Development of molecular genetic research tools of wild Oryza

National Institute of Genetics, JapanYutaka Sato

211026 ANRRC

Wild Oryza genetic resources at NIGCollected during 1950s to 1980s.

Accumulating genome information of wild Oryza attracts interests of many researchers.

NIG wild Oryza resources1,700 accessions covering 21 wild Oryza species

Origin of cultivated rice

wildO. barthii

domesticatedO. glaberrima

Africa wildO. rufipogon

domesticatedO. sativa

Asia

161116, CB79@Prey Noup dist., Sihanouk, Cambodia

O. rufipogon, perennial type

O. rufipogon, annual type

Oryza rufipogon

Photos from Dr Nonomura

<Purpose>Promotion of the usage of wild Oryza genetic resources

(for Bio-Circular-Green researches)

More researchers use wild Oryza genetic resources.

>Genome information>Molecular genetic research tools

Acceleration of wild Oryza research.

<Purpose>Promotion of the usage of wild Oryza genetic resources

(for Bio-Circular-Green researches)

>Genome information>Molecular genetic research tools

Development of genetic transformation method for wild Oryza research.

Procedure for genetic transformation of wild Oryza

1. Put tags when flowers bloomed.

2. Harvest 8-11 after flowering.

3. Excise immature embryo.

4. Agrobacterium infection.

5. Culture on media.

6. Regeneration of plantlet.

W2184, O. glumaepatula, AANB, O. sativa, AA

8 – 11 DAP Ovary

8 – 11 DAP spikelets

Type1 Type4 Type7 Type8japonica indica

Embryo resting nN6C CCMC nN6C CCMC

culture nN6C CCMC nN6C CCMCPre-

regeneration - NBPRC NBPRC -

regeneration N6R RNM RNM N6Rrooting N6F MSI MSI N6F

Modified from Hiei and Komari, 2008, Nature Protocol

Conditions for immature embryo culture and regeneration

Species genometotal

accessions tested

Callus induction regeneration accessions

(%)*1O. barthii AA 10 8 7 70%O. glumaepatula AA 18 17 14 83%O. longistaminata AA 1 1 1 100%O. meridionalis AA 15 13 7 47%O. rufipogon AA 44 39 28 67%O. punctata (2X) BB 8 8 5 63%O. eichingeri CC 2 1 1 50%O. officinalis CC 16 7 4 25%O. rhizomatis CC 3 3 2 67%O. australiensis EE 10 5 0 0%O. brachyantha FF 7 6 4 57%O. granulata GG 4 2 0 0%O. meyeriana GG 2 1 0 0%O. minuta BBCC 12 10 5 42%O. punctata (4X) BBCC 8 4 2 25%O. alta CCDD 3 2 1 33%O. grandiglumis CCDD 7 6 6 86%O. latifolia CCDD 10 4 2 20%O. longiglumis HHJJ 9 1 1 11%O. ridleyi HHJJ 3 0 0 0%

total 192 138 90*1, number of regenerated accessions / number of tested accessions

Most of species in wild Oryza were regenerated

Species genome Regenerated accessions

total accessions

tested

GFP-signal observed

accessionsaccessions

(%)*2

O. barthii AA 7 6 3 50%O. glumaepatula AA 14 14 13 93%O. longistaminata AA 1 1 1 100%O. meridionalis AA 7 7 1 14%O. rufipogon AA 28 26 17 65%O. punctata (2X) BB 5 4 3 75%O. eichingeri CC 1 1 1 100%O. officinalis CC 4 2 0 0%O. rhizomatis CC 2 2 0 0%O. brachyantha FF 4 4 3 75%O. minuta BBCC 5 3 2 67%O. punctata (4X) BBCC 2 2 2 100%O. alta CCDD 1 1 1 100%O. grandiglumis CCDD 6 1 0 0%O. latifolia CCDD 2 1 0 0%O. longiglumis HHJJ 1 1 0 0%

total 90 76 47

Most of species in wild rice were infected by Agrobacterium tumefaciens

*2, number of GFP-signal observed accessions/ number of tested accessions embryo

Agrobacteriuminfection

selection

regeneration

O. brachyantha, FF, W1711

O. glumaepatula, AA, W2160

Transgenic

Transgenic plants expressing GFP in the background of Oryza brachyantha (FF)

WT

regeneratedO. brachyantha, FF, W1711

regeneratedO. brachyantha, FF, W1711

Transgenic WT

Genome editing can promote usage of wild Oryza genetic resources for breeding.

Collaborators

Toshihiro Kumamaru (Kyushu Univ.)Takahiko Kubo (Kyushu Univ.)Hideshi Yasui (Kyushu Univ.)Yoshiyuki Yasui (Kyushu Univ.)

Ken-ichi Nonomura (National Institute of Genetics)Katsutoshi Tsuda (National Institute of Genetics)Misuzu Nosaka (National Institute of Genetics)Toshiya Suzuki (National Institute of Genetics)Kim Nhung Ta (National Institute of Genetics)Yuri Yoshida (National Institute of Genetics)Ayumi Agata (National Institute of Genetics)

This work was supported by NBRP Fundamental Technology Upgrading Program (AMED Japan).