Fertilization and uniparental chromosome elimination during crosses with maize haploid inducers

Abstract

Producing maternal haploids via a male inducer can greatly accelerate maize (Zea mays) breeding process. However, the mechanism underlying haploid formation remains unclear. In this study, we constructed two inducer lines containing cytogenetic marker B chromosome or alien centromeric histone H3 variant-yellow fluorescent protein vector to investigate the mechanism. The two inducer lines as the pollinators were crossed with a hybrid ZhengDan958. B chromosomes were detected in F1 haploids at a low frequency, which was direct evidence to support the occurrence of selective chromosome elimination during haploid formation. We found that most of the inducer chromosomes were eliminated in haploid embryonic cells during the first week after pollination. The gradual elimination of chromosomes was also detected in the endosperm of defective kernels, although it occurred only in some endosperm cells as late as 15 d after pollination. We also performed a genome-wide identification of single nucleotide polymorphism markers in the inducers, noninducer inbred lines, and 42 derived haploids using a 50K single nucleotide polymorphism array. We found that an approximately 44-Mb heterozygous fragment from the male parent was detected in a single haploid, which further supported the occurrence of paternal introgression. Our results suggest that selective elimination of uniparental chromosomes leads to the formation of haploid and possible defective kernels in maize as well, which is accompanied with unusual paternal introgression in haploid cells.

Figure 1.

Experimental flow chart for developing and identifying haploid induction lines containing B chromosome and CENH3-YFP. The CAU5 with a high HIR of 8.79% was the recurrent parent, while HiII^YFP and B73+B were used as the donor parents, respectively. YFP signals or Bs were detected in the high HIR lines in each generation.

Figure 2.

Identification of YFP signals and B chromosomes in the haploid induction lines. A, Metaphase chromosomes were hybridized with ZmBs probe (red) from the CAU^B line. Bar = 10 μm. B, YFP signals from the CAU^YFP line were observed directly under epifluorescence microscope. Bar = 10 μm. Diploid (C-1) and Haploid (C-2) was offspring from ZD958×CAU^B. Bar = 1 cm.

Figure 3.

Karyotyping of the somatic chromosomes of progeny from the cross of ZD958 × CAU^B probed with CentC (green) and ZmBs (red). A, Diploid without B chromosome. B, Diploid with two B chromosomes. C, Haploid without B chromosome. D, Haploid with one B chromosome. E and E’, Haploid with one abnormal B chromosome. F, Haploid with two B chromosomes. Arrows in B and D to and F denote the B chromosome(s). Bars = 10 μm.

Figure 4.

Ploidy determination in the 7- and 10-DAP embryos of ZD958×CAU^B by the number of 45S rDNA and/or Cent4 signals. A to D2 are 7 DAP, and E to E2 are 10 DAP. FISH was conducted with 45S rDNA (red) and ZmBs (yellow). A and A’, Diploid with two B chromosomes. B and B’, Diploid without the B chromosome. C and C’, Haploid without the B chromosome. D, A wide-angle view of the mixoploid embryo at 7 DAP. Further enlarged cell clusters with 45S rDNA (red) and ZmBs (yellow) are shown in the inset of D1 and D2. E, A wide-angle view of the embryo at 10 DAP. Further enlarged cell clusters with Cent4 (red) and 45S rDNA (green) are shown in E1 and E2. Bars = 10 μm in A to C, A’ to C’, D1, D2, E1, and E2. Bars = 1 mm in D and E.

Figure 5.

Ploidy detection of aborted endosperm at different days after pollination. A to E are 13 DAP, F and G are 5 DAP, and H1 to H3 are 9 to 15 DAP. A, The first one was normal kernel and the others were defective kernels with different sizes. B, Enlarged view of the normal kernel in A with embryo (outlined with red), and the ovary wall was removed. C, Enlarged view of the defective kernel in A without embryo, and the ovary wall was removed. Bars = 500 μm. D, YFP signals in the endosperm of normal kernel. E, YFP signals in the endosperm of defective kernel. F, Spread of aborted endosperm at 5 DAP with three 45S rDNA (red, arrows) and two ZmBs (yellow). G, Interphase nucleus of an aborted endosperm at 5 DAP with three 45S rDNA (red) and three Cent4 (green). H1, Interphase nucleus of an aborted endosperm with two 45S rDNA (green) and two Cent4 (red). H2, An aborted endosperm with three 45S rDNA (green) and two Cent4 (red, arrows). H3, An aborted endosperm showed mixoploid. One cell showed three 45S rDNA (green) and three Cent4 (red); another cell showed two 45S rDNA (green, arrows) and three Cent4 (red). Bars = 10 μm

Figure 6.

Relative expression level of CENH3 in ZD958, CAU5, and B73 from different positions within CENH3 CDS. Fourteen-day-old seedlings grown in a greenhouse were sampled for gene expression analysis. The expression level was determined by qRT-PCR. Actin was used as the internal standard.

Figure 7.

The polymorphisms of SSR markers among haploid plants derived from the cross Z58 × CAU5. The lanes from 1 to 6 were CAU5, Z58, B18, B27, B33, and B7, respectively. B18, B27, B33, and B7 are haploids derived from the parents Z58 and CAU5. A, Polymorphism detected by marker X10. B, Polymorphism detected by marker X35. C, Polymorphism detected by marker umc1317. [See online article for color version of this figure.]

Figure 8.

Schematic illustration of the fate of fertilized embryo sac with B chromosomes (without considering the mechanism of B chromosomes accumulation). Most embryo sacs developed into diploid kernels with B chromosomes. A small number of them developed into haploid kernels with (seldom) or without B chromosome. The rest of embryos became defective kernels. The development of zygotes is complicated, and it is yet to be confirmed whether the embryo will be produced