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Progress in Perennial Rice Breeding and Genetics
Fengyi Hu
Food Crops Research Institute,YAAS22 Sept. WaggaWagga, Australia
Introduction • Soil erosion in uplands of southeast Asia has been a serious problem that led to the project of developing perennial upland rice at IRRI (IRRI 1989)
The idea of developing perennial rice for erosion control
Development of perennial upland rice has been proposed by several authors (IRRI 1989; Wagoner, 1990; Xiu, 1995; Schmit, 1996; Tao et al., 2000, 2001; Sacks, 2001;)
Cultivars of rice is usual annual food crop after long time domestication by farmer and breeding procedure by breeder or geneticist.
The donor(s) for prerenniality?
The donor(s) for prerenniality?
All over the world growth of O. sativa is Annual
The donor(s) for prerenniality?
• O. longistaminata is the logical donor for perenniality from its feature of rhizome as compared to other wild rice species (O. officinalis, O. rhizomatious, O. australiensis)
Oryza Species, the Species Complex, Chrom.,Genome group and Distribution
Oryza species, the species complexes, chromosome number, genome group and distribution
Section Chromosome Genome Distribution
Complex Number group Species
Oryza O.sativa complex O.sativa L. 24 AA Worldwide O.nivara Sharma et Shastry 24 AA Tropical and Sub.Asia O.rufipogon Griff 24 AA Tropical and Sub.Asia O.meridionalis Ng 24 AmAm Tropical Australia O.glumaepatula Steud. 24 AglAgl South America O.glaberrima Steud. 24 AgAg Africa(mainly West) O.barthii A.Chev. 24 AgAg Africa O.longistaminata Chev.et Roehr 24 AlAl Africa
O.officinalis complex O.officinalis Wall ex Watt 24 CC Tropical and Sub.Asia O.minuta Presl.et Presl. 48 BBCC Philippines O.eichingeri Peter 24 CC Sri Lanka,Africa O.rhizomatis Vaughan 24 CC Sri Lanka O.punctata Kotschy ex Steud. 24,48 BB,BBCC Africa O.latifolia Desv. 48 CCDD Latin America O.alta Swallen 48 CCDD Latin America O.grandiglumis (Doell) Prod. 48 CCDD South America O.australiensis Domin 24 EE Australia
Ridleyanae Tateoka O.brachyantha Chev.et Roehr. 24 FF Africa
O.schlechteri Pilger 48 HHKK Papua New Guinea O.ridleyi complex O.ridleyi Hook.f. 48 HHJJ SE Asia O.longiglumis Jansen 48 HHJJ Irian Jaya,Indonesia
Granulata O.meyeriana Roschev. complex O.meyeriana Baill 24 GG SE Asia
O.granulata Nees et Arn.ex Watt 24 GG S.and SE Asia
The Diagram of Evolution of Wild Species among AA Genome of Rice
Gondawanaland Common ancestor
South and Southeast Asia Tropical Africa O. rufipogon O . longistaminata
O. nivar O. barthii
Indica ---- Japonica O . glaberrima
Parallel evolution
Comparing the O. longistaminata and O. rufipogon
O. longistaminata (AA genome)
Photo was cited from Vaughan (1994)
O. rufipogon (AA genome)Photo was cited from Vaughan (1994)Tufted and scrambling herb, stolon
O. longistaminata• Long anther
• Self-incompatibility
• Allogamy
• Rhizomatous stem
• Bacterial Blight resistance(Xa21)
• Nematode resistance
• …….
The Oryza longistaminata. A: The panicle of the O. longistaminata; B: The performance of O. longistaminata in field; C, D: the strong Rhizome of O. longistaminata.
