Progress in Perennial Rice Breeding and Genetics

Fengyi Hu

Food Crops Research Institute,YAAS 22 Sept. WaggaWagga, Australia • Soil erosion in uplands of Introduction 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;) The donor(s) for prerenniality?

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?

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 complexes, chromosome number, genome group and distribution Oryza Species, the Species Complex, Chrom.,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) O. rufipogon (AA genome) Photo was cited from Vaughan (1994) Photo was cited from Vaughan (1994) Tufted and scrambling herb, stolon O. longistaminata C A • Long anther • Self- incompatibility • Allogamy • Rhizomatous B D stem • Bacterial Blight resistance(Xa21) • Nematode The Oryza longistaminata. A: The panicle of the O. resistance longistaminata; B: The performance of O. longistaminata in field; C, D: the strong Rhizome • ……. of O. longistaminata. 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. graminicala was 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 longistaminata Photo was cited from Vaughan (1994) The Strategy for Perennial Rice Breeding

RD23 O. longistaminata

Progeny derived from F 1 hope to by Self-intercross, back cross, transgenic and MAS with diversity germplasm of rice F1 plant of RD23_Longi

Rhizome

F1 plant in greenhouse Problems for the PR Breeding and genetics

1, Less F1 progeny from O. longistaminata up 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. Problems for the PR Breeding and genetics

2, Lethal gene The 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, S gene) 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 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. Progress in PR

There are two stages for our results:

Phase I is from 1997-2004; Phase II is from 2005-now. Progress in Phase I

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 F1 plant 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. Rhizome

This is a important material for development Cultivar of PR or PUR. F 1 Progress in Phase I

Segregation of rhizome trait in the

F2 population based on field experiment

Rhizome presence absence Numbers 121 106 2 X (9:7) 0.8011

Result : Two Dominant Complementary, Rhz2, Rhz3, Genes for Rhizome Expression in O. Longistaminata Progress in Phase I PCR-base Molecular Genetic Map

0.0 RM428 0.0 RM485 0.0 RM60 0.0 RM551 0.0 RM159 0.0 RM133 13.1 RM323 9.2 OSR17 6.4 RM122 6.2 RM435 10.6 RM518 OSR35 19.0 RM283 15.1 OSR14 15.2 RM231 11.1 13.7 RM170 RM261 21.6 RM2530(6) 25.5 RM279 19.9 RM5883(10) 20.6 27.3 RM13 26.8 RM587 26.6 OSR2 31.8 RM423 32.4 RM185 RM272 34.9 RM405 RM510 32.3 36.8 RM555 44.0 OSR13 43.9 RM142 37.6 45.7 RM292 52.1 Rhz2 48.4 RM119 RM174 Rhz3 52.4 RM249 56.8 53.4 OSR16 50.6 55.6 RM204 60.0 OSR9 57.8 RM273 58.0 RM509 57.8 RM36 67.3 RM6420 66.5 RM158 62.9 RM322 65.7 RM252 66.5 RM251 74.9 RM314 70.5 RM71 75.1 RM282 78.3 RM317 RM253 87.8 RM306 79.9 RM164 77.6 83.4 RM300 84.0 RM4551 83.0 RM402 97.5 RM237 89.5 RM338 93.9 RM291 RM276 109.5 RM297 96.9 RM341 86.6 104.3 RM349 101.7 RM163 114.7 RM302 98.3 RM136 106.1 OSR15 107.3 RM161 105.4 RM5818 118.1 RM212 112.7 RM327 116.2 RM156 107.2 RM348 117.0 RM421 118.3 RM319 116.4 RM127 122.4 RM265 123.5 RM280 128.5 RM6309 128.5 RM315 133.6 RM4626 132.7 RM528 138.1 RM263 142.7 RM6097 138.9 RM274 143.4 RM4509 145.9 OSR23 145.8 OSR31 148.6 RM2421 151.0 RM87 154.9 RM529 157.5 RM240 157.3 RM55 157.4 RM176 165.0 RM1(1) 163.6 RM334 162.7 RM345 167.9 RM2525 167.6 RM31 RM166 174.5 OSR21 177.7 180.9 RM4612 190.1 RM213 202.7 RM208 203.1 RM114 206.4 RM207 214.6 OSR26 224.2 RM442 www.gramene.org Progress in Phase I

Chromosome 3 Chromosome 4 Mapping for Rhz2 0.0 RM60 0.0 RM551 and Rhz3 15.2 RM231 19.9 RM5883(10) 10.6 RM518 44.0 OSR13 52.1 Rhz2 20.6 RM261 The molecular 53.4 OSR16 57.8 RM36 32.4 RM185 66.5 RM251 mapping of Rhizome 75.1 RM282 84.0 RM4551 43.9 RM142 89.5 RM338 48.4 RM119 gene Rhz2 and Rhz3 was 50.6 Rhz3 57.8 RM273 116.2 RM156 mapped to the interval 65.7 RM252 133.6 RM4626 between markers OSR16 142.7 RM6097 145.8 OSR31 78.3 RM317 157.3 RM55 (1.3 cM) and OSR13 (8.1 165.0 RM1(1) 167.9 RM2525 cM) on rice chromosome 180.9 RM4612 104.3 RM349 106.1 OSR15 4 and Rhz2 located 203.1 RM114 107.2 RM348 between RM119 (2.2 cM) 116.4 RM127 224.2 RM442 123.5 RM280 and RM273 (7.4 cM) on chromosome 3. Progress in Phase II

