We isolated the entire sequence of the coding region of Waxy gene of a non-waxy accession of Job’s tears (Coix lacryma-jobi) by PCR-based methods. We also compared the entire sequences of the gene between two non-waxy accessions and three waxy cultivars and found a 275-bp deletion in the coding region (exons 10–11) of this gene specific to waxy cultivars. We showed by PCR genotyping that this deletion is commonly found in Japanese and Korean cultivars and confirmed that this deletion resulted in lack of Wx protein. We also confirmed that this polymorphism of the gene co-segregates with phenotypes in endosperm and pollen. These results suggest that this PCR-based marker will be useful in breeding of Job’s tears and that genetic information obtained in other grass species will be also useful in genetics and breeding of Job’s tears.
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We thank Mr. T. Funakoshi, Genebank of Hiroshima Center for Promotion of Agriculture and Forestry for providing us with C. lacryma-jobi germplasm and also thank Dr. T. Itani, Faculty of Life and Environmental Sciences, Prefectural University of Hiroshima, for providing us with rice seeds. We also thank Dr. H. Nakayama, National Agricultural Experiment Station for Kyushu Okinawa Region, for providing technical information on SDS-PAGE of Wx protein.
Takehiro Hachiken and Yuya Masunaga contributed equally to this work.
Electronic supplementary material
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Phylogenetic tree constructed based on full-length amino acid sequences of Waxy gene in cereals. The tree was constructed by the neighbor-joining method. Coix-lacryma-jobi: Job’s tears in the present study. Sorghum bicolor : sorghum (U23945), Zea mays: maize (NM_001111531), Oryza sativa: rice (AF515483), Hordeum vulgare: barley (AF486514), Triticum aestivum: wheat (AF286320), Secale cereale: rye (FJ491377). (PPT 182 kb)
SDS-PAGE of Wx protein. 1: Hoshiyutaka, non-waxy rice cultivar as a control. 2: Tanchomochi, waxy rice cultivar as a control. M: size marker (Protein Ladder Marker), 3: Non-waxy wild accession var. lacryma-jobi from Takasago, Hyogo, Japan. 4: Hatojiro. 5: Obanazawa landrace. 6: Okayama landrace. 7: Miyagi landrace 1. 8, 9: Gyeonggi-do. 10: Kyushu 1. 11: Non-waxy wild accession var. lacryma-jobi from Suzuhari, Hiroshima, Japan (see Table 1). N and W stand for non-waxy and waxy, respectively. (PPT 149 kb)
Pollen staining examined by microscope. A: Pollens staining dark blue (Individual F2-59). B: Pollens staining reddish-brown (Individual F2-14). C. Mixture of pollens staining reddish-brown and those staining dark blue (Individual F2-91). See the detail in text and Table 2. Non-staining pollens were regarded as sterile pollens and excluded from counting. Scale is shown in each photograph. (PPT 282 kb)
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Hachiken, T., Masunaga, Y., Ishii, Y. et al. Deletion commonly found in Waxy gene of Japanese and Korean cultivars of Job’s tears (Coix lacryma-jobi L.). Mol Breeding 30, 1747–1756 (2012). https://doi.org/10.1007/s11032-012-9758-z
- Coix lacryma-jobi
- Job’s tears
- PCR-based marker
- Waxy gene