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The polyembryo gene (OsPE) in rice

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Abstract

T-DNA insertional mutagenesis is one of the most important approaches for gene discovery and cloning. A fertile polyembryo mutant generated by T-DNA/Ds insertion in Oryza sativa, cv. Basmati 370 showed twin or triple seedlings at a frequency of 15–20%. T-DNA insertion was confirmed by 950 bp hpt gene amplification in the promoter region of the candidate gene. The annotated protein corresponding to the OsPE candidate gene has been reported as a hypothetical protein in O. sativa. OsPE gene lacked functional homologs in other species. No OsPE paralog was found in rice. No conserved domains were found in the protein coded by OsPE. RT-PCR showed the expression of OsPE gene in Basmati 370 shoots. Full-length OsPE gene was cloned in Basmati 370. The combined use of Southern blot, genome walking, TAIL-PCR, RT-PCR techniques, and bioinformatics led to the identification of a candidate gene controlling the multiple embryos in rice. There is gain of function, i.e., multiple embryos in the seeds in the knockout mutant OsPE whereas its wild-type allele strictly controls single embryo per seed. The seeds with multiple embryos are distributed at random in the rice mutant panicle. The origin of multiple embryos, whether apomictic, zygotic or both is under investigation.

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Acknowledgements

We thank Dr. Kuldeep Singh, PAU Ludhiana for providing rice SSRs for BSA. We also thank Dr. K. S. Gill, WSU for his thoughtful review and suggestions in improving this article. The financial assistance by CSIR (India) to A.B and NSF (USA) to S.K.F. and H. S. D. is highly acknowledged.

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Authors and Affiliations

  1. Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, 247667, Uttarakhand, India

    Anju Puri, P. Osman Basha, Mankesh Kumar, Deepak Rajpurohit, Gursharn S. Randhawa & Harcharan S. Dhaliwal

  2. Department of Plant Sciences, North Dakota State University, Fargo, ND, USA

    Shahryar F. Kianian & Anantharama Rishi

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  1. Anju Puri
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  2. P. Osman Basha
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  3. Mankesh Kumar
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  4. Deepak Rajpurohit
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  5. Gursharn S. Randhawa
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  6. Shahryar F. Kianian
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  7. Anantharama Rishi
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  8. Harcharan S. Dhaliwal
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Corresponding author

Correspondence to Harcharan S. Dhaliwal.

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Fig. S1

Germination of OsPE mutant and Basmati 370 on 80 ppm hygromycin solution. a Polyembryonic mutant b Basmati 370 (DOC 2922 kb)

Fig. S2

PCR amplification of 950 bp hpt fragment in insertional mutants Insertional Mutants (lanes 1,2,4); OsPE (lane 3); Basmati 370 (lane 5); lane M: molecular weight marker 1 Kb (DOC 48 kb)

Fig. S3

Schematic diagram of HmRDs construct used for insertional mutagenesis of Basmati 370 (DOC 32 kb)

Fig. S4

Meiosis in PMC of OsPE, a Diakinesis 12IIs; b Metaphase-I, 12IIs; c Telophase-I; d Tetrad stage (DOC 434 kb)

Fig. S5

T-DNA right border sequences with three T-RB nested primers in color (DOC 29 kb)

Fig. S6

Primers (DOC 33 kb)

Fig. S7

T-DNA flanking sequence using T-RB2 (T-DNA based primer) for OsPE mutant (DOC 29 kb)

Fig. S8

T-DNA flanking sequence using AP2 for OsPE mutant (DOC 29 kb)

Fig. S9

T-DNA flanking sequence using T-RB3 (Tertiary TAIL- PCR product) for OsPE mutant (DOC 29 kb)

Fig. S10

BLAST result of T-DNA flanking sequence using T-RB2 (Genome Walking PCR) showing 94% identity with rice cultivar Nipponbare at Chromosome 3 (DOC 30 kb)

Fig. S11

BLAST result of T-DNA flanking sequence using AP2 (Genome Walking PCR) showing 87% identity with rice cultivar Nipponbare at Chromosome 3 (DOC 31 kb)

Fig. S12

BLAST result of T-DNA flanking sequence using T-RB3 (TAIL-PCR) showing 92% identity with rice cultivar Nipponbare at Chromosome 3 (DOC 30 kb)

Fig. S13

Sequence of candidate gene OsPE including the promoter region in O. sativa cv. Nipponbare genomic DNA Chromosome 3 (DOC 33 kb)

Fig. S14

Diagrammatic representation of T-DNA insertion on chromosome 3 in Polyembryo mutant of Basmati 370 (DOC 35 kb)

Fig. S15

Diagrammatic representation of designing rice genome specific primers and expected product size in Polyembryony mutant of Basmati 370 (DOC 36 kb)

Fig. S16

Protein BLAST of O. sativa (Os03g0241300) with Arabidopsis, Z. mays, and V. vinifera (DOC 37 kb)

Table S1

Primer combination of rice genome specific and T-DNA specific PCR and expected product size in Basmati 370 and OsPE (DOC 39 kb)

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Puri, A., Basha, P.O., Kumar, M. et al. The polyembryo gene (OsPE) in rice. Funct Integr Genomics 10, 359–366 (2010). https://doi.org/10.1007/s10142-009-0139-6

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  • DOI: https://doi.org/10.1007/s10142-009-0139-6

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