The 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 Progress in Phase I

The QTLs of Rhizome traits mapping on Chromomsome

Chr1 Chr3 Chr4 RM428 RM60 RM551 RM323 RM518 RM283 RM231 RM2530(6) RM5883(10) RM261 OSR2 RM185

RM272

RBD RIL

TN RM142

OSR13 RDW

RBD RBN

RIN

RIL

RL RN RM292 TN Rhz2 RM119 OSR16 Rhz3 RM36 RM273 RM158 RM252

RM251 RBN

RIN

RN RL RM282 RM317

RM306 RM4551

RIL RL RM237 RM338 RM297 RM349 RM302 OSR15 RM212 RM156 RM348 RM319 RM127 RM265 RM280 RM315 RM4626 RM6097 OSR23 OSR31 RL RN RBD RM529 RM55

RM1(1) RBN RIL RIN RM2525

RM4612 TN RDW

RM114 Progress in Phase I

The QTLs of Rhizome traits mapping on Chromomsome

Chr5 Chr6 Chr7 Chr10

RM159 RM133 RM427 RM216 RM122 RM435 RM481 OSR35 RM170 RM125 RM13 RM587 RM180 RM467 RM405 RM510 RM214

RM320

RIL

RN RL RM249 RM11 RM271 RM509 RM204 OSR22 RM269 RM6420 RM2826

RM314 RBD RM336 RIL RM253 RL RM164 RM234 RM228 RM402 RM18 RM291 RM276 RM47 RM333

RM163 RM136 RM134

RBD

RIN

RIL RN RL RM5818 RM118 RM496

RM161 RBD

RIL RL TN RM590 RM421

RM6309 RL RN RBD RM528 RM274 RM4509 RBN RIL RIN RM87 RM176 RM334 RM345 TN RDW RM31 OSR21 Progress in Phase I Comparative mapping (Rice and Sorghum)

1 2 3

Sorghum LG F RM428 RM485 RM60 Sorghum LG C OSR17 pSB367 RM323 OSR14 RM231 R944 RM283 RM6883 RM3530 pSB107 pSB088 OSR2 RM279 Rhz2 (M5) RM272 RM423 RM555 pSB300A OSR13 (M5) RM292 pSB201 Sorghum QRbd2 LG A RM174 OSR16 OSR9 RM36 pSB050 pSB193 pSB614 RM322 (M5) 10 RM158 RM251 (M3) RM71 QRl1 QRn3 RM282 RZ776 Csu173 RZ284 (M3) RM300 RM5551 RM216 CDO686 RM306 RM338 pSB613 QRbn2 pSB341 RM237 (M3) RM341 (M4)

RM297 RM302 RM327 pSB637b RM212 (M4) RM156 RM467 RM319 RM265 RG157 RM315 RM4626 RM263 RM7097 RM271 OSR23 pSB038 OSR31 RM3421 RM529 SHO68 RM240 RM55 CDO98 QRn10 RM1 Csu111a RM3525 RM269 (M1) pSB512 (M4) RM166 RM5612 RM228 RG437

RM213 RM333 pSB094 RM208 RM114 RM207 RM496 OSR26 RM590 RM442 PNAS, 2003, 100:4050-4054 Progress in Phase I Comparative mapping (Rice and Sorghum)

4 5 6 7

RM159 RM133 RM427

Sorghum RM122 RM435 RM551 LG D RM481 OSR35 RM170 Rhz3 RM518 QRn7 RM125 RM261 RM13 RM587 RZ69 RM180 RM405 RM185 RM510 Sorghum RM214 LG B

RM142 pSB428a RM320 RM119 RM249 (M2) RM204 RM11 pSB188 RM509 OSR22 RM273 (M2) QRl7 RM3826 pSB077 RM7420 RM252 RM336 R1245 RZ740a RM314 Sorghum RM253 RM164 LG I RM317 Sorghum RM402 RZ395 RM276 RM234 LG G RM18 RM291 RM47 QRn5 RM136 RM134 RM163 RM118 RM349 pSB445 RM5818 RM161 QRi6 OSR15 RM348 pSB355 R1436 (M6) RM421 RM127 pSB069 RM280 CDO17 RM7309 RM528 RZ612 RM274 RM5509

RM87

RM176 RM334 RM345 RM31 QRin6 OSR21 Progress in Phase I Rhz2 and Rhz3 gene registration Progress in Phase I

The BC2F1 Individual Progress in Phase II

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 F2 progeny 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

