Maize Genetics Cooperation Newsletter vol 85 2011

 

 

Characterization of two regulators of aleurone mottling—summary of research initiated at the University of Wisconsin.

 

            --Goncalves Butruille M, Stinard PS, Sachs MM, and Kermicle JL

 

            Kernel pigmentation in certain accessions of open-pollinated varieties from the Four Corners region of Southwest US is exceptional in that mottled aleurone is true breeding.  However, F1 kernels resulting from outcrosses with various stocks, including r1 testers, are fully colored.  A provisional test of inheritance was performed by crossing two such collections, Osage and Kokoma, to R1-sc:124 in W22 background.  F2 kernel progeny gave a 63:1 ratio of full color to dark and light mottled as expected for a three gene difference (Table 1).  (See Stinard et al., this MNL, for companion article.)  This outcome would result if the accessions carried three recessive factors relative to the R1-sc:124 stock: two regulators of mottling, provisionally termed mot1 and mot2, and a responsive r1 haplotype.  The r1 haplotype in these accessions proved to be of the R1-d class, which is subject to dilution by dominant modifiers (Stinard PS and Sachs MM, 2002. J Hered 93: 421-428).

 

            An r1-g mot1 mot2 tester was developed by backcrossing the Hopi mottled variants for four generations to a W22 inbred conversion of r1-g(Stadler), a colorless seed and plant haplotype. The tester was selfed and crossed during each generation of introgression to a true breeding mottled stock to confirm the presence of the mot factors.

 

            Twelve R1-d haplotypes isolated from geographic locations spread across western North America, plus the genetic stock R1-d(Arapaho), were chosen to further characterize interaction between the R1-d class of haplotypes and the mot factors.  The 13 R1-d stocks, each carried in W22 background, were crossed to the r1-g mot1 mot2 tester to make an F1. These F1s (genotype R1-d r1-g; Mot1 mot1; Mot2 mot2) were then reciprocally crossed to the r1-g mot1 mot2 tester.  In backcrosses with the F1 as female, six of the haplotypes showed a ratio of 4 colorless (cl) to 3 dark mottled (DMT) to 1 light mottled (LMT) expected if three factors were segregating (Table 2). Only 3 ears out of 28 crosses had chi-square values showing deviation from this ratio. The remaining seven haplotypes did not segregate for an obvious light mottled class when the R1-d haplotype-carrying parent was used as a female, indicating a lack of sensitivity of the R1-d haplotype to the mot factors in female backcrosses. These crosses showed a 1:1 segregation for colorless to dark mottled (Table 3).  Only 3 ears out of 34 had chi-square tests that showed significant deviation from a 1:1 ratio. However, the male backcrosses of all twelve haplotypes showed sensitivity of the R1-d haplotype to the mot factors.

 

            Since we know from the male backcrosses that these R1-d haplotypes are capable of responding to the mot factors, imprinting or a dosage effect is indicated to have caused stronger expression of the aleurone color of these R1-d accessions when passed through the female in the presence of the mot factors.  We call this the “strong imprinting response.”  On the other hand, the R1-d haplotypes responding to the mot factors when passed through the female can be said to have a null or weak response to imprinting on the female gametophyte, i. e. a “weak imprinting response.”  Although dosage effects have not been completely ruled out, we believe that imprinting differences are involved due to the effects of the mot factors on R1-r(standard) in classic imprinting experiments (data not shown).  Therefore, we tentatively conclude that R1-d haplotype-specific imprinting responses in interaction with the mot factors are responsible for the differences observed in the female backcrosses.

 

            In all crosses where R1-d is inherited through the male gametophyte (cross: [r1-g mot1 mot2] X [R1-d r1-g; Mot1 mot1; Mot2 mot2]), a wider variance in the color classes is observed than when R1-d is inherited through the female. A distinct medium mottling (MMT) class appears, and the dark mottled class is not as strong as when the R1-d haplotype is inherited from the female parent. This indicates that the “weak imprinting response” R1-d haplotypes do have a slight response to imprinting rather than a completely null response.  Thus the mot factors can be used to differentiate between R1-d haplotypes that respond strongly and weakly to imprinting.

