Abstract
Production of maternal haploids using a conspecific haploid inducer is routine and highly efficient in maize. However, the underlying mechanism of haploid induction (HI) is unclear. We develop a method to isolate three nuclei from a pollen grain and four microspores from a tetrad for whole-genome sequencing. A high rate of aneuploidy is observed at the three-nucleus stage (6/22 pollens) rather than at the tetrad stage (1/72 microspores) in one HI line CAU5. Frequent aneuploidy is also observed in another two inducer lines, but not in two regular lines, which implies that HI may be associated with pollen aneuploidy. We further sequence the individual embryos and endosperms of 88 maize kernels crossing between regular and inducer lines. Genome-wide elimination of the CAU5-derived chromosome is identified in eight of 81 embryos. Together, these results suggest that continuous chromosome fragmentation occurring post meiosis in the gametophyte may cause haploidy of the embryo.
Highlights
Production of maternal haploids using a conspecific haploid inducer is routine and highly efficient in maize
We proposed that a continuous large-scale chromosome fragmentation probably initiates around the mitotic stage of pollen development, and could be the cause of subsequent chromosome elimination during kernel development, which was supported by comparison of single-sperm sequencing results from two regular lines (B73 and Chang7-2) and two inducer lines (B73-inducer and CHOI3) (Table 1), we can not absolutely rule out the possibility that the sperm-derived genome may initiate fragmentation during embryonic mitosis to gradually give rise to haploid embryo
We proposed a model for haploid induction (HI) in which chromosome elimination is not accomplished at one stroke and may be initiated by chromosome defects occurring in the male gamete
Summary
Production of maternal haploids using a conspecific haploid inducer is routine and highly efficient in maize. We develop a method to isolate three nuclei from a pollen grain and four microspores from a tetrad for whole-genome sequencing. We make further efforts to improve the method for isolating the three nuclei from one mature pollen grain of maize for whole-genome sequencing. Combining the single embryo and endosperm sequencing of the hybrid kernel between regular (Zheng58) and inducer (CAU5) lines at 9 days after pollination (DAP), we conclude that chromosome fragmentation starting around the mitotic stage of pollen development is a probable key factor for HI. The proportion of pollen grains with low viability and no viability was higher, and the high viability class was much less abundant than in the regular lines (Fig. 1c, d; Supplementary Fig. 4c, d). 66 nuclei from 22 mature pollen grains of CAU5 were isolated, amplified, and whole genome sequenced
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