A
B D
C
Additional useful features from O. longistaminata
• A saturated molecular linkage map was constructed (Causse, 1994, Wilson, 1999).• Xa21, resistance to Bacterial Blight has been cloned with map-based (Khush, 1990; Song, 1995).• Resistance to tungro viruses has been verified (Angeles E.R. 1998). • Resistance to root knot nematode M. graminicalawas reported (Imelda R. Soriaano, 1999).• A saturated molecular linkage map based on PCR markers (Hu, 2003)• ……
Goal
• Exploit the possibility of using perenniality from O. longistaminata for development of cultivars of perennial upland rice (PUR) or perennial rice (PR)
Oryza longistaminataPhoto was cited from Vaughan (1994)
The Strategy for Perennial Rice Breeding
F1
RD23O. longistaminata
Progeny derived from F1
by Self-intercross, back cross, transgenic and MAS with diversity germplasm of rice
hope to
x
F1 plant of RD23_Longi
F1
RhizomeF1 plant in greenhouse
Problems for the PR Breeding and genetics
1, Less F1 progeny from O. longistaminataup to now, there are ~6 cases reported for
obtaining the F1 progeny since it is difficult to obtain the F1 plant in the procedure of interspecific hybrid between O. sativa and O. longistaminata.
among these cases, although F1 plant can get, most of these absence Rhizome. Including CIRAD’s (Ghesquiere, 1991), IRRI’s(Bara, 2000) and Japanese (Maekawa, 1997), and others not so clear.
2, Lethal geneThe Rhizome present is Linkage to lethal
gene with D1 and D2; (Chu and Oka 1970; Ghesquiere, 1991)
results to obtain the progeny lack with Rhizome, means the Rhizome is usual lost in the progeny lines during the selection for breeding purpose.
Problems for the PR Breeding and genetics
3, The reproductive barriers (Hybrid Sterility, Sgene)
This is a popular phenomenon between the cultivar and its wild species, even between two sub-species, Indica and Japonica of O. sativa.
It is also a main factor for obtaining the progeny that combine the favorable traits/gene from receiptor and donors. For example, the yield components.
Problems for the PR Breeding and genetics
4, Photoperiod sensitivity
From Vegetative growth to productive growth, the day-light is important for the short day-light plant, including rice as it derived from tropical zone.
The progeny with strong ability of vegetative growth of O. longistaminata is main factor to lack selection for PR.
Problems for the PR Breeding and genetics
5, Seed Dormancy
Problems for the PR Breeding and genetics
6, Shatter grains
Problems for the PR Breeding and genetics
7, awn
Problems for the PR Breeding and genetics
How to overcome these problems?
All of these problems are the negative effect for PR improvement.
One of is utilization the traditional methods, such as Self-intercross, Backcross, then select the useful progeny.
Other one is understanding the genetic mechanism of these factor, specially for Rhizome, then using the MAS, Transgenic Strategy for cultivars of PR improvement.
There are two stages for our results:
Phase I is from 1997-2004;Phase II is from 2005-now.
Progress in PR
During First stages(1997-2005), there are 6 results has been obtained.
1, Obtaining the F1 plant derived RD23/O. longistaminata. (here, RD23 is an Indica type cultivar of rice)
2, From F2 and the molecular mapping shown that the Two Dominant Complementary Genes (Rhz2 and Rhz3) for Rhizome Expression in O. Longistaminata and mapped on chr3 and chr4, respectively.
3, Rhz2 and Rhz3 have been registered on the International Rice Genetic Committee.
4, The plant of BC2F1 with Rhizome has been obtained.5, The Rhizome related traits has been QTLs analysis.6, The first PCR-based molecular genetic map has been
constructed.
Progress in Phase I
The F1 plant of the RD23/O. longistaminata cross was obtained by direct hybridization followed by embryo rescue and had 32.5% pollen fertility, indehiscent anthers, rhizomes that were intermediate in size, and abundance between the parents.
This is a important material for development Cultivar of PR or PUR.