 

            We hypothesize that one of the mot factors, arbitrarily named mot2 on this basis, has a stronger effect on seed color than the other.  The expected distribution of classes should be 4 cl (r1-g r1-g) : 2 DMT (R1-d r1-g; Mot1 mot1; Mot2 mot2 or R1-d r1-g; mot1 mot1; Mot2 mot2) : 1 MMT (R1-d r1-g; Mot1 mot1; mot2 mot2) : 1 LMT (R1-d r1-g; mot1 mot1; mot2 mot2) if mot2 has a stronger effect (see Figure 1). The chi-square tests for this ratio were non-significant for all but 4 out of 72 ears (data not shown).  So we conclude that Mot2 has a stronger effect on aleurone color than Mot1.  Both imprinting-sensitive and insensitive R1-d haplotypes show light mottling of the aleurone when inherited through the male gametophyte in the presence of both mot factors, but full color when the wild type Mot alleles are present.  This must be due to a compensation effect by the wild type Mot alleles that intensifies color (or prevents color reduction) even in the absence of imprinting, when R1-d is passed through the male gametophyte.  At the same time, this shows that the segregation ratio of 1:1 for the R1-d imprinting-sensitive haplotypes when R1-d is passed through the female (Table 5) is not due to them not being able to respond to the mot factors, but rather imprinting provides an alternative route to increasing r1 gene expression.

 

            Another interesting aspect of the mot factors’ effect on these geographic and tribal R1-d haplotypes is the differential effect on seed and seedling colors. The R1-d class of r1 haplotypes typically shows strong pigmentation of the aleurone and also of the scutellum and germinating roots.  We observed that medium mottled kernels from the male testcross, [r1-g mot1 mot2] X [R1-d r1-g; Mot1 mot1; Mot2 mot2], had colored scutella and produced seedling root color when germinated, but the light mottled kernels had colorless scutella and produced seedlings with green roots (Figure 2).  We conclude that only one of the factors is needed to produce color in the scutellum and roots. This factor does not cause strong seed color and therefore must be mot1, since mot2 was designated as the factor causing stronger seed color.  We hypothesize that a color class distribution of both seed and seedling should be as follows: 1 LMT/Green : 1 MMT/Red : 1 DMT/Green : 1DMT/Red, where Green and Red refer to plant colors, if two mot factors are segregating but these two mot factors have different effects on plant color.  These classes would correspond to: 1 LMT/Green = R1-d r1-g; mot1 mot1; mot2 mot2; 1 MMT/Red = R1-d r1-g; Mot1 mot1; mot2 mot2; 1 DMT/Green = R1-d r1-g; mot1 mot1; Mot2 mot2; and 1 DMT/Red = R1-d r1-g; Mot1 mot1; Mot2 mot2.  This ratio was generally observed when kernels were germinated and plant colors were scored (Table 4).  A few seedlings in two unexpected classes were also observed: LMT/Red and MMT/Green.  One possible explanation for these unexpected classes is heterofertilization; other possibilities include kernel color misclassifications and the presence of other as yet uncharacterized modifiers.  Chi-square tests of counts of seedlings grown from colored kernels of male testcrosses of four R1-d accessions, with two ears each, showed no deviation from the expected ratios for five out of the eight ears (Table 4).

 

            In summary, the true-breeding mottled phenotype observed in Southwestern Native American accessions of maize results from the interaction of a permissive R1-d haplotype with two mottling factors, mot1 and mot2.  For mottling to occur, the R1-d haplotype must be homozygous, or heterozygous with a colorless r1 haplotype, and mot1 and mot2 must be homozygous.  The R1-d haplotypes studied can be grouped into two classes based on phenotype in the presence of mot1 and mot2 when crossed reciprocally with r1-g mot1 mot2 testers:  (1) Weak imprinting response R1-d haplotypes produce light mottled kernels in the presence of mot1 and mot2 regardless of whether the R1-d haplotype is transmitted through the male or female gametophyte.  These are the R1-d haplotypes present in true-breeding light mottled lines.  (2) Strong imprinting response R1-d haplotypes produce dark mottled kernels when transmitted through the female, but light mottled kernels when transmitted through the male in the presence of mot1 and mot2.  Table 5 summarizes the origins of the various R1-d haplotypes studied and their pattern of imprintability observed in combination with homozygous mot1 mot2.  Note that an imprinting effect rather than an endosperm dosage effect was inferred on the basis of tests made using R1-r(standard).  More direct imprinting tests using R1-d(Arapaho) are in progress.

 

            Four of the R1-d haplotypes characterized for imprinting response were analyzed molecularly by Walker and Panavas (2001. Genetics 159:1201-1215), two strong responders and two weak responders (Table 5).  No differences were observed between the two types at the gross structural level—all four haplotypes showed the same structural features typical of R1-d haplotypes:  a q gene, an intact S2 gene, and a truncated S1 gene missing 5’ noncoding sequences.  The molecular basis for the difference in imprinting responses remains a question.

