Maize Genetics Cooperation Newsletter vol 81 2007

An r1 haplotype‑specific aleurone color enhancer expressed only in female outcrosses

‑‑Stinard, PS

Members of the Fcu/Arv system of r1 haplotype‑specific aleurone color enhancers increase aleurone pigmentation in crosses to specific weakly pigmented r1 haplotypes (Kermicle, MNL 77:52; Stinard, MNL 77:77-79). The enhancers characterized to date have mapped to the same location on chromosome 2, and express equally well in male and female outcrosses to susceptible testers (Stinard, MNL 78:63-64; MNL 79:45; MNL 80:31; MNL 80:33). However, during the course of propagating an accession (R1‑r(Venezuela459#16039)) from the Brink r1 haplotype collection donated to the Stock Center by Jerry Kermicle, aberrant ratios appeared in crosses to tester stocks, indicating the possible presence of multiple enhancers. The ratios were not simple Mendelian ratios, and differed depending on the direction of the cross. 1:1 ratios of colored to pale kernels, or ears with completely pale kernels, were invariably obtained when a segregating Arv parent was used as the male parent, but when used as a female, ratios were either 1:1, or showed an excess of colored kernels. Subsequent generations of outcrossing allowed the separation of two enhancers, one (Arv‑V459A) that behaves as previously characterized enhancers, producing colored kernels in both male and female outcrosses, and one (Arv‑V459B) that produces colored kernels in female outcrosses, but only pale kernels in male outcrosses. The ratios of colored and pale kernels on ears of female outcrosses of plants heterozygous for both enhancers deviate from 3:1 ratios and indicate linkage, with an approximate separation of 25 centiMorgans.

Data from testcrosses are presented in Tables 1, 2, and 3; and linkage data are summarized in Table 4. All stocks are in a W22 inbred background, and tests for Arv status were conducted using testers and backgrounds carrying responsive r1 haplotypes (either R1-r(Venezuela459#16039) or R1-r(Venezuela559-PI302355)).

Data presented in Table 1 summarize the results of reciprocal crosses of plants carrying Arv‑V459A and/or Arv‑V459B with arv testers. The Arv parents were grown from the male outcross to an arv tester of a plant heterozygous for Arv‑V459A and homozygous for Arv‑V459B. Under the model presented above, colored kernels from such a cross would be expected to carry both Arv‑V459A and Arv‑V459B, and pale kernels would be expected to carry Arv‑V459B only. The results of reciprocal crosses of such progeny bear this out. All plants grown from colored kernels produced an excess (from a 1:1 ratio) of colored kernels in female outcrosses, and a 1:1 ratio of colored to pale kernels in male outcrosses. This result can be explained if both Arv‑V459A and Arv‑V459B are expressed in female outcrosses, but only Arv‑V459A is expressed in male outcrosses. All plants grown from pale kernels produced a 1:1 ratio of colored to pale kernels in female outcrosses, and only pale kernels in male outcrosses. Again, this result can be explained if Arv‑V459B is expressed in female, but not male, outcrosses. The kernel counts of female outcrosses of plants heterozygous for both Arv‑V459A and Arv‑V459B showed an excess of colored kernels from a 1:1 ratio, but significantly less than a 3:1 ratio (p < 0.05, calculations not shown), suggesting linkage in coupling of these two factors.

Data presented in Table 2 summarize the results of reciprocal crosses of plants carrying Arv‑V459B with arv testers. The Arv‑V459B parents were grown from kernels from the self-

Table 1. Counts of colored (Cl) and pale kernels from reciprocal crosses of plants carrying Arv‑V459A and Arv‑V459B with arv testers. Families 2005‑603 and 2005‑604 were planted from the cross: arv X Arv‑V459A/+; Arv‑V459B. Plants crossed in 2005‑603 were grown from colored kernels, and plants crossed in 2005‑604 were grown from pale kernels.