Progress in Phase I
F1
Rhizome
F1 plant
Progress in Phase I
Segregation of rhizome trait in the F2 population based on field experiment
absence
0.8011X2(9:7)
106121NumberspresenceRhizome
Result :Two Dominant Complementary, Rhz2, Rhz3, Genes for Rhizome Expression in O. Longistaminata
PCR-base Molecular Genetic Map
Progress in Phase I
RM4280.0RM32313.1RM28319.0RM2530(6)21.6OSR226.6RM27232.3RM29245.7
RM15866.5
RM30687.8RM23797.5RM297109.5RM302114.7RM212118.1RM319118.3RM265122.4RM315128.5OSR23145.9RM529154.9
RM4850.0OSR179.2OSR1415.1RM27925.5RM42331.8RM55536.8
RM17456.8OSR960.0RM32262.9RM7170.5RM30083.4
RM34196.9
RM327112.7
RM263138.1RM2421148.6RM240157.5
RM166177.7
RM213190.1RM208202.7RM207206.4OSR26214.6
RM600.0
RM23115.2RM5883(10)19.9
OSR1344.0Rhz252.1OSR1653.4RM3657.8RM25166.5RM28275.1RM455184.0RM33889.5
RM156116.2
RM4626133.6RM6097142.7OSR31145.8RM55157.3RM1(1)165.0RM2525167.9RM4612180.9
RM114203.1
RM442224.2
RM5510.0RM51810.6RM26120.6RM18532.4RM14243.9RM11948.4Rhz350.6RM27357.8RM25265.7RM31778.3
RM349104.3OSR15106.1RM348107.2RM127116.4RM280123.5
RM1590.0RM1226.4OSR3511.1
RM1327.3RM40534.9
RM24952.4RM50958.0
RM16479.9
RM29193.9RM163101.7RM161107.3RM421117.0
RM274138.9
RM87151.0RM334163.6RM31167.6
RM1330.0RM4356.2RM17013.7
RM58726.8RM51037.6
RM20455.6RM642067.3RM31474.9RM25377.6RM40283.0RM27686.6RM13698.3RM5818105.4
RM6309128.5RM528132.7RM4509143.4
RM176157.4RM345162.7OSR21174.5
www.gramene.org
Mapping for Rhz2and Rhz3
The molecular mapping of Rhizome gene Rhz2 and Rhz3 was mapped to the interval between markers OSR16 (1.3 cM) and OSR13 (8.1 cM) on rice chromosome 4 and Rhz2 located between RM119 (2.2 cM) and RM273 (7.4 cM) on chromosome 3.
Progress in Phase I
RM5510.0
RM51810.6
RM26120.6
RM18532.4
RM14243.9RM11948.4Rhz350.6RM27357.8
RM25265.7
RM31778.3
RM349104.3OSR15106.1RM348107.2RM127116.4
RM280123.5
Chromosome 4
RM600.0
RM23115.2RM5883(10)19.9
OSR1344.0Rhz252.1OSR1653.4RM3657.8RM25166.5RM28275.1RM455184.0RM33889.5
RM156116.2
RM4626133.6RM6097142.7OSR31145.8RM55157.3RM1(1)165.0RM2525167.9RM4612180.9
RM114203.1
RM442224.2
Chromosome 3
Progress in Phase IIThe segregation and recombination of genotype of
two dominant complementary genes, Rhz2 and Rhz3 (9:7 model of Mendelian)
AB Ab aB ab
AB AABB AABb AaBB AaBb
Ab AABb AAbb AaBb Aabb
aB AaBB AaBb aaBB AaBb
ab AaBb Aabb aaBb aabb
AB Ab aB ab
AB AABB AABb AaBB AaBb
Ab AABb AAbb AaBb Aabb
aB AaBB AaBb aaBB AaBb
ab AaBb Aabb aaBb aabb
The QTLs of Rhizome traits mapping on Chromomsome
Progress in Phase I
RM428RM323RM283RM2530(6)OSR2RM272RM292
RM158
