Review: Breeding and Molecular Research of Some …...Review: Breeding and Molecular Research of...
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Review: Breeding and Molecular Research of Some Tropical Grasses in Japan
Hitoshi [email protected]
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