Chromosome 3 Chromosome 4

0.0 RM60 0.0 RM551 15.2 RM231 19.9 RM5883(10) 10.6 RM518 44.0 OSR13 52.1 Rhz2 20.6 RM261 53.4 OSR16 RM14603 57.8 RM36 2.1 32.4 RM185 66.5 RM251 Rhz2 75.1 RM282 1.1 84.0 RM4551 OSR16 43.9 RM142 RM119 89.5 RM338 48.4 RM119 0.4 50.6 Rhz3 Rhz3 57.8 RM273 0.3 116.2 RM156 RM1700 65.7 RM252 133.6 RM4626 0 142.7 RM6097 ~35kb 145.8 OSR31 78.3 RM317 9.528kb 157.3 RM55 165.0 RM1(1) 167.9 RM2525 180.9 RM4612 104.3 RM349 106.1 OSR15 203.1 RM114 107.2 RM348 116.4 RM127 224.2 RM442 123.5 RM280 Progress in Phase II Microarray

1a 1b 1c 2a 2b 2c 3a 3b 3c 4a 4b 4c 5a 5b 5c 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 The results will be for the rhizome prevalent gene set were accepted by Plant further performed biology of BMC Progress in Phase II Microarray

80 Up-regulated Genes 70 Down-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. longistaminata is first report. Progress in Phase II

Breeding lines • Two rhizome gene locus are heterozygote in O. Longi (AaBb); • 5% grain filling; • Normal pollen fertility • Strong Rhizome • Less cultivar- like plant type BC1 RLR504 • Short awn Progress in Phase II

Breeding lines • 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 200808_11 BC1 plant Progress in Phase II

Breeding lines (F2) AaBb AaBB AABb AABB

Two rhizome gene locus are heterozygote; Normal grain filling; Strong rhizome presence

36-1 F2 plant AaBb Progress in Phase II

Breeding lines

• 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 Breeding lines

34-31 AABb • One rhizome gene locus is heterozygote, other one is homozygote • Normal grain filling; • Normal pollen fertility • Strong rhizome presence • Self-compatibility Progress in Phase II Breeding lines

• Two rhizome gene locus are homozygote • • Normal grain filling; • Normal pollen fertility • Strong rhizome presence • Self-compatibility 14-2 AABB Progress in Phase II

Breeding lines

• 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 Progress in Phase II

Breeding lines

• Two rhizome gene locus are homozygote; • 75% grain filling; • Normal pollen fertility • Rhizome absence • Self-compatibility

10-25 F2 plant Progress in Phase II

Breeding lines

• Rhz2 is heterozygote, Rhz3 is homozygote; • 70% grain filling; • Normal pollen fertility • Rhizome absence • Self-compatibility • Cultivar-like plant type • Short awn 12-38 F2 plant Progress in Phase II

Spikelet fertility improvement

Rang: 0%-90% above

0% ~2% ~10% ~30% >50% >90% Progress in Phase II • NILs with Rhizome construction

0% ~2% ~10% ~30% >50% >90%

F4 F5 Fn NILs with Rhziome Progress in Phase II Reproductive Ability after three times for cutting of F2 population

Aug., Sep., Oct., 2007 2007 2007

Feb., Apr., 2008 2008

After Aug., Aug., What happen? 2008 Dec. 2009?? 2010??? 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 Progress in Phase II(2010)

• Genetic study on perenniality of rice • Perennial rice breeding • Other perennial crops screening in Kunming Progress in Phase II(2010)

Two rhizome gene cloning 1, Whole genome de novo sequence of O. longistaminata (finished and data analysis on going) 2, Fine mapping 3, Candidate gene of Rhz2 and Rhz3 4, Transformation for candidate genes Whole genome sequence of O. longistaminata

70X coverage the physical map of O. longistaminata Transgenic results

RNAi AABB as and receiptor Overpress

Si mil ar t o Zi nc- f i nger pr ot ei n RICE_13428 3 Os03t 0216000- 01 KNUCKLES, Zi nc f i nger , C2H2- t ype Progress in Phase II (2010)

Perennial Rice Breeding 1, NILs 2, Obtained the materials with Rhizome in F3, F6, F7 with different genotype 3, Problem? 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 MAS

22-93(36), AaBB

22-93(18), aaBB Breeding materials for PR

F3 lines from 34-31 (AABb) Two plants, AABb, AAbb MAS

34-31(4-29), AAbb

34-31(6-12), AABb Breeding materials for PR • F6 and F7 lines from NILs (AABB)

F6 F7 Progress in Phase II (2010)

• Problems and prospect Next step

Rhizome gene Rhz2, Rhz3 cloning

Hope to understand the genetics of Rhizome and using transgenic method for perennial rice breeding. 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 step International Cooperation

2009, in field at experimental station in Sanya, China Next step

Washington State University: perennial wheat

University of Manitoba: (potentially) perennial rye, wheat

Yunnan Academy of Agricultural Sciences: perennial rice 云南农科院，多年生水陆稻

FFI-CRC: perennial wheat 澳大利亚美国土地研究所 The Land Institute: perennial sorghum, sunflower, wheat, +. International Network of Perennial Crops 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 Thanks to…. The UR Group Peng Xu Dr. E. Sacks Xiangneng Deng Prof. Dayun Tao Jiawu Zhou Jin Li Wei Deng Qiong Li Lijuan Li Yang Yu Wenting Wan Thank you for your attention!