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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.

Wheat (Triticum aestivum L.) is one of the most important staple crops globally. Doubled haploid technology enables rapid development of pure lines and has been extended from maize to several other crop species. A key step in DH breeding is the identification of haploids from diploids, which requires accurate and convenient phenotypic markers. In this study, we generated two wheat haploid inducers carrying different markers by a one-step strategy. One harbored a dual fluorescent marker system consisting of eGFP and TagRFP, the other carried a RUBY reporter. Both markers enabled near 100% accuracy of haploid identification at the immature embryo, mature embryo, and germinating seedling stages. Moreover, both lines consistently exhibited a high and stable haploid induction rate (∼20%). This study not only provides efficient wheat haploid inducers but also establishes a convenient pipeline for developing haploid induction systems in other crop species.

Doubled haploid (DH) technology becomes more routinely applied in maize hybrid breeding. However, some issues in haploid induction and identification persist, requiring resolution to optimize DH production. Our objective was to implement simultaneous marker-assisted selection (MAS) for qhir1 ( MTL/ZmPLA1/NLD ) and qhir8 ( ZmDMP ) using TaqMan assay in F 2 generation of four BHI306-derived tropical × temperate inducer families. We also aimed to assess their haploid induction rate (HIR) in the F 3 generation as a phenotypic response to MAS. We highlighted remarkable increases in HIR of each inducer family. Genotypes carrying qhir1 and qhir8 exhibited 1 – 3-fold higher haploid frequency than those carrying only qhir1 . Additionally, the qhir1 marker was employed for verifying putative haploid seedlings at 7 days after planting. Flow cytometric analysis served as the gold standard test to assess the accuracy of the R1-nj and the qhir1 marker. The qhir1 marker showed high accuracy and may be integrated in multiple haploid identifications at early seedling stage succeeding pre-haploid sorting via R1-nj marker.

Background Anthocyanins are widely applied as a marker for haploid identification after haploid induction in maize. However, the factors affecting anthocyanin biosynthesis in immature embryos and the genes regulating this process remain unclear. Results In this study, we analyzed the influence of genetic background of the male and female parents, embryo age and light exposure on anthocyanin accumulation in embryos. The results showed that light exposure was the most crucial factor enhancing the pigmentation of immature embryos. The identification accuracy of haploid embryos reached 96.4% after light exposure, but was only 11.0% following dark treatment. The total anthocyanin content was 7-fold higher in immature embryos cultured for 24 h under light conditions compared to embryos cultured in the dark. Transcriptome analysis revealed that the differentially expressed genes between immature embryos cultured for 24 h in dark and light chambers were significantly enriched in the pathways of flavonoid, flavone, flavonol and anthocyanin biosynthesis. Among the genes involved in anthocyanin biosynthesis, five up-regulated genes were identified: F3H , DFR , ANS , F3′H and the MYB transcription factor-encoding gene C1 . The expression patterns of 14 selected genes were confirmed using quantitative reverse transcription-polymerase chain reaction. Conclusion Light is the most important factor facilitating anthocyanin accumulation in immature embryos. After 24 h of exposure to light, the expression levels of the structural genes F3H , DFR , ANS , F3′H and transcription factor gene C1 were significantly up-regulated. This study provides new insight into the factors and key genes regulating anthocyanin biosynthesis in immature embryos, and supports improved efficiency of immature haploid embryo selection during doubled haploid breeding of maize.

Multiple factors can affect the R1-nj purple kernel expression and seed set, reducing its efficiency in identifying haploids in maize. The complex interaction among the haploid inducer (HI), source germplasm (SG), and season (S) is inevitable in in vivo maize haploid induction but could be used through compatibility and stability tests. We tested five HI genotypes on 25 distinct source germplasm in two different seasons of tropical savanna in Thailand. The dry season was more suitable than the rainy season for haploid induction. We noticed varying degrees of R1-nj inhibition among the 25 tropical source germplasm, with some of them exhibiting significant issues with the R1-nj purple kernel expression. Therefore, using the R1-nj alone may not provide accurate ploidy identification in maize. Despite the intense R1-nj expression, haploid inducer BHI306 showed poor stability and compatibility with tropical source germplasm for pollination rate and seed set during the rainy season. The GGE biplot suggested KHI42 and KHI64 as the most compatible haploid inducers under their respective two different mega-source germplasm for the pollination rate and R1-nj seed set. These findings can guide breeders in selecting the most compatible and stable haploid inducers under varying conditions.

Doubled haploid (DH) technology accelerates maize breeding by generating completely homozygous lines within two generations, but its efficiency depends on reliable haploid identification markers. The Purple plant 1 (Pl-1) root pigmentation marker has emerged as a promising alternative to R1-Navajo (R1-nj), which suffers from frequent suppression in tropical germplasm. This study evaluated Pl-1 expression and seedling vigor in 298 diverse Thai maize genotypes across four market classes under controlled growth chamber conditions (24 ± 1 °C, 85–90% humidity, and standardized lighting), followed by testcross analysis with 89 representative genotypes crossed with BHI306 to distinguish between allelic absence and epistatic suppression mechanisms. Complete absence of Pl-1 expression was observed in 99.3% of Thai genotypes, contrasting with consistent expression in the temperate-derived control (BHI306). Testcross F1 progeny from 89 Thai × BHI306 crosses exhibited intermediate expression levels (1.57–2.05) across all market classes, confirming allelic absence rather than suppressor-mediated inhibition. Substantial genetic diversity was detected in seedling vigor traits independent of Pl-1 status, with root length varying 43-fold and fresh weight 20-fold, showing highly significant genotypic effects. The uniform lack of Pl-1 expression across Thai germplasm eliminates background interference, highlighting its utility as a complementary marker when introgressed from inducer lines. These findings establish the genetic foundation for implementing optimized DH breeding strategies in tropical maize through marker-assisted backcrossing approaches.

