High frequency DNA rearrangement at qγ27 creates a novel allele for Quality Protein Maize breeding

Abstract

Copy number variation (CNV) is a major source of genetic variation and often contributes to phenotypic variation in maize. The duplication at the 27-kDa γ-zein locus (qγ27) is essential to convert soft endosperm into hard endosperm in quality protein maize (QPM). This duplication is unstable and generally produces CNV at this locus. We conducted genetic experiments designed to directly measure DNA rearrangement frequencies occurring in males and females of different genetic backgrounds. The average frequency with which the duplication rearranges to single copies is 1.27 × 10^-3 and varies among different lines. A triplication of γ27 gene was screened and showed a better potential than the duplication for the future QPM breeding. Our results highlight a novel approach to directly determine the frequency of DNA rearrangements, in this case resulting in CNV at the qγ27 locus. Furthermore, this provides a highly effective way to test suitable parents in QPM breeding.

Keywords: Agricultural genetics; Genome duplication.

Fig. 1. Genetic designation of measurement of DNA rearrangement frequency at the γ27 locus.

a Structure of the γ27 locus. B73 bears the Ra allele, Mo17 and K0326Y the Saa allele, and W22 and A188 the Sab allele. The K-D line bears a 1.38-Mb deletion at the locus, entirely removing the γ27 locus. The junction ends of the deletion are shown in an enlarged window, wherein four genes near the two ends are indicated. GRMZM2G117230 and GRMZM2G003179 are located outside of the deletion and retain in the K-D genome, whereas GRMZM2G138689 and GRMZM2G318319 reside in the deletion, thereby missing in K-D; b SDS-PAGE analysis of zein proteins in K0326Y and K-D. Total zein from 200 μg of corn flour was loaded in each lane. Each subgroup of zeins is indicated beside the gel. M, protein markers from top to bottom correspond to 25, 20, and 15 kDa; c Screening rearranged events by PCR amplification of genomic DNA from crosses of different inbred lines with K-D. A rearranged event is indicated by a red box. 0707-L (2387 bp) and 0707-S (464 bp) are the larger and smaller PCR bands amplified from ARID4-L and ARID-S, respectively; d Diagrammatic representation of nonallelic homologous recombination mediated DNA rearrangement at the γ27 locus for the Sab allele. ➀, ➁, and ➂ indicate the locations that DNA rearrangement may occur at this locus.

Fig. 2. Structural analysis of DNA rearrangement at the γ27 locus.

a–d Structure of the γ27 locus duplication. Nine pairs of polymorphisms (SNP1/1*, SNP2/2*, SNP3/3*, 0707-1S/L, SNP4/4*, SNP5/5*, SNP6/6*, SNP7/7*, and SNP8/8*) for analysis of rearranged sites are indicated. Based on SNP detection, the regions wherein rearrangement occurred were drawn in dotted lines. The number of events for each region was indicated in the bracket beside. a K0326Y; b W22; c A188; d Mo17.

Fig. 3. Screening rearranged alleles with a triplication of γ27 gene in UniformMu stocks.

a Quantitative analysis of γ27 copies in different UniformMu stocks by genomic real-time PCR. The single-copy gene Pbf was used as the internal control; b SDS-PAGE analysis of zein proteins in UniformMu stocks with one, two and three copies of γ27 gene. The level of γ27 protein is generally proportional to the copy number. The accession number for each stock is indicated above the corresponding lane. Each subgroup of zeins is indicated beside the gel. 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; c The ratios of the A (γ27-A) and B (γ27-B) copies of γ27 gene in different stocks. The PCR products amplified from the leaf genomic DNA with the γ27 gene primers and cloned for sequencing, and the genomic DNA numbers of two γ27 alleles are expected to be 1:1 (expectation rate). χ²_0.05 <3.84; d Allelic expression of γ27 gene in different lines. The triplet endosperm is formed by the fusion of one sperm of the male parent with the two polar nuclei of the female parent, and W22 contains two copies of γ27 gene while XF134 contains one copy, the cDNA numbers of two γ27 alleles (female from W22 and male from XF134) are expected to be 4:1 (expectation rate). χ²_0.05 < 3.84.

Fig. 4. PB phenotypes in Ra, Sab, and Rabb endosperm cells.

a Ra; b Sab; c Rabb; d Statistical analysis of PB densities in Ra, Sab and Rabb endosperm cells. It is calculated by the distance of two neighboring PBs.

Fig. 5. Endosperm modification in αRNAi by Rabb.

a Ear phenotypes of αRNAi, Rabb and αRNAi-M (modified αRNAi by Rabb); b SDS-PAGE analysis of zein proteins in αRNAi, Rabb and αRNAi-M. Three kernels for each genotype were analyzed. Each subgroup of zeins is indicated beside the gel. 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.

Fig. 6. The Rabb allele increases the number and size of PBs in αRNAi-M.

a, b TEM of αRNAi (a) and αRNAi-M (b) endosperm cells at ×1000 magnification. c, d TEM of αRNAi (c) and αRNAi-M (d) endosperm cells at ×2000 magnification. SG, starch granule; PB, protein body; RER, rough endoplasmic reticulum; e Statistical analysis of the number of PBs in a field of view at ×1000 magnification. **P < 0.01 as determined by Student’s t-test; f Statistical analysis of PB diameters in αRNAi and αRNAi-M. *P < 0.05 as determined by Student’s t-test.