 

            The mot factors themselves have differential effects on intensity of aleurone and plant color produced by all R1-d haplotypes studied.  Seedlings grown from kernels carrying an R1-d haplotype and homozygous or heterozygous for the Mot1 allele produce plant color regardless of mot2 genotype; homozygous mot1 seedlings are green regardless of mot2 genotype.  Kernels carrying an R1-d haplotype and the Mot2 allele and homozygous for mot1 are more darkly mottled than kernels carrying an R1-d haplotype and the Mot1 allele and homozygous for mot2—this interaction is most evident when R1-d is transmitted through the male.  These differential interactions are the basis for distinguishing between the two mot factors.

 

            We used a bulk segregant analysis in an attempt to map the three factors involved in the aleurone mottling and seedling color effects. Seeds from 5 F2 ears of Navajo Robin’s Egg Corn (NREC) crossed to R1-sc:124 showing the 63:1 ratio of full color to mottled were germinated, plus one plant from each parental and one from F1 seed. Leaf discs from two to five of the F3 plants from each color class were pooled to produce a bulked DNA sample. The MaizeSNP50 Illumina Corn Chip was used for genotyping. Table 6 shows some statistics on data resulting from the analysis. We expected that polymorphic markers between bulked samples will be genetically linked to the color factors. Since these three factors act as recessive genes, we expect the colored pool to more likely show heterozygous calls for the markers in the linked region while the mottled pooled samples show homozygous calls, the same calls as for the mottling parent NREC.  By comparing the 5 paired pools, F1 and 2 parental lines using excel filters, we found three regions linked to the mottling effect as expected. The r1 gene position was confirmed by this analysis (Table 7) and is contained in the smaller interval detected on chromosome 10.  mot1 and mot2 were located to two other segments on chromosomes 3 and 4. Which factor is located on which chromosome is not known, since this population was not large enough to allow their phenotypic discrimination. Their physical positions on chromosome 3 and chromosome 4 are shown in Table 7. Exact genetic positions are not provided, but the approximate size of each genetic interval is suggested.


Figure 1.  Male backcross of a heterozygous R1-d:Arapaho r1-g Mot1 mot1 Mot2 mot2 plant to an r1-g mot1 mot2 tester.  Colored kernels are in three classes: dark mottled, medium mottled and light mottled in a 2:1:1 ratio. Seedlings grown from medium mottled kernels were red, half of the seedlings grown from dark mottled kernels were red, and seedlings grown from light mottled kernels were green, indicating that the weak kernel mottling factor, Mot1, is responsible for induction of typical R1-d seedling color. Other R1-d geographic alleles showed variation in seedling pigmentation in response to the mottling factors.

 


Figure 2.  Seedling pigmentation phenotypes illustrating interactions between R1-d haplotypes and mottling factors mot1 and mot2.  (A) Green seedlings grown from R1-d:Arapaho mot1 mot2 kernels.  (B) Red seedlings grown from R1-d:Arapaho Mot1 Mot2 kernels.

 


Table 1.  Kernel counts from F2 ears of the cross of W22 R1-sc:124 X Hopi mottled accessions.  DMT = full colored and dark mottled.  LMT = light mottled.  Chi-square for 63 DMT : 1 LMT.

 

Source

R1-d haplotype

DMT

LMT

chi-square

significance

GB 871-1

Osage

452

9

0.455

NS

GB 871-2

Osage

570

11

0.413

NS

GB 872-1

Kokoma

446

6

0.162

NS

GB 872-2

Kokoma

456

6

0.209

NS

GB 872-3

Kokoma

454

11

1.950

NS

GB 872-4

Kokoma

446

10

1.179

NS

GB 872-5

Kokoma

448

8

0.109

NS

GB 872-6

Kokoma

533

10

0.275

NS

 


Table 2.  Kernel counts of female backcrosses of R1-d haplotypes showing “weak imprinting response” (light mottled kernels segregating).  Cross:  [R1-d r1-g; Mot1 mot2; Mot2 mot2] X [r1-g mot1 mot2].  cl = colorless kernels.  Chi-square for 4 cl : 3 DMT : 1 LMT.

 