Doubled haploid technology is widely applied in maize. The haploid inducer lines play critical roles in doubled haploid breeding. We report the development of specialized haploid inducer lines that enhance the purple pigmentation of crossing immature embryos. During the development of haploid inducer lines, two breeding populations derived from the CAU3/S23 and CAU5/S23 were used. Molecular marker-assisted selection for both qhir1 and qhir8 was used from BC1F1 to BC1F4. Evaluation of the candidate individuals in each generation was carried out by pollinating to the tester of ZD958. Individuals with fast and clear pigmentation of the crossing immature embryos, high number of haploids per ear, and high haploid induction rate were considered as candidates. Finally, three new haploid inducer lines (CS1, CS2, and CS3) were developed. The first two (CS1 and CS2) were from the CAU3/S23, with a haploid induction rate of 8.29%–13.25% and 11.54%–15.54%, respectively. Meanwhile, the CS3 was from the CAU5/S23. Its haploid induction rate was 8.14%–12.28%. In comparison with the donor haploid inducer lines, the 24-h purple embryo rates of the newly developed haploid inducer lines were improved by 10%–20%, with a ~90% accuracy for the identification of haploid immature embryos. These new haploid inducer lines will further improve the efficiency of doubled haploid breeding of maize.

Doubled haploid technology, which is used to rapidly purify genetic resources, is one of the key technologies in modern maize breeding. In a previous study, the major quantitative trait locus qhir1 , which influences in vivo haploid induction, was narrowed down to a 243-kb region, which made it feasible to use marker-assisted selection (MAS) for inducer development. Recently, a new method was developed for haploid identification using oil content (OC). The objective of this study was to develop high oil inducer lines using MAS of the qhir1 locus. We constructed an F 2 population, two backcross populations that were backcrossed to the inducer CAU5 (BC 1 F 1 -CAU5) and the high oil inbred line GY923 (BC 1 F 1 -GY923), respectively, which was derived from the cross GY923 × CAU5, and subjected continuous selfing to develop high oil inducer lines. In each cycle, three different parameters including kernel OC, marker genotype at qhir1 and haploid induction rate (HIR) were used for pedigree selection. Three candidate high oil inducer lines were developed, with an OC of approximately 8.5 %, an HIR of approximately 8 % and superior agronomic performance, which are suitable values for the application of these lines to haploid identification by OC. Our results confirm the notion that HIR selection combined with MAS for qhir1 is an effective approach to haploid inducer breeding. In addition, we determined that the accuracy of haploid identification by OC is influenced by the female germplasm resource and the high oil inducer and that appropriate critical points for OC can balance the false discovery rate and false negative rate.

Doubled haploid (DH) technology is an efficient strategy for producing completely homozygous lines for breeding programs. Mutations in the MATRILINEAL (MTL) phospholipase trigger intraspecific haploid induction in cereals. Although an in vivo haploid induction system based on OsMTL-edited plants has been established in rice (Oryza sativa), DH technology is still limited by other factors, such as haploid identification, which is one of the essential steps required for DH technology. In the study, we addressed this technical challenge by integrating specific molecular and phenotypic markers into rice haploid inducers. We first generated large fragment insertion or deletion mutations within the OsMTL gene and designed a pair of primers flanking the mutational sites to be used as the specific and universal molecular markers between wild-type and Osmtl plants. Next, we screened for hairy leaf as a single dominant trait and integrated it into specific molecular marker-based haploid inducers using the cross and self-cross method. When crossing cytoplasmic male sterile lines with these haploid inducers, we utilized the specific InDel marker and hairy leaf phenotypic marker to identify putative haploids (or double haploids). These putative haploids were further confirmed through ploidy and phenotypic analysis, demonstrating the high efficiency of haploid identification using these markers. The haploid induction rate (HIR) of the developed specific molecular and phenotypic marker-based haploid inducers ranged from 3.7% to 12.5%. We have achieved successful integration of distinct molecular and phenotypic markers into rice haploid inducers. Our advanced marker-based system has significantly enhanced the accuracy of haploid identification, thereby expediting the adoption of DH technology in rice breeding.

Haploid breeding can shorten the breeding period of new maize varieties and is an important means to increase maize yield. In the breeding program, a large number of haploid seeds need to be screened, and this step is mainly achieved manually, which hinders the industrialization of haploid maize breeding. This article aims to develop a multispectral camera to identify the haploid seeds automatically. The camera was manufactured by replacing narrow-band filters of the ordinary CCD camera, and the RGB, 405 nm, 980 nm and 1050 nm images of haploid or diploid seeds were simultaneously captured (the characteristic wavelengths were determined according to color and high-oil markers of maize). The performance was tested using four maize varieties with the two genetic markers. The results show that the developed multispectral camera significantly improved the recognition accuracy of haploid maize seeds to 92.33%, 97.33%, 97% and 93.33% for the TYD1903, TYD1904, TYD1907 and TYD1908 varieties, respectively. The cameras in the near-infrared region (wavelengths of 980 nm and 1050 nm) achieved better performance for the varieties of high-oil marker, with an increase of 0.84% and 1.5%, respectively. These results demonstrate the strong potential of the multispectral imaging technology in the haploid seed identification of maize.