	Kernel No.; Female OC			Kernel No.; Male OC
Arv parent	Cl	pale	1:1 c^2	Cl	pale	1:1 c^2	Arv status
source: 2004‑830‑8/827‑6; Cl planted
2005‑603‑1	247	115	48.133^1	146	153	0.164	A + B
2005‑603‑2	213	142	14.200^1	145	132	0.610	A + B
2005‑603‑4	162	124	5.049^1	147	151	0.054	A + B
2005‑603‑6	170	112	11.929^1	99	74	3.613	A + B
2005‑603‑7	186	100	25.860^1	124	122	0.016	A + B
2005‑603‑8	217	121	27.266^1	112	125	0.713	A + B
2005‑603‑9	220	105	40.692^1	85	114	4.226	A + B
source: 2004‑830‑8/827‑6; pale planted
2005‑604‑1	89	87	0.023	0	309		B
2005‑604‑6	171	166	0.074	0	248		B
2005‑604‑7	147	139	0.224	0	287		B
2005‑604‑9	221	186	3.010	0	317		B
2005‑604‑10	183	155	2.320	0	285		B
2005‑604‑11	147	168	1.400	0	191		B
2005‑604‑12	120	155	4.455	0	340		B

Two‑sided c² value significant at p = 0.05 level.

Table 2. Counts of colored (Cl) and pale kernels from reciprocal crosses of plants carrying Arv‑V459B with arv testers. Families 2005‑605 and 2006‑606 were planted from the cross: [Arv‑V459B/+] selfed. Plants crossed in 2005‑605 were grown from pale kernels, and plants crossed in 2006‑606 were grown from colored kernels.

	Kernel No.; Female OC			Kernel No.; Male OC
Arv parent	Cl	pale	1:1 c^2	Cl	pale	Arv status
source: 2004‑827‑7@; pale planted
2005‑605‑4	47	36	1.458	0	263	B
2005‑605‑5	103	89	1.021	0	216	B
2005‑605‑6	0	66		0	312	‑
2005‑605‑8	119	128	0.328	0	333	B
2005‑605‑11	66	51	1.923	0	254	B
2005‑605‑12	49	62	1.523	0	196	B
source: 2004‑827‑7@; Cl planted
2005‑606‑1	165	0		0	351	B
2005‑606‑3	45	41	0.186	0	250	B
2005‑606‑4	79	78	0.006	0	262	B
2005‑606‑5	27	31	0.276	0	335	B
2005‑606‑6	60	56	0.138	0	284	B
2005‑606‑9	139	0		0	378	B
2005‑606‑10	74	91	1.752	0	280	B
2005‑606‑11	54	0		0	333	B
2005‑606‑12	48	44	0.174	0	297	B

pollination of a plant heterozygous for Arv‑V459B. Colored kernels from such a cross would be expected to be either heterozygous or homozygous for Arv‑V459B, with one copy of Arv‑V459B transmitted through the female; pale kernels would be expected to carry one copy of Arv‑V459B transmitted through the male, or be homozygous arv. The results of reciprocal crosses of such progeny bear this out. Plants grown from pale kernels produced either 1:1 ratios of colored to pale kernels (parent heterozygous for Arv‑V459B), or only pale kernels (parent homozygous for arv) in female outcrosses; and only pale kernels in male outcrosses. Kernels grown from colored kernels produced either all colored kernels (parent homozygous for Arv‑V459B) or 1:1 ratios of colored to pale kernels (parent heterozygous for Arv‑V459B) in female outcrosses, and only pale kernels in male outcrosses.

Tests were carried out another generation in order to confirm these inheritance patterns. The data presented in Table 3 summarize the results of these tests. The plants tested in family 2005P‑94 were grown from colored kernels from the male outcross to arv of a plant heterozygous for both Arv‑V459A and Arv‑V459B. All such plants would be expected to carry Arv‑V459A, and half should also carry Arv‑V459B. The female outcrosses of 3 out of 7 plants tested had ratios of colored to pale kernels that did not deviate significantly from 1:1, indicating parental heterozygosity for Arv‑V459A only, and the remaining 4 plants had an excess of colored kernels (but not a 3:1 ratio) in female outcrosses, indicating heterozygosity for both Arv‑V459A and Arv‑V459B. The male outcrosses of 3 plants segregated 1:1 for colored to pale kernels as expected. The remaining 4 plants were not outcrossed as males.

The plants tested in family 2005P‑95 were grown from colored kernels from the female outcross to arv of a plant heterozygous for Arv‑V459B. All such plants would be expected to be heterozygous for Arv‑V459B. The female outcrosses of all plants tested segregated 1:1 for colored to pale kernels, and the male outcrosses produced pale kernels only, as expected.

Table 3. Counts of colored (Cl) and pale kernels from reciprocal crosses of plants carrying Arv‑V459A and Arv‑V459B with arv testers. Family 2005P‑94 was planted from the cross: arv X Arv‑V459A/+; Arv‑V459B/+. Family 2005P‑95 was planted from the cross: Arv‑V459B/+ X arv. Both families were grown from colored kernels.

	Kernel No.; Female OC			Kernel No.; Male OC
Arv parent	Cl	pale	1:1 c^2	Cl	pale	1:1 c^2	Arv status
source: 2005‑622‑2/603‑6; Cl planted
2005P‑94‑2	107	107	0	45	60	2.143	A
2005P‑94‑3	146	71	25.922^1	‑	‑		A + B
2005P‑94‑4	128	119	0.328	33	40	0.671	A
2005P‑94‑5	93	110	1.424	40	46	0.419	A
2005P‑94‑7	157	117	5.839^1	‑	‑		A + B
2005P‑94‑8	135	88	9.906^1	‑	‑		A + B
2005P‑94‑10	120	85	5.976^1	‑	‑		A + B
Source: 2005‑604‑6/619‑2; Cl planted
2005P‑95‑2	147	132	0.806	0	161		B
2005P‑95‑3	82	94	0.818	0	52		B
2005P‑95‑4	135	112	2.142	0	67		B
2005P‑95‑6	108	130	2.034	0	47		B
2005P‑95‑7	101	101	0	0	106		B

Two‑sided c² value significant at p = 0.05 level.

All female outcrosses of plants heterozygous for both Arv‑V459A and Arv‑V459B in coupling produced ears with ratios of colored to pale kernels deviating from the 3:1 ratio expected of independent segregation (p < 0.05, calculations not shown). Kernel counts from all such ears examined are summarized in Table 4. A chi‑square test of homogeneity revealed that the kernel color ratios are reasonably homogeneous in this data set, being homogeneous at the p = 0.01 level, but not at p = 0.05. Since the phenotypes of the parental class carrying both Arv‑V459A and Arv‑V459B cannot be distinguished from the two crossover classes carrying the individual factors, the number of individuals in this parental class was approximated as being equal to the parental class lacking both factors, namely the pale kernel class. Based on this assumption, the map distance between these two factors is calculated to be 25.2 +/‑ 0.8 centiMorgans. Direct multi‑point linkage tests will be conducted with factors on chromosome 2 in order to verify this result and to better place both factors.

Table 4. Summary of counts of colored (Cl) and pale kernels from the cross: Arv‑V459A/+; Arv‑V459B/+ X arv.

	Kernel counts
Arv parent	Cl	pale	freq. of Cl
2005‑603‑1	247	115	0.68
2005‑603‑2	213	142	0.60
2005‑603‑4	162	124	0.57
2005‑603‑6	170	112	0.60
2005‑603‑7	186	100	0.65
2005‑603‑8	217	121	0.64
2005‑603‑9	220	105	0.68
2005P‑94‑3	146	71	0.67
2005P‑94‑7	157	117	0.57
2005P‑94‑8	135	88	0.61
2005P‑94‑10	120	85	0.59
Totals	1973	1180	0.63

Homogeneity c² = 22.767, df = 10.

Data are homogeneous at the p = 0.01 level, but not at the p = 0.05 level.

Linkage between Arv‑V459A and Arv‑V459B = 25.2 +/‑ 0.8 cM.

Tests have not yet been performed to determine whether the differential expression of Arv‑V459B is due to a dosage effect or to imprinting—this will be the subject of further study. Mapping will be conducted with chromosome 2 markers to determine which (if either) of these two factors maps to the same position as Fcu, and to determine a more precise location for the second factor.

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