Source

R1-d haplotype

PI number

cl

DMT

LMT

chi-square

significance

GB 645

Arizona-1

PI213729

206

186

54

3.453

NS

GB 645

Arizona-1

PI213729

202

161

39

3.221

NS

GB 645

Arizona-1

PI213729

233

186

41

5.83

NS

GB 645

Arizona-1

PI213729

147

102

29

1.46

NS

GB 645

Arizona-1

PI213729

172

141

48

0.817

NS

GB 652

Arizona-2

PI213738

207

158

65

2.718

NS

GB 652

Arizona-2

PI213738

244

195

113

32.464

P<.001

GB 652

Arizona-2

PI213738

171

142

45

0.806

NS

GB 652

Arizona-2

PI213738

178

143

36

2.29

NS

GB 651

Canada

PI214199

188

256

69

39.707

P<.001

GB 651

Canada

PI214199

115

96

21

3.147

NS

GB 651

Canada

PI214199

194

144

53

0.407

NS

GB 651

Canada

PI214199

185

136

47

0.056

NS

GB 651

Canada

PI214199

228

148

52

1.931

NS

GB 656

New Mexico-3

PI218150

166

111

41

0.918

NS

GB 656

New Mexico-3

PI218150

136

98

23

3.057

NS

GB 656

New Mexico-3

PI218150

160

130

50

1.961

NS

GB 656

New Mexico-3

PI218150

138

114

38

0.676

NS

GB 656

New Mexico-3

PI218150

158

142

44

2.473

NS

GB 658

New Mexico-5

PI218169

190

144

57

1.552

NS

GB 658

New Mexico-5

PI218169

121

130

34

8.322

P<.05

GB 658

New Mexico-5

PI218169

121

80

38

3.262

NS

GB 658

New Mexico-5

PI218169

210

153

58

0.701

NS

GB 658

New Mexico-5

PI218169

206

172

58

1.327

NS

GB 659

New Mexico-6

PI218173

160

115

50

2.59

NS

GB 659

New Mexico-6

PI218173

146

114

52

5.051

NS

GB 659

New Mexico-6

PI218173

156

97

38

2.178

NS

GB 659

New Mexico-6

PI218173

168

130

40

0.209

NS

 


Table 3.  Kernel counts of female backcrosses of R1-d haplotypes showing “strong imprinting response” (no light mottled kernels segregating).  Cross:  [R1-d r1-g; Mot1 mot2; Mot2 mot2] X [r1-g mot1 mot2].  Chi-square for 1 cl : 1 DMT.  (The few LMT kernels not included in chi-square tests.)

 

Source

R1-d haplotype

PI number

cl

DMT

LMT

chi-square

significance

GB 648

Iowa

PI217411

186

192

0

0.095

NS

GB 648

Iowa

PI217411

263

215

2

4.82

P<.05

GB 648

Iowa

PI217411

214

177

 

3.501

NS

GB 648

Iowa

PI217411

183

161

2

1.407

NS

GB 648

Iowa

PI217411

231

216

 

0.503

NS

GB653

N Dakota

PI213807

168

162

1

0.109

NS

GB653

N Dakota

PI213807

192

172

 

1.099

NS

GB653

N Dakota

PI213807

151

130

1

1.569

NS

GB653

N Dakota

PI213807

152

129

 

1.883

NS

GB653

N Dakota

PI213807

108

110

 

0.018

NS

GB 647

Oklahoma

PI213756

133

131

 

0.015

NS

GB 647

Oklahoma

PI213756

280

287

 

0.086

NS

GB 647

Oklahoma

PI213756

186

179

 

0.134

NS

GB 647

Oklahoma

PI213756

238

230

2

0.137

NS

GB 649

S Dakota 1

PI213779

194

179

2

0.603

NS

GB 649

S Dakota 1

PI213779

120

122

 

0.017

NS

GB 649

S Dakota 1

PI213779

209

221

3

0.335

NS

GB 649

S Dakota 1

PI213779

233

273

2

3.162

NS

GB650

Washington-1

PI217488

143

129

 

0.721

NS

GB650

Washington-1

PI217488

198

235

8

3.162

NS

GB650

Washington-1

PI217488

218

246

1

1.69

NS

GB650

Washington-1

PI217488

217

238

1

0.969

NS

GB650

Washington-1

PI217488

279

261

2

0.6

NS

GB660

Washington-2

PI217489

256

229

5

1.503

NS

GB660

Washington-2

PI217489

168

151

3

0.906

NS

GB660

Washington-2

PI217489

148

200

3

7.77

P<.01

GB660

Washington-2

PI217489

192

187

 

0.066

NS

GB660

Washington-2

PI217489

92

124

 

4.741

P<.05

GB 873

Arapaho

 

240

206

 

2.592

NS

GB 873

Arapaho

 

291

278

2

0.297

NS

GB 873

Arapaho

 

225

230

 

0.055

NS

GB 873

Arapaho

 

229

226

 

0.02

NS

GB 873

Arapaho

 

256

260

3

0.031

NS

GB 873

Arapaho

 

219

230

 

0.269

NS

 


Table 4.  Seedling phenotypes for colored kernels from test crosses: [r1-g mot1 mot2] X [R1-d r1-g; Mot1 mot1; Mot2 mot2].  Chi-square for 1 LMT/Green : 1 MMT/Red : 1 DMT/Green : 1 DMT/Red.  The few seedlings in unexpected classes were not included in chi-square calculations.

 

R1-d haplotype

PI number

imprinting