Gene duplication confers enhanced expression of 27-kDa γ-zein for endosperm modification in quality protein maize

Abstract

The maize opaque2 (o2) mutant has a high nutritional value but it develops a chalky endosperm that limits its practical use. Genetic selection for o2 modifiers can convert the normally chalky endosperm of the mutant into a hard, vitreous phenotype, yielding what is known as quality protein maize (QPM). Previous studies have shown that enhanced expression of 27-kDa γ-zein in QPM is essential for endosperm modification. Taking advantage of genome-wide association study analysis of a natural population, linkage mapping analysis of a recombinant inbred line population, and map-based cloning, we identified a quantitative trait locus (qγ27) affecting expression of 27-kDa γ-zein. qγ27 was mapped to the same region as the major o2 modifier (o2 modifier1) on chromosome 7 near the 27-kDa γ-zein locus. qγ27 resulted from a 15.26-kb duplication at the 27-kDa γ-zein locus, which increases the level of gene expression. This duplication occurred before maize domestication; however, the gene structure of qγ27 appears to be unstable and the DNA rearrangement frequently occurs at this locus. Because enhanced expression of 27-kDa γ-zein is critical for endosperm modification in QPM, qγ27 is expected to be under artificial selection. This discovery provides a useful molecular marker that can be used to accelerate QPM breeding.

Keywords: QPM; artificial selection; endosperm; gene duplication; o2 modifiers.

Fig. S1.

SDS/PAGE analysis of zein proteins in QPM K0326Y, B73, and their reciprocal crosses. Total zein from 200 μg of corn flour was loaded in each lane. Because of the abundance, the levels of 27-kDa γ-zein were directly compared on SDS/PAGE based the intensity of Coomassie staining. The level of 27-kDa γ-zein is proportional to the genetic dosage of QPM (K0326Y > K0326Y × B73 > B73 × K0326Y > B73). The 27-kDa γ-zein is indicated by the arrow.

Fig. 1.

Silencing of Pbf in QPM. (A) Ear phenotypes of K0326Y and K0326Y × o2;PbfRNAi/+. In K0326Y × o2;PbfRNAi/+, half of the progeny inherited the PbfRNAi transgene and expressed a reduced level of 27-kDa γ-zein and exhibited an opaque phenotype. Two representative vitreous and opaque seeds are indicated by asterisks and arrowheads. (B) Truncated seed phenotypes. The left two seeds not inheriting the PbfRNAi are vitreous, whereas the right two positive seeds are opaque. (C) The seeds inheriting the PbfRNAi expressed a decreased level of 27-kDa γ-zein. The 27-kDa γ-zein is indicated by the arrow.

Fig. S2.

Allelic expression of 27-kDa γ-zein gene in different lines. (A) XF134 expressing a shorter 27-kDa γ-zein. (B) An 18-bp deletion in XF134-γ27. (C) Allelic examination of 27-kDa γ-zein gene expression in XF134, B73, K0326Y, and Mo17. Because the endosperm is triploid (two from the female and one from the male), the cDNA numbers of two 27-kDa γ-zein alleles are expected to be 2:1 (expectation rate), if the parent lines don’t have qγ27 or qγ27 acts in a trans manner. Superscript “a” represents χ²_0.05 = 3.84.

Fig. 2.

GWAS and linkage mapping of qγ27. (A) SDS/PAGE analysis of zein proteins from different inbred lines. K0326Y QPM, Mo17, B97, CML322, CML333, Ky21, and MS71 expressed higher levels of 27-kDa γ-zein than B73, CML103, CML228, CML247, Ki3, A619, and Nc350. The 27-kDa γ-zein is indicated by the arrow. Total zein from 200 μg of corn flour was loaded in each lane. M, protein markers from top to bottom correspond to 25, 20, and 15 kDa. The zein protein analysis was the same in all of the following investigations. (B) Manhattan plot of the 27-kDa γ-zein levels. The numbers on the horizontal axis indicate the maize chromosomes. (C) SDS/PAGE analysis of zein proteins in different IBM lines. The 27-kDa γ-zein is expressed at a higher level in lines 181, 184, 187, 188, and 192 than in 182, 183, 185, 186, 189, 190, and 191, which are similar to levels of their parent lines Mo17 and B73, respectively. (D) Linkage analysis of the 27-kDa γ-zein expression. (Left) The logarithm of the odds (LOD) score profile with the permutation threshold indicated by the horizontal line; right, the LOD score profile for the enlarged chromosome 7 region.

