021614 yang-rui li--research and development priorities for sugar industry of china

Research and Development

Priorities for Sugar Industry of

China: Recent Research Highlights

Dr. Yang-Rui Li, Professor and Director

Sugarcane Research Center

Chinese Academy of Agricultural Sciences

Guangxi Academy of Agricultural Sciences

International Conclave on Sugar Crops & SugarFest

2014

Sweeteners and Green Energy from Sugar Crops:

Emerging Technologies

February 15-17, 2014, Lucknow-India

Distribution of Sugar Crops in China

1 Guangxi 2 Yunnan 3 Guangdong

4 Hainan 5 Hunan 6 Sichuan

7 Jiangxi 8 Fujian 9 Guizhou

① ③

②

②

④

⑤

⑥ ⑦

⑧ ⑨

Major Sugarcane

Growing Area in

Mainland China

Sugar industry for 2012/2013 in China

• Total sugar:13.07 million tons;

• Cane sugar is 11.98 million tons;

• Beet sugar 1.09 million tons.

• Sugar mills: total 293 among them 248 for sugarcane and 45 mills for sugar beet.

Su

gar

pro

du

cti

on

(M

illi

on

To

ns)

Milling year

Sugar Production in China

0

2

4

6

8

10

12

14

16

98/99 99/00 00/01 01/02 02/03 03/04 04/05 05/06 06/07 07/08 08/09 09/10 10/11 11/12 12/13

Cane sugar Beet sugar Tatol sugar

The cane sugar output (million ton) in the

major provinces in China since 1998-99

Year Total Guangxi Yunnan Guang- Hainan Others

dong

2002/03 9.40 5.61 1.89 1.17 0.42 0.31

2003/04 9.43 5.88 1.95 0.99 0.41 0.20

2004/05 8.57 5.32 1.59 0.90 0.39 0.37

2005/06 8.01 5.38 1.41 0.92 0.19 0.12

2006/07 10.75 7.09 1.83 1.28 0.38 0.17

2007/08 13.68 9.41 2.16 1.45 0.52 0.17

2008/09 11.53 7.64 2.23 1.06 0.46 0.14

2009/10 10.07 7.10 1.71 0.86 0.32 0.08

2010/11 9.66 6.73 1.76 0.87 0.23 0.07

2011/12 10.51 6.94 2.01 1.15 0.31 0.10

2012/13 11.98 7.93 2.24 1.21 0.50 0.10

Challenge for Sugar

Industry in China

1. More than 80% of sugarcane is grown in

rain-fed upland fields, and easily affected by

drought.

2. Cost increase for sugarcane production

due to fast increase in labor, fertilizers,

chemicals and other supplies.

• Cost escalation

Labor cost: 20-30% increase yearly.

Cane harvest: 99% manual operation;

100-150 Yuan (USD$16.5-24.8) per ton.

Fertilizer & chemical: 30% increase; especially

50-70% increase for potassium fertilizer.

3. Singleness of sugarcane variety

It is suffered easily by biotic and abiotic

stresses, such as low temperature, insect

pest , diseases, etc.

Serious damage

by frost

4. Pests and diseases

Ratoon stunting disease

Smut

Shoot rot

Borer Thripid

Aphis

Scarab

Longhorn beetle

Sugarcane germplasm innovation

Future research priorities of

Chinese sugar industry


• Wild germplasm such as S. Spontaneum,

Erianthus, Narenga distributes in various

locations of China.

• Incorporate wild germplasm such as Saccharum

spontaneum L., Erianthus arundinacius (Retz.)

Jesws. and Narenga porphyrocoma (Hance)

Bor. into the commercial breeding parents for

sugarcane improvement.

SRC-CAAS/GXAAS has a sugarcane cross breeding base in Sanya city of Hainan province (18°35′ N, 109°68′E).

E. arundinaceus - S. spontaneum complex (AS complex) creating

E. arundinaceus × S. spontaneum

15 crosses, 8000 seedlings

Progeny detection : Phenotype + molecular (SSR-PCR\SRAP-PCR)+cytology

AS complex (GXAS)

AS complex F1 (GXASF1)

AS complex BC1 (GXASBC1)

Backcross: S. hybrid

Backcross: S. hybrid

Germplasm innovation with S. spontaneum and E. arundinaceus

AS complex: GXAS07-6-1 (GXA87-36 × GXS79-9)

1：Female:GXA87-36 2：Male: GXS79-9 3： GXAS07-6-1

400

300

200

100

1 2 3 1 2 3 1 2 3 M

SSR identification

mSSCIR36 mSSCIR67 mSSCIR19



AS complex:

GXAS07-6-1

×

GT02-761


AS complex: GXAS07-6-1 Progeny

chromosome observation

GXS79-9（2n=64)

GXA87-36（2n=60) GXAS07-6-1（2n=62)

GXASF1 08-2-33 (2n=80)

GXASF1 08-1-10 (2n=76)

GXASF1 08-3-1 (2n=76)

Deduced transmission pattern: n + n

Chromosome transmission of AS complex

AS complex genomic in situ hybridization (GISH)

50×Blocking 80×Blocking

Green: S. spontaneum

Red: E. arundinaceus


Similarity coefficient between GXASF1 and male parent

♀YT93-159

♂and F1 ♀GT01-53 ♂and F1 ♀GT02-761

♂and F1

GXAS07-6-1 0.488 GXAS07-6-1 0.535 GXAS07-6-1 0.531

08-1-1 0.753 08-2-1 0.713 08-3-1 0.723

08-1-2 0.736 08-2-2 0.748 08-3-2 0.746

08-1-3 0.719 08-2-5 0.742 08-3-3 0.750

08-1-4 0.701 08-2-6 0.717 08-3-5 0.753

08-1-5 0.737 08-2-10 0.754 08-3-6 0.730

08-1-6 0.738 08-2-22 0.734 08-3-7 0.736

08-1-7 0.725 08-2-24 0.725 08-3-8 0.717

08-1-8 0.718 08-2-33 0.703 08-3-10 0.733

08-1-9 0.726 08-2-37 0.742 08-3-11 0.712

08-1-10 0.720 08-2-52 0.707 08-3-12 0.729

08-1-11 0.712 08-2-54 0.731 08-3-13 0.736

08-3-15 0.713

Genetic background of AS complex F1 progeny:

Molecular Marker Assisted Selection


M 1 2 3 4 5 6 M

AFLP genetic

background of

GXASBC1

1：GXASF1 08-1-11

2, 5：Co649

3： GXASBC1: GXASF1 08-

1-11 × Co649

4：GXASF1 08-1-1

6： GXASBC1: GXASF1 08-

1-1 × Co649

500

400

300

200


DNA inheritance of E. arundinaceus and S. spontaneum in

their progenies of different generation

4.1 genetic relationship of GXAS07-6-1 and its parents by SRAP

600

500

400

300

200

100

M 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 M

1：Male: GXA87-36 2：Female: GXS79-9 3： GXAS07-6-1


24 particular loci of female parent GXA87-36 (E.

arundinaceus) all pass to both F1 and BC1.

16 particular loci of male parent GXS79-9 (S. spontaneum)

pass 16 to F1, 15 to BC1.

DNA inheritance of E. arundinaceus and S.

spontaneum in their progenies of different

generations


Major traits investigation of GXASBC1

GXASBC1 11-1-11 GXASBC1 11-3-9

Stalk number: 14

Stalk diameter: 2.2

Brix: 19

Stalk number: 6

Stalk diameter: 2.5

Brix: 23


Design more BC2, BC3 crosses by good BC1 or

BC2 clones;

Keep tracking molecular marker in breeding

process;

Keep tracking chromosome transmission in

breeding process;

Work on going:


Germplasm exchanges

Germplasm exchanges with different

countries are also our priority.

We hope to improve the genetic

variation of sugarcane crossing parents

through utilizing the germplasm from

different countries.

The accumulation of multiple

germplasm would increase

heterogeneous in the hybrids.

Sugarcane breeding and

variety propagation

Producing high productivity, high sugar,

strongly resistant and nitrogen efficient

sugarcane cultivars is one of our major

priority in our sugarcane breeding

program.

Ratoon ability is also one of the

important selections for sugarcane

improvement in China.

Released in 2010

Cross: YC94-46×ROC22

Early maturity, high yield, high

sucrose content, good smut

resistance, strong ratoon

ability, strong tillering, good

cold tolerance

GT29（ GT02-761）

New sugarcane varieties

Released in 2011

Cross: YT91-976×ROC1

Early maturity, high yield,

high sucrose content, good

cold and drought tolerance,

good ratoon ability

GT32 (GT02-208)


Released in 2011

Cross: ZZ92-126×CP72-2086

Mid maturity, high yield, good

ratoon ability, good drought

tolerance

GT37 (GT03-2357)


Released in 2013

Cross:YT85-177 × CP84-

1198

Early maturity, high sucrose

content, high yield, good

smut resistance, good cold

and drought tolerance,

good ratoon ability

GT40 (GT02-1156)


Released in 2013

Cross: ROC22 × GT92-66

High yield , high sucrose

content, good ratoon ability,

good disease resistance,

good drought tolerance,

broad adaptability

GT42 (GT04-1001)


Development of low cost

drought resistant cultivation

technologies

Model for applying

vinasse in sugarcane:

Changli Farm, Shangsi

County

② ①

③ ④

Use vanasse as liquid fertilizer

Vinasse application Conventional management

• Recently, we have successfully used vinasse

for making granule fertilizer, which is good for

storage, transportation and application.

• Combining popularization of the two

technologies will completely avoid vanasse

from pollution, and recycle the nutrient into

agricultural fields.

（3）Water-saving

irrigation

（3）Water-saving irrigation

Spray irrigation

Drip irrigation

Trash mulching

PC-Based fertilization

（6）Comprehensive pests control

Sexual attractant application

Cuban fly application

•Borer control

Trichogramma application

Longhorn beetle control

▲Beauveria

bassiana control

▲Manual control ▲Good food

•Larva

Field trapping

•Adult:In May to early June

•Field trapping

• Good for many pests.

Light trapping

• Only good for young larva

Chemical control

Healthy seedcane production

Sterilized stem tip is used for tissue culture

Healthy seedcane production

1st year healthy seedcane Good performance of healthy seedcane

（10）Machine operation

It is urgent to develop mechanization for

sugarcane production;

Large scale of farm will be necessary for

mechanization;

Machine operation must match with farming

practice.

New standard for millable cane should be made

and used to promote the mechanization

development.

Biological nitrogen fixation in sugarcane

• We are trying to develop methods for detecting the

bacterial nifH gene expression in sugarcane stems

based on the established associative sugarcane-

diazotroph systems, use qRT-PCR to measure the nifH

expression activity in sugarcane stems from 25 cultivars

grown under conditions suitable for associative N2

fixation, and use RT-PCR to amplify the nifH

transcriptomes from cultivars showed high nifH

expression activity.

Biological nitrogen fixation in

sugarcane

Morphological identification (22000×)

L03: Klebsiella plantica

rod shaped bacterium with

multiple flagella, size:

(0.3~0.5)μm×(0.9~1.2)μm

A01:Pantoea agglomonarens

Rod, single and pairwise

aligment, multiple flagella;

size:(0.6~0.8)μm×(1.8~2.5)μm

L09: Klebsiella axytoca

rod shaped bacterium with

multiple flagella, size:

(0.3~0.5)μm×(0.9~1.2)μm

Endophytic N-fixing bacteria invasion and

colonization

E. coli S17-1pir strain with

pFAJ1819 vector (GFP gene)

pFAJ1819 vector was transferred into

nitrogen-fixing bacterium,

Klebsiella plantica (L03)

Strain with GFP

Day 1

root hair zone

Day 2

root cells

Day 4

root column Day 7

stalk parenchyma

Day 8

leaf Kranz

Root cap Root hair zone Lateral root

formation

Root cortex Leaf sheath cells

Detection of N-fixing bacterium, Microbacterium sp. (16SH)

contained GFP gene colonization in sugarcane under a laser

scanning confocal microscopy

Tissue cultured seedling of ROC22 inoculated with N-fixing bacterium,

Klebsiella plantica (L03) under a fluorescence microscopy

Proteomic analysis of interaction between N-fixing bacterium

and sugarcane

GT21, Control

pH4-7，loaded 300μg/gel，silver stained

1 1

4

3

17

4

3

10 6

5

2 2

5

6 9

8

7 7

8

9

10 13

15

14

12

11 11

12

13 14

15

17

16 16

GT21 inoculated with Klebsiella sp.

for 20 days up-regulated：1 (spot #10);

down-regulated: 10 (#1,2,3,4,6,7,8,11,13,15;

novel: 4 (#5,9,14,17)

AB1881

22.1)Azo

hydrom

onas la

taEU0

48175.1

)Uncult

ured bac

terium

EU5425

78.1)Ide

onella d

echlora

tans

AB1881

21.1)Azo

hydrom

onas au

stralica

SRSnifH

120609

CT51

SRSnifH

120609

CT33

EU048169

.1)Uncu

ltured ba

cterium

AJ50531

5.1)Sinor

hizobium

sp.

JX081993.1)Ensifer adhaerens

EU586055.1)Uncultured bacterium

AB188120.1)Pelomonas saccharophila


FJ008185.1)Uncultured soil bacterium

AP