RM306RM237RM297RM302RM212RM319RM265RM315
OSR23RM529
RL
RIL
RM60
RM231RM5883(10)
OSR13Rhz2OSR16RM36RM251RM282RM4551RM338
RM156
RM4626RM6097OSR31RM55RM1(1)RM2525
RM4612
RM114
RBD
RIL
TNR
L
RN
RBN
RIN
RM551
RM518RM261
RM185RM142RM119Rhz3RM273RM252RM317
RM349OSR15RM348RM127RM280
RL
RN
RBD
RBN
RIL
RIN
TN
RD
W
RL RN RBD
RBN RIL RIN
TN RDW
Chr1 Chr3 Chr4
Progress in Phase IThe QTLs of Rhizome traits mapping on Chromomsome
RM159RM122OSR35
RM13RM405
RM249RM509
RM164
RM291RM163RM161RM421
RM274
RM87
RM334RM31
RL
RN
RIL
RIN
RBD
Chr5RM133RM435RM170
RM587
RM510
RM204RM6420RM314RM253RM402RM276RM136RM5818
RM6309RM528RM4509
RM176RM345
OSR21
RL
RBD
RIL
TN
RM427RM481
RM125RM180RM214RM320RM11OSR22RM2826RM336RM234RM18RM47RM134RM118
RL
RBD
RIL
RM216
RM467
RM271RM269
RM228
RM333
RM496RM590
RL
RN
RIL
RL RN RBD
RBN RIL RIN
TN RDW
Chr6 Chr7 Chr10
Progress in Phase I
RM485
OSR17OSR14
RM279
RM423RM555
RM174OSR9RM322
RM71
RM300
RM341
RM327
RM263
RM3421
RM240
RM166
RM213
RM208RM207
OSR26
2Sorghum
LG F
pSB367
pSB107
pSB201
pSB193
pSB341(M4)
pSB637b(M4)
pSB038
pSB512 (M4)
pSB094
RG157
Csu173
CDO686
RG437
RM216
RM467
RM271
RM269
RM228
RM333
RM496RM590
10
SHO68
Csu111a(M1)
CDO98 QRn10
RM428
RM323RM283RM3530OSR2
RM272
RM292
RM158
RM306
RM237
RM297RM302RM212RM319RM265RM315
OSR23
RM529
1
RZ776(M3)
pSB614(M3)
pSB613(M3)
SorghumLG A
QRl1
RM60
RM231RM6883
OSR13
OSR16RM36
RM251
RM282
RM5551RM338
RM156
RM4626
RM7097OSR31
RM55
RM1RM3525
RM5612
RM114
RM442
3
SorghumLG C
pSB088(M5)
pSB300A(M5)
pSB050(M5)
RZ284
R944
Rhz2
QRn3
QRbd2
QRbn2
Comparative mapping (Rice and Sorghum)
PNAS, 2003, 100:4050-4054
Progress in Phase I
Rhz3
RM427
RM481
RM125
RM180
RM214
RM320RM11OSR22
RM3826
RM336
RM234RM18RM47RM134RM118
7
SorghumLG B
RZ395
pSB077
R1245
RM159
RM122OSR35
RM13
RM405
RM249
RM509
RM164
RM291
RM163
RM161
RM421
RM274
RM87
RM334RM31
5
SorghumLG G
R1436
pSB445
pSB069
RM133
RM435
RM170
RM587
RM510
RM204
RM7420
RM314RM253RM402RM276
RM136
RM5818
RM7309RM528
RM5509
RM176
RM345
OSR21
6
SorghumLG I
pSB355(M6)
CDO17
RZ612
RM551
RM518
RM261
RM185
RM142RM119
RM273
RM252
RM317
RM349OSR15RM348
RM127
RM280
4
SorghumLG D
pSB428a(M2)pSB188(M2)
RZ69
RZ740a
QRl7
QRi6QRn5
QRn7
QRin6
Comparative mapping (Rice and Sorghum)
Progress in Phase IRhz2 and Rhz3 gene registration
The BC2F1 Individual
Progress in Phase I
During in the phase II, the action for PR breeding and genetics are in progress.
The fine mapping of Rhizome genes, Rhz2 and Rhz3, are on the way;
A lots of the breeding lines for PR purpose have been evaluated in field;
The pollen grain fertility loci QTL analysis of F2progeny was detected.
