Review: Breeding and Molecular Research of Some Tropical Grasses in Japan

Hitoshi NakagawaHitoshi.nakagawa@crl.hpk.co.jp

Hamamatsu Photonics K. K., Central Research Laboratory

5000 Hirakuchi, Hamakita-ku, Hamamatsu, Shizuoka, Japan

IFTBC (MBFT) 2019Lake Buena Vista, FL, USA, March 24-27, 2019

Dawn of forage grass research in Japan: Kaoru Ebara (1970) Japanese Journal of Grassland Sciences (Kyushu Branch) 1: 7-13 (in Japanese)

1935- : Hokkaido National Agricultural Experiment Station; Corn Breeding Project (MAF) in Nagano Prefectural Agricultural Experiment Station

1) Breeding of hybrid corn2) Teosinte, Sorghum, Barnyard millet3) Alfalfa, White clover, Red clover, Timothy, Orchardgrass,

Reed canarygrass, fescues, Bentgrass

After the WWII: Increasing needs for beef and dairy food production

1) 1947: Establishment of Forage Cultivation Laboratory in National Livestock Research Station (NLRS)

2) 1952: Establishment of Forage Crop Laboratory in Kyushu National Agricultural Experiment Station (Grassland Division)

3) 1953: Grassland Division in NLRS

Sub-tropical

(No frost)

Kyushu Natl. Agric. Expt.Stn. (Kumamoto):rhodesgrass, guineagrassannual use, Elianthus,Sorghum

Natl. Grassland ResearchInstitute (Nishinasuno) BasicRes.; Guineagrass (apomixis)Zoysia, Elianthus

Tropical Agriculture Research Center (TARC;JIRCAS), Okinawa Branch; Ishigaki Island,Guineagrass (apomixis), Elianthus

Shikoku Natl. Agric.Expt. Stn. (Zentsuji)Germplasmintroduction andevaluation

Okinawa Pref. Liv.Research Ctr.Guineagrass, Bracharia

rhodesgrass, signalgrass

40N

32N

24N

35N

28NKagoshima Pref. Agric.Expt. Stn. Bahiagrass,Dhahlisgrass,rhodesgrass annualuse

Cool temperate

Warm temperate

Aichi Pref. Agric. Res. Stn.Colored Guineagrass

Warm temperate and subtropical Areas where tropical grasses are investigated and cultivated in Japan

1955-: Bermudagrass (Cynodon dactylon), Dahlisgrass (Paspalm dilatatum), Bahiagrass (Paspalum notatum) (Supported by Dr. G. Burton, USDA)

1) Cytological Study of Dahlisgrass2) Anther culture of Dahlisgrass for the breeding of apomictic species

→Failed1964-: UJNA (United States and Japan Conference on the Development

and Utilization of Natural Resources), Forage Seed Production PanelUS-side Co-Chairs: Dr. Austin Campbell (USDA, Beltsville) and Dr. Reed

Barker (Oregon State University); Dr. Michael Casler (USDA, Madison) and Dr. Bryan Kindiger (USDA, Grazinglands Research Laboratory)

1968-: Joining OECD Forage seed Scheme

Dawn of tropical grass research in Japan: Toyokazu Yamada (1970) Japanese Journal of Grassland Sciences (Kyushu Branch) 1: 1-6 (in Japanese)

Africa CollectionSadao HOJITO and ToshizoHORIBATA (1982) Plant exploration, collection and introduction from Africa, Nekken Shiryo 58, 120 pp. (in Japanese)

From 1971 to 1973, Hojito and Horibata made excursions to East Africa. Specifically, in Kenya, Tanzania, Uganda, and Ethiopia, they collected ca. 2,000 accessions of tropical grasses. The collection is named as the “Africa collection”

Reg. Institution Breeding Method & material

Bahiagrass (Paspalum notatum)1) Nanpu 1969 Kyushu Natl.

AESMaternal selection from introductions from USA.(2n=20; Sexual)

2) Shinmoe 1973 Kagoshima PAES

Synthetic variety selected from 70 collected lines of Pensacola-type (2n=20; Sexual)

3) Nangoku 1985 Kagoshima PAES

Synthesize 5 clones from “Pensacola 64-P”(2n=20; Sexual)

4) Nan-ou 1994 Kagoshima PAES

Mass selection from tetraploid USA lines (2n=40: Apomictic)

Dallisgrass (Paspalum dilatatum)1) Natsugumo 1981 Kyushu Natl.

AESMass selection from domestic population in Southern Kyushu, Japan. (2n=50; Apomictic)Problem: ergot → difficulty in seed production

Tropical grass cultivars registered in Japan from 1980 to 2018.