Fig. 3.

Map-based cloning of qγ27. (A) Location of qγ27 on the physical map by GWAS. (B) Location of qγ27 on the physical map by the linkage analysis of 280 IBM individuals. (C) Fine-mapping of qγ27 using the population of (Mo17 × B73) × B73 with 6912 seeds. The numbers under each bar indicate the number of recombinants between qγ27 and the molecular marker. (D) Genotypes and phenotypes of the recombinants. (E) Mo17 scaffold 4130. (F) BACs 432 and 378 are overlapped and yield a continuous 258-kb sequence.

Fig. S3.

Phenotyping of 27-kDa γ-zein expression in the F₁BC₁ population of (Mo17 × B73) × B73. Two representative gels are shown. “H” and “L” indicate high and low level of 27-kDa γ-zein.

Fig. 4.

Sequence alignment of the two haplotypes of B73 and Mo17. (A) The contiguous sequence of the Mo17-γ27 locus determined in this study was aligned with B73 orthologous region. The small and long black bars indicate the 50-kDa and 27-kDa γ-zein locus, respectively. The red bar in Mo17 represents the duplicated fragment of the 27-kDa γ-zein locus. Sequence homology between the two inbreds is connected by vertical gray areas. Scale markers for both contiguous sequences are indicated. (B) Gene duplication of the 27-kDa γ-zein locus in Mo17. The solid boxes represent genes. Compared with B73, the duplicated region is illustrated in red. Genes 1, 2, 3, and 4 represent the 27-kDa γ-zein gene, GRMZM2G565441, GRMZM2G138976, and GRMZM5G873335, respectively.

Fig. S4.

Copy-number variation and gene expression. (A) Southern blot of 27-kDa γ-zein gene in B73, Mo17, and K0326. (B) Quantitative expression analysis of 27-kDa γ-zein gene, GRMZM2G138976, and GRMZM5G873335 in B73 and Mo17. Because Mo17 contains the duplication, RNA transcript levels of the three genes are from their two copies. **P < 0.01.

Fig. S5.

Sequence alignment of 27-kDa γ-zein gene (copies) from B73 and Mo17. The ORF region of 27-kDa γ-zein gene is indicated by an asterisk.

Fig. S5.

Sequence alignment of 27-kDa γ-zein gene (copies) from B73 and Mo17. The ORF region of 27-kDa γ-zein gene is indicated by an asterisk.

Fig. S5.

Sequence alignment of 27-kDa γ-zein gene (copies) from B73 and Mo17. The ORF region of 27-kDa γ-zein gene is indicated by an asterisk.

Fig. 5.

Association of the elevated expression of 27-kDa γ-zein and the gene duplication. (A) SDS/PAGE analysis of zein proteins in different QPM lines. All QPMs accumulate significantly higher levels of 27-kDa γ-zein than the wild-type inbred CM105 and its o2 mutant CM105o2. The 27-kDa γ-zein is indicated by the arrow. (B) There is gene duplication at the 27-kDa γ-zein locus in all QPMs. The size of each band is indicated beside. M, DNA markers.

Fig. S6.

Gene duplication of the 27-kDa γ-zein locus in CM105Mo2. (A) SDS/PAGE analysis of zein proteins in the isogenic lines CM105, CM105o2, and CM105Mo2. The QPM line CM105Mo2 accumulates a significantly higher level of 27-kDa γ-zein than the other two lines. (B) Absence and presence of the duplication in the three isogenic lines.

Fig. S7.

Polymorphism of A and B copies of 27-kDa γ-zein gene. B73, Mo17, and K0326Y bearing the respective Ra, Saa, and Saa allele, show a unique peak across the CDS, whereas the standard W22 and A188 bearing the Sab allele exhibit the double peaks at the polymorphic sites. A special stock of W22, W22-Rb, has the only B copy resulting from DNA rearrangement or deduplication at the 27-kDa γ-zein locus.

Fig. 6.

Comparison of 27-kDa γ-zein expression in the wild-type W22 and W22-Rb stock. (A) Comparatively lower accumulation of 27-kDa γ-zein in W22-Rb stock. (B) Absence of the duplication in the W22-Rb stock.

Fig. S8.

Gene duplication of the 27-kDa γ-zein locus in Z. mays ssp. Parviglumis. All accessions but Ames28084 and Ames 28101 have the same duplication of the 27-kDa γ-zein locus as Mo17.