Progress in Perennial Rice Breeding and Genetics...O.eichingeri Peter 24 CC Sri Lanka,Africa...

Progress in Perennial Rice

Breeding and Genetics

Fengyi Hu

Food Crops Research Institute,YAAS

22 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?


All over the world growth of O. sativa is Annual


• 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 Am

Am

Tropical Australia

O.glumaepatula Steud. 24 Agl

Agl

South America

O.glaberrima Steud. 24 AgA

g Africa(mainly West)

O.barthii A.Chev. 24 AgA

g Africa

O.longistaminata Chev.et Roehr 24 AlA

l 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

RD23

O. 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

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.

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.


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.


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.


5, Seed Dormancy


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

F

1

Rhizome

F1 plant

Progress in Phase I

Segregation of rhizome trait in the

F2 population based on field experiment

absence

0.8011X2(9:7)

106121Numbers

presenceRhizome

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

RM30687.8RM23797.5RM297109.5RM302114.7RM212118.1RM319118.3RM265122.4RM315128.5

OSR23145.9

RM529154.9

RM4850.0

OSR179.2OSR1415.1RM27925.5RM42331.8RM55536.8

RM17456.8OSR960.0RM32262.9RM7170.5

RM30083.4

RM34196.9

RM327112.7

RM263138.1

RM2421148.6

RM240157.5

RM166177.7

RM213190.1

RM208202.7RM207206.4OSR26214.6

RM600.0

RM23115.2RM5883(10)19.9

OSR1344.0Rhz252.1OSR1653.4RM3657.8RM25166.5RM28275.1RM455184.0RM33889.5

RM156116.2

RM4626133.6RM6097142.7OSR31145.8RM55157.3RM1(1)165.0RM2525167.9

RM4612180.9

RM114203.1

RM442224.2

RM5510.0

RM51810.6

RM26120.6

RM18532.4RM14243.9RM11948.4Rhz350.6RM27357.8RM25265.7

RM31778.3

RM349104.3OSR15106.1RM348107.2RM127116.4RM280123.5

RM1590.0RM1226.4OSR3511.1

RM1327.3RM40534.9

RM24952.4RM50958.0

RM16479.9

RM29193.9RM163101.7RM161107.3

RM421117.0

RM274138.9

RM87151.0

RM334163.6RM31167.6

RM1330.0RM4356.2RM17013.7

RM58726.8

RM51037.6

RM20455.6RM642067.3RM31474.9RM25377.6RM40283.0RM27686.6RM13698.3RM5818105.4

RM6309128.5RM528132.7

RM4509143.4

RM176157.4RM345162.7

OSR21174.5

www.gramene.org

Mapping for Rhz2

and 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

RM14243.9RM11948.4

Rhz350.6

RM27357.8

RM25265.7

RM31778.3

RM349104.3OSR15106.1RM348107.2

RM127116.4

RM280123.5

Chromosome 4

RM600.0

RM23115.2RM5883(10)19.9

OSR1344.0

Rhz252.1OSR1653.4RM3657.8RM25166.5RM28275.1RM455184.0RM33889.5

RM156116.2

RM4626133.6RM6097142.7OSR31145.8RM55157.3RM1(1)165.0RM2525167.9

RM4612180.9

RM114203.1

RM442224.2

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

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

RM428

RM323RM283RM2530(6)OSR2RM272

RM292

RM158

RM306RM237RM297RM302RM212RM319RM265RM315

OSR23

RM529

RL

RIL

RM60

RM231RM5883(10)

OSR13Rhz2OSR16RM36RM251RM282RM4551RM338

RM156

RM4626RM6097OSR31

RM55RM1(1)RM2525

RM4612

RM114

RB

D

RIL

TN

RL

RN

RB

N

RIN

RM551

RM518

RM261

RM185

RM142RM119Rhz3RM273RM252

RM317

RM349OSR15RM348RM127RM280

RL

RN

RB

D

RB

N

RIL

RIN

TN

RD

W

RL RN RBD

RBN RIL RIN

TN RDW

Chr1 Chr3 Chr4

Progress in Phase I

The QTLs of Rhizome traits mapping on Chromomsome

RM159RM122OSR35

RM13

RM405

RM249RM509

RM164

RM291RM163RM161

RM421

RM274

RM87

RM334RM31

RL

RN

RIL

RIN

RB

D

Chr5

RM133RM435

RM170

RM587

RM510

RM204RM6420RM314RM253RM402RM276

RM136RM5818

RM6309RM528

RM4509

RM176RM345

OSR21

RL

RB

D

RIL

TN

RM427

RM481

RM125RM180RM214RM320RM11OSR22RM2826RM336RM234RM18RM47RM134RM118

RL

RB

D

RIL

RM216

RM467

RM271

RM269

RM228

RM333

RM496RM590

RL

RN

RIL

RL RN RBD

RBN RIL RIN

TN RDW

Chr6 Chr7 Chr10

Progress in Phase I

RM485

OSR17

OSR14

RM279

RM423

RM555

RM174

OSR9RM322

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

RM496

RM590

10

SHO68

Csu111a

(M1)