Progress in Phase II
F2 Individual in Field for Rhizome Evaluation and Mapping
Progress in Phase II
F2 plant in field for Rhizome genes fine mapping and breeding lines selection
Progress in Phase II
Experiment: determination of Rhizome expression with 3 replications with random plot design for by cutting method
Progress in Phase II
Result of fine mapping renewed
Progress in Phase II
RM5510.0
RM51810.6
RM26120.6
RM18532.4
RM14243.9RM11948.4 Rhz350.6RM27357.8RM25265.7
RM31778.3
RM349104.3OSR15106.1RM348107.2RM127116.4RM280123.5
Chromosome 4
RM600.0RM23115.2RM5883(10)19.9
OSR1344.0Rhz252.1OSR1653.4RM3657.8RM25166.5RM28275.1RM455184.0RM33889.5
RM156116.2RM4626133.6RM6097142.7OSR31145.8RM55157.3RM1(1)165.0RM2525167.9RM4612180.9
RM114203.1
RM442224.2
Chromosome 3
RM14603Rhz2OSR16
1.12.1
RM119Rhz3RM17000
0.30.4
9.528kb~35kb
Progress in Phase II Microarray
In this study, the specific gene expression patterns across five tissues in O. longistaminata, especially in the rhizome were characterized by using the Affymetrix microarray platform. Results showed that the different gene sets were determined exclusively expressed in five tissues, 58 and 61 genes were identified as prevalent sets in rhizome tip and internode respectively. Cis-element analysis and co-localization of rhizome related QTLs for the rhizome prevalent gene set were further performed
1a 1b 1c 2a 2b 2c 3a 3b 3c 4a 4b 4c 5a 5b 5c
The results will be accepted by Plant biology of BMC
Progress in Phase II Microarray
0
10
20
30
40
50
60
70
80Up-regulated GenesDown-regulated Genes
Functional classification of the differentially expressed genes in the rhizome tip in comparison with shoot tip.
Progress in Phase II Fosmid library
• The library consists of 110,000 clones, which has insertion with an average size of about 43kb and represents 10 genome equivalents and is no bias (10X). The results indicate that the fosmid library has high quality and deep coverage that is sufficient for target gene isolation, physical mapping,gene functional analysis and so on. The Fosmid library of O. longistaminatais first report.
Breeding lines
Progress in Phase II
BC1 RLR504
• Two rhizome gene locus are heterozygote in O. Longi (AaBb);
• 5% grain filling;
• Normal pollen fertility
• Strong Rhizome
• Less cultivar-like plant type
• Short awn
Breeding lines
Progress in Phase II
BC1 RLR540
• Two rhizome gene locus are heterozygote in O. Longi (AaBb);
• 5% grain filling;
• Normal pollen fertility
• Strong Rhizome
• Less cultivar-like plant type
• Short awn
Breeding lines
Progress in Phase II
200808_11 BC1 plant
• Two rhizome gene locus are homozygote in O. Longi (AABB);
• 75% grain filling;
• Normal pollen fertility
• Strong Rhizome
• Self-compatibility
• Less cultivar-like plant type
• Short awn
Breeding lines (F2) AaBb AaBB AABb AABB
Progress in Phase II
36-1 F2 plant AaBb
Two rhizome gene locus are heterozygote;
Normal grain filling;
Strong rhizome presence
Breeding lines
Progress in Phase II
• One rhizome gene locus is heterozygote, other one is homozygote
• Normal grain filling;
• Normal pollen fertility
• Strong rhizome presence
• Self-compatibility
22-93 F2 plant AaBB
Progress in Phase II
34-31 AABb • One rhizome gene locus is heterozygote, other one is homozygote
• Normal grain filling;
• Normal pollen fertility
• Strong rhizome presence
• Self-compatibility
Breeding lines
Progress in Phase II
14-2 AABB
• Two rhizome gene locus are homozygote
•• Normal grain filling;
• Normal pollen fertility
• Strong rhizome presence
• Self-compatibility
Breeding lines
Breeding lines
Progress in Phase II
• Two rhizome gene locus are homozygote in O. longi;
• 75% grain filling;
• Normal pollen fertility
• Rhizome absence
• Self-compatibility
• Awn less
6-28 F2 pant
Breeding lines
Progress in Phase II
• Two rhizome gene locus are homozygote;
• 75% grain filling;
• Normal pollen fertility
• Rhizome absence
• Self-compatibility
10-25 F2 plant
Breeding lines
Progress in Phase II
• Rhz2 is heterozygote, Rhz3 is homozygote;
• 70% grain filling;
• Normal pollen fertility
• Rhizome absence
• Self-compatibility
• Cultivar-like plant type
• Short awn12-38 F2 plant
Spikelet fertility improvement
Progress in Phase II
Rang:
0%-90% above
0% ~2% ~10% ~30% >50% >90%
• NILs with Rhizome constructionProgress in Phase II
0% ~2% ~10% ~30% >50% >90%
F2
F3
F4F5
Fn
NILs with Rhziome
Reproductive Ability after three times for cutting of F2 population
Progress in Phase II
Aug., 2007
Feb., 2008
Aug., 2008
Sep., 2007
Apr., 2008
After Aug., What happen? Dec. 2009?? 2010???
