The RUBY reporter enables efficient haploid identification in maize and tomato

Abstract

Summary 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.

Genetic basis of maize maternal haploid induction beyond MATRILINEAL and ZmDMP

“…(2022) recently developed an efficient and accurate (99.1% accuracy) haploid selection marker through co-expression of two transcription factor genes ( ZmC1 and ZmR2 ) in novel haploid inducer line called ‘Maize Anthocyanin Gene InduCer 1 (MAGIC1)’. Shortly after, Wang et al. (2023) showed that expression of RUBY enabled 100% accuracy of haploid identification based on deep betalain pigmentation.…”

“…For instance, Chen et al (2022) recently developed an efficient and accurate (99.1% accuracy) haploid selection marker through co-expression of two transcription factor genes (ZmC1 and ZmR2) in novel haploid inducer line called 'Maize Anthocyanin Gene InduCer 1 (MAGIC1)'. Shortly after, Wang et al (2023) showed that expression of RUBY enabled 100% accuracy of haploid identification based on deep betalain pigmentation. The development of a group of inducers with numerous anthocyanin markers and HIRs above 12.0% allowed for effective haploid generation and screening (Rotarenco et al, 2010).…”

Haploid identification in maize

“…The upgraded version, MAGIC2, could identify haploids from diploids due to differential anthocyanin accumulation in immature embryos, coleoptiles, sheaths, roots, leaves, and dry seeds. Instead of anthocyanin synthesis pathway for haploid selection, Wang et al (2023) utilized the RUBY reporter, a betalain biosynthesis system, as a new biomarker. The expression of RUBY resulted in deep betalain pigmentation in maize embryos as early as 10 DAP and enabled 100% accuracy of immature haploid embryo identification.…”

CRISPR/Cas Technology Revolutionizes Crop Breeding

“…Importantly, a fast technique for visual screening of wheat haploids was developed by combining the haploid inducer generated by editing TaMTL and embryo-specific anthocyanin markers, providing a promising strategy for a large-scale haploid inducer in different crops [151]. Recently, a RUBY reporter system, a background-independent and efficient marker for haploid identification, has been established, which enables easy and accurate haploid identification in maize and tomato, which will be promising in double haploid breeding in different crops [152].…”