Name Reg. Institution Breeding Method & materialRhodesgrass (Chloris gayana Kunth)1) Hatsunatsu 1988 Kyushu

Natl. AES9 maternal lines selected from 2x exotic germplasms(2n=20)

2) Asatsuyu 1995 Kagoshima PAES

7 clones selected from 2x exotic germplasm (2n=20)

3) Ryokufu 2006 Kagoshima PAES

8 clones by recurrent selection (3 times) from “Hatsunatsu” etc. (2n=20)

Registered cultivars registered in Japan from 1980 to 2018.

Guineagrass (Panicum maximum Jacq.)

1) Natsukaze 1986 Kyushu Natl. AES

An apomictic hybrid of a diploid sexual line GR297 (Africa Collection)) (2n=4x =32; Apomictic)

2) Natsuyutaka 1989 Kyushu Natl. AES

Line selection from 57 lines collected from Africa (Including Africa Collection) (2n=4x =32; Apomictic)

3) Natsukomaki 2002 Kyushu Natl. AES

Line selection for machinery tolerance (2n=4x =32; Apomictic)

Name Reg. Institution Breeding Method & material

4) Paikaji 2007 Okinawa PLES

Open-pollinated hybrid between sexual tetraploid and unknown pollinator (2n=4x =32; Apomictic)

5) Umaku 2012 Okinawa PLES

Line selection for high yield machinery tolerance (2n=4x =32; Apomictic)

6) Nekken No. 1 (Noh PL 1 guineagrass)

1993 TARC Doubled Chromosome Number of a sexual diploid line GR297 (AfricaCollection) (2n=4x =32; Sexual)

Colored guineagrass (P. coloratum L.)1) Tamidori 1988 Aichi PAES Mass selection from cv. Solai (2n=4x

=32; Sexual)2) Tayutaka 1992 Aichi PAES Mass selection from Kabrabra type

germplasm (2n=4x =32; Sexual)

Registered cultivars registered in Japan from 1980 to 2018.

Cytology, breeding and molecular analysis

Tropical grass species include polyploids; without identifying the chromosome number and reproductive methods, no breeding strategy, no molecular research

Rhodesgrass is most popular tropical grass in the grassland of Japan (mainly in subtropical Okinawa Islands).

(1) There are both diploid (2n=20: early maturing) and tetraploid (2n=40: very late maturing) types(2)Why seed fertility of commercial seed is low? (less than

30%)(3) Tetraploids are apomictic? (Brown and Emery 1958;

Hutton 1961)

Chloris gayana: rhodesgrass1) Cytology

CSIRO germplasm

Chromosome number and reproductive method of Rhodes grass (Chloris gayana Kunth)

Species Chromosome Number

(2n)

Reproductive Method

Species Chromosome Number

(2n)

Reproductive Method

pilosa 20 Self-poll radiate 40 Self-poll

roxburghiana 20 Self-poll (?) distichophylla 80 Self-poll

virgata 20 Self-poll ciliata 80 Self-pollacicularis 40 Self-poll castilloniana 100 Self-pollbarbata 40 Self-poll truncata 120 Self-polldivaricata 40 Self-poll Rhodesgrass 20 Out-crosspolydactyla 40 Self-poll (?) Rhodesgrass 40 Out-cross

pycnothrix 40 Self-poll

All observed Chloris species are sexual: roxurghiana and polydactylla exhibited low seed fertilities by both open-and self-pollination with paper bag; Rodesgrass exhibited low seed fertilities by self-pollination, 0-10 %.Nakagawa, H. et al. (1987) Chromosome number, reproductive method and morphological characteristics of Chloris species. J. Japan. Grassl. Sci. 33:191-205

Chloris gayana: rhodesgrass2) Breeding

Chloris gayana Kunth: Rhodesgrass3) Molecular analysis

(1) Ubi BE et al. (2000): AFLP Variation in Tetraptoid Cultivars of Rhodcsgrass(Cloris gayana Kunth). Grassland Science 46: 242-248,

1. Variation within and among 5 tetraploid cultivars of rhodesgrass was assessed based on AFLP analysis.

2. The results of this study show that AFLP analysis can be an efficient technique for the genetic characterization of rhedesgrass cultivars with potential applications for breeding purposes.

(2) Ubi BE et al. (2003) Genetic Diversity in Diploid Cultivars of Rhodesgrass Determined on the Basis of Amplified Fragment Length Polymorphism Markers, Crop Sci. 43: 1516–15221. Genetic diversity of 13 diploid cultivars through 237 AFLP marker analysis.2. Cluster analysis revealed: (1) Group 1 (recent Japanese cvs and their African

source population); (2) Group 2 (African cvs); (3) Group 3 (one African cv). 3. The genetic diversity within recent Japanese cvs was comparable to the diversity

within old African cvs and no evidence of a reduced genetic base because of breeding efforts

2) Panicum maximum Jacq.: Guineagrass1) Establishing apomixis breeding

A pioneer in Japan: Late Dr. Kosuke Nakajima, NGRI, MAFF, Japan

Dr. Yves Savidan, L’ ORSTOM, Adiopodoume, Cote d’Ivore

Dr. Wayne W. Hanna, USDA, Coastal plain expt. Stn., Tifton, GA, USA

Dr. Wayne W. Hanna

Dr. Yves Savidan

Exploration and collection of guineagrass

1. S. Hojito and T. Horibata: Ethiopia, Uganda, Kenya, Tanzania (1971-73): 140 accessions (included only one sexual accession: GR297)K. Nakajima developed cv. Natukaze (Norin No. 1

guineagrass) using GR2971. K. Nakajima: Tanzania (1981)2. K. Nakashima and H. Nakagawa: Tanzania (1989)

Tanzania is a center of origin of Panicum maximum.

Sexual and apomictic embryo sacs by Clearing method

Nakagawa, H (1990) JARQ 24:163-168

Antipodal cells

Polar nuclei

EggSexual embryo sac Apomictic embryo sac

Molecular analysis and isolation of apomixis gene(s) toward the permanent hybrid generation

Molecular analysis of apomixis

Construction of Genetic Map and mapping of

an apomixis Gene By linkage analysis

Construction of BAC Library

Determination of mRNAsBy using genetic information

And the sequence

Isolation of an apomixis gene

Breeding of Permanent Hybrid in maize, sorghum, rice and wheat by using an apomixis gene (Asexual revolution)

cDNAmicroarray

GR297

Diploid・Sexual

An Introduction from Tanzania Tetraploid・Apomictic

Natsukaze（Norin No. 1）

Tetraploid・Apomictic

Pseudo-test cross Hybrid population

1）71 plants; 2）1,233 plants

Maternal line, Noh PL 1 (Nekken No. 1）

Tetraploid・sexual

Chromosome doubling and selection Hybridization

Hybridization

Development of Hybrid population for linkage analysis

Apomictic plants and sexual plants segregate into 1 : 1 ratio

The genome sizes estimated with flow cytometry showed that the diploid GR-297 was 499 Mbp, and tetraploid Noh PL 1, Natsukaze and Natsuyutaka posses 995, 1023 and 1045 Mbp, respectively. （Akiyama et al. Grassld. Sci. 2008)

Micro-array analysis: Photos of the ovaries from immature panicles of 2 guineagrass cultivars, “Natsukaze (4x Apomixis)” and “Noh PL 1 (4x Sexual)”.Five ovaries were collected at the same time during the early and middle developmental stages from immature panicles and were used for RT-PCR and Q-PCR analysis. The scale bar is 1mm. (Yamada-Akiyama et al 2009,Journal of Plant Physiology, 166(7):750-761)

Development of apospory initial cells

Sexual

Apomictic

4608 Probes on microarray = cDNA clones from immature

panicles of two apomictic strains Ku5954 and GM64-3A.

Target RNA = pistils from an apomictic strain OKI64.

Detection of expressed genes in apomictic pistils by microarray(common expression genes in both apomictic strains)

Homology search: Sequencing and Screening by homology search based on the public database

Matched data = putative function genes and/or apomictic librariesUnmatched data = novel genes

Expression analysis:Comparison between an apomictic and a sexual cultivars by RT-PCR and Q-PCR

Aposporous ovary-specific expressed genes

Microarray analysis:

4608spots→394spots

394clones→196ESTs →30ESTs

9 ovary-specific ESTs → 3 aposporous specific ESTs (expressed sequence tag)

ResultsOf the 4608 probes in the microarray, 3 ESTs

revealed aposporous ovary-specific genes expressed in the early developmental stage, suggesting that these could be apomixis-related genes.

Yamada-Akiyama et al 2009,Journal of Plant Physiology, 166(7):750-761

Molecular analysis and isolation of apomixis gene(s) toward the permanent hybrid generation

Molecular analysis of apomixis

Construction of Genetic Map and mapping of

an apomixis Gene By linkage analysis

Construction of BAC Library

Determination of mRNAsBy using genetic information

And the sequence

× Isolation of an apomixis gene

Breeding of Permanent Hybrid in maize, sorghum, rice and wheat by using an apomixis gene (Asexual revolution)

FinishedFinished

No recombination!!

cDNAmicroarray

Mutation inductionby gamma-ray and ion beams

↓

Linkage map of Bracharia: Collaborated research with Embrapa, BrazilTaikuwa et al. (2016) Japanese Society of Grassland Science, Grassland Science, 62

Apospory specific genome region

Micro array: Dr. HitomiYamada-Akiyama

FISH: Dr. Yukio Akiyama Dr. Wayne and

Barabara Hanna AFLP marker: Dr. Masumi Ebina

STS marker: Dr. Manabu Takahara

Dr. Tadashi Takamizo

Thank you