CDO98 QRn10

RM428

RM323

RM283RM3530

OSR2

RM272

RM292

RM158

RM306

RM237

RM297RM302RM212RM319RM265

RM315

OSR23

RM529

1

RZ776

(M3)

pSB614

(M3)

pSB613

(M3)

Sorghum

LG A

QRl1

RM60

RM231

RM6883

OSR13

OSR16RM36

RM251

RM282

RM5551

RM338

RM156

RM4626

RM7097OSR31

RM55

RM1RM3525

RM5612

RM114

RM442

3

Sorghum

LG 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

RM320

RM11

OSR22

RM3826

RM336

RM234RM18

RM47

RM134

RM118

7

Sorghum

LG B

RZ395

pSB077

R1245

RM159

RM122

OSR35

RM13

RM405

RM249

RM509

RM164

RM291

RM163

RM161

RM421

RM274

RM87

RM334

RM31

5

Sorghum

LG G

R1436

pSB445

pSB069

RM133

RM435

RM170

RM587

RM510

RM204

RM7420

RM314

RM253

RM402

RM276

RM136

RM5818

RM7309

RM528

RM5509

RM176

RM345

OSR21

6

Sorghum

LG I

pSB355

(M6)

CDO17

RZ612

RM551

RM518

RM261

RM185

RM142

RM119

RM273

RM252

RM317

RM349

OSR15RM348

RM127

RM280

4

Sorghum

LG D

pSB428a

(M2)

pSB188

(M2)

RZ69

RZ740a

QRl7

QRi6

QRn5

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 F2

progeny was detected.


F2 Individual in Field for Rhizome Evaluation and Mapping


F2 plant in field for Rhizome genes fine mapping and breeding lines selection


Experiment: determination of Rhizome expression with 3 replications with random plot design for by cutting method


Result of fine mapping renewed


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

RM14603

Rhz2

OSR161.1

2.1

RM119

Rhz3

RM1700

0

0.3

0.4

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

80

Up-regulated Genes

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. longistaminatais first report.

Breeding lines


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


BC1 RLR540

• Two rhizome

gene locus are

heterozygote in

O. Longi (AaBb);


• Normal pollen

fertility

• Strong Rhizome

• Less cultivar-

like plant type

• Short awn

Breeding lines


200808_11 BC1 plant

• Two rhizome gene

locus are homozygote

in O. Longi (AABB);


• Normal pollen

fertility

• Strong Rhizome

• Self-compatibility

• Less cultivar-like

plant type

• Short awn

Breeding lines (F2) AaBb AaBB AABb AABB


36-1 F2 plant AaBb

Two rhizome

gene locus are

heterozygote;

Normal grain

filling;

Strong rhizome

presence

Breeding lines


• One rhizome gene

locus is heterozygote,

other one is

homozygote

• Normal grain filling;

• Normal pollen fertility

• Strong rhizome

presence


22-93 F2 plant AaBB


34-31 AABb • One rhizome gene

locus is heterozygote,

other one is

homozygote



• Strong rhizome

presence


Breeding lines


14-2 AABB



•



• Strong rhizome

presence


Breeding lines

Breeding lines




in O. longi;



• Rhizome absence


• Awn less

6-28 F2 pant

Breeding lines



locus are homozygote;



• Rhizome absence


10-25 F2 plant

Breeding lines


• Rhz2 is heterozygote,

Rhz3 is homozygote;



• Rhizome absence


• Cultivar-like plant type

• Short awn12-38 F2 plant

Spikelet fertility improvement


Rang:

0%-90%

above

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

• NILs with Rhizome construction


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

F2

F3

F4

F5

Fn

NILs with Rhziome

Reproductive Ability after three times for cutting of F2 population


Aug.,

2007

Feb.,

2008

Aug.,

2008

Sep.,

2007

Apr.,

2008

After Aug.,

What happen?

Dec. 2009??

2010???

Oct.,

2007


F3 population from 34-31(F2) used to reproduced ability

test from 2008 to now


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 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

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- 01Si mi l ar t o Zi nc- f i nger pr ot ei n

KNUCKLES, Zi nc f i nger , C2H2- t ype

RNAi

and

Overpress

AABB as

receiptor

Perennial Rice Breeding

1, NILs

2, Obtained the materials with Rhizome in F3,

F6, F7 with different genotype

3, 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


34-31(6-12), AABb

34-31(4-29), AAbb

MAS

• F6 and F7 lines from NILs (AABB)


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 step

International Cooperation

2009, in field at experimental station in Sanya, China

Next step

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

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 Group

Peng Xu

Xiangneng Deng

Jiawu Zhou

Jin Li

Wei Deng

Qiong Li

Lijuan Li

Yang Yu

Wenting Wan

Thank you for your attention!

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