The RUBY reporter enables efficient haploid identification in maize and tomato.

In vivo haploid induction has been extended from maize to monocotyledonous plants like rice, wheat, millet and dicotyledonous plants such as tomato, rapeseed, tobacco and cabbage. Accurate identification of haploids is a crucial step of doubled haploid technology, where a useful identification marker is very pivotal. R1-nj is an extensively used visual marker for haploid identification in maize. RFP and eGFP have been shown to be feasible in identifying haploid. However, these methods are either limited to specific species, or require specific equipment. It still lacks an efficient visual marker that is practical across different crop species. In this study, we introduced the RUBY reporter, a betalain biosynthesis system, into maize and tomato haploid inducers as a new marker for haploid identification. Results showed that expression of RUBY could result in deep betalain pigmentation in maize embryos as early as 10 days after pollination, and enabled 100% accuracy of immature haploid embryo identification. Further investigation in tomato revealed that the new marker led to deep red pigmentation in radicles and haploids can be identified easily and accurately. The results demonstrated that the RUBY reporter is a background-independent and efficient marker for haploid identification and would be promising in doubled haploid breeding across different crop species.

Genome-wide association and genomic prediction for resistance to southern corn rust in DH and testcross populations.

Southern corn rust (SCR), caused by Puccinia polysora Underw, is a destructive disease that can severely reduce grain yield in maize (Zea mays L.). Owing to P. polysora being multi-racial, it is very important to explore more resistance genes and develop more efficient selection approaches in maize breeding programs. Here, four Doubled Haploid (DH) populations with 384 accessions originated from selected parents and their 903 testcross hybrids were used to perform genome-wide association (GWAS). Three GWAS processes included the additive model in the DH panel, additive and dominant models in the hybrid panel. As a result, five loci were detected on chromosomes 1, 7, 8, 8, and 10, with P-values ranging from 4.83×10-7 to 2.46×10-41. In all association analyses, a highly significant locus on chromosome 10 was detected, which was tight chained with the known SCR resistance gene RPPC and RPPK. Genomic prediction (GP), has been proven to be effective in plant breeding. In our study, several models were performed to explore predictive ability in hybrid populations for SCR resistance, including extended GBLUP with different genetic matrices, maker based prediction models, and mixed models with QTL as fixed factors. For GBLUP models, the prediction accuracies ranged from 0.56-0.60. Compared with traditional prediction only with additive effect, prediction ability was significantly improved by adding additive-by-additive effect (P-value< 0.05). For maker based models, the accuracy of BayesA and BayesB was 0.65, 8% higher than other models (i.e., RRBLUP, BRR, BL, BayesC). Finally, by adding QTL into the mixed linear prediction model, the accuracy can be further improved to 0.67, especially for the G_A model, the prediction performance can be increased by 11.67%. The prediction accuracy of the BayesB model can be further improved significantly by adding QTL information (P-value< 0.05). This study will provide important valuable information for understanding the genetic architecture and the application of GP for SCR in maize breeding.

ZmCOI2a and ZmCOI2b redundantly regulate anther dehiscence and gametophytic male fertility in maize.

In higher plants, the generation and release of viable pollen from anthers is vital for double fertilization and the initiation of seed development. Thus, the characterization of genes related to pollen development and anther dehiscence in plants is of great significance. The F-box protein COI1 plays a crucial role in the jasmonate (JA) signaling pathway and interacts with many JAZ family proteins in the presence of jasmonoyl-isoleucine (JA-Ile) or coronatine (COR). The mutation of AtCOI1 in Arabidopsis leads to defective anther dehiscence and male sterility (MS), although COI has not been shown to affect fertility in Zea mays (maize). Here we identified two genes, ZmCOI2a and ZmCOI2b, that redundantly regulate gametophytic male fertility. Both ZmCOI2a and ZmCOI2b are highly homologous and constitutively expressed in all tissues tested. Subcellular localization revealed that ZmCOI2a and ZmCOI2b were located in the nucleus. The coi2a coi2b double mutant, generated by CRISPR/Cas9, had non-dehiscent anthers, delayed anther development and MS. In addition, coi2a coi2b male gametes could not be transmitted to the next generation because of severe defects in pollen germination. The JA content of coi2a coi2b anthers was unaltered compared with those of the wild type, and the exogenous application of JA could not rescue the fertility defects of coi2a coi2b. Transcriptome analysis showed that the expression of genes involving the JA signaling transduction pathway, including ZmJAZ3, ZmJAZ4, ZmJAZ5 and ZmJAZ15, was affected in coi2a coi2b. However, yeast two-hybrid assays showed that ZmJAZs interacted with ZmCOI1s, but not with ZmCOI2s. In conclusion, ZmCOI2a and ZmCOI2b redundantly regulate anther dehiscence and gametophytic male fertility in maize.

A DMP-triggered in vivo maternal haploid induction system in the dicotyledonous Arabidopsis.

Doubled haploid technology using inducer lines carrying mutations in ZmPLA1/MTL/NLD and ZmDMP1-4 has revolutionized traditional maize breeding. ZmPLA1/MTL/NLD is conserved in monocots and has been used to extend the system from maize to other monocots5-7, but no functional orthologue has been identified in dicots, while ZmDMP-like genes exist in both monocots and dicots4,8,9. Here, we report that loss-of-function mutations in the Arabidopsis thaliana ZmDMP-like genes AtDMP8 and AtDMP9 induce maternal haploids, with an average haploid induction rate of 2.1 ± 1.1%. In addition, to facilitate haploid seed identification in dicots, we established an efficient FAST-Red fluorescent marker-based haploid identification system that enables the identification of haploid seeds with >90% accuracy. These results show that mutations in DMP genes also trigger haploid induction in dicots. The conserved expression patterns and amino acid sequences of ZmDMP-like genes in dicots suggest that DMP mutations could be used to develop in vivo haploid induction systems in dicots.