121-133.

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