Oct., 2007
Progress in Phase II
F3 population from 34-31(F2) used to reproduced ability test from 2008 to now
Progress in Phase II Reproductive Ability after three times for cutting
2008 2009 2010
• Genetic study on perenniality of rice• Perennial rice breeding• Other perennial crops screening in Kunming
Progress in Phase II(2010)
Two rhizome gene cloning1, Whole genome de novo sequence of O.
longistaminata (finished and data analysis on going)
2, Fine mapping3, Candidate gene of Rhz2 and Rhz34, Transformation for candidate genes
Progress in Phase II(2010)
Whole genome sequence of O. longistaminata
70X coverage the physical map of O. longistaminata
Transgenic results
RI CE_13428 3 Os03t 0216000- 01 Si mi l ar t o Zi nc- f i nger pr ot ei nKNUCKLES, Zi nc f i nger , C2H2- t ype
RNAi
and
Overpress
AABB as receiptor
Perennial Rice Breeding1, NILs2, Obtained the materials with Rhizome in F3,
F6, F7 with different genotype3, Problem?
Progress in Phase II (2010)
NILs of RD23/O. longistaminata
• F7 population with 336 family lines
• Rhizome with normal pollen fertility
• Larger varieties of agronomic traits: plant high, tiller,
• Favorable genes mining, stem borer-resistance
Breeding materials for PR
F3 lines from 22-93(AaBB)
Two plants, AaBB, aaBB
22-93(18), aaBB
22-93(36), AaBB
MAS
F3 lines from 34-31 (AABb)Two plants, AABb, AAbb
Breeding materials for PR
34-31(6-12), AABb
34-31(4-29), AAbb
MAS
• F6 and F7 lines from NILs (AABB)Breeding materials for PR
F6
F7F6
• Problems and prospect
Progress in Phase II (2010)
Rhizome gene Rhz2, Rhz3 cloning
Hope to understand the genetics of Rhizome and using transgenic method for perennial rice breeding.
Next step
Next step
Large population (F3, F4, F5 ) in MAS with 4 different genotypes: AaBB, AABb, AaBb and AABB.
Hope to select the plant with rhizome and good fertility panicle.
Next step
The individuals with Rhizome and Fertility after selected screening both Upland and Aerobic land.
Next stepInternational Cooperation
2009, in field at experimental station in Sanya, China
Next stepWashington State University: perennial wheat
The Land Institute: perennial sorghum, sunflower, wheat, +.
University of Manitoba: (potentially) perennial rye, wheat
Yunnan Academy of Agricultural Sciences: perennial rice
FFI-CRC: perennial wheat
云南农科院,多年生水陆稻
澳大利亚美国土地研究所
Washington State University: perennial wheat
The Land Institute: perennial sorghum, sunflower, wheat, +.
University of Manitoba: (potentially) perennial rye, wheat
Yunnan Academy of Agricultural Sciences: perennial rice
FFI-CRC: perennial wheat
International Network of Perennial Crops
云南农科院,多年生水陆稻
澳大利亚美国土地研究所
NATURE|Vol 456|4 December 2008
可能改变世界的5个作物科学家
Acknowledgment
Financial support from
• National Natural Science Foundation of China
• The Land Institute
Dr. E. Sacks
Prof. Dayun Tao
Thanks to….The UR GroupPeng Xu
Xiangneng Deng
Jiawu Zhou
Jin Li
Wei Deng
Qiong Li
Lijuan Li
Yang Yu
Wenting Wan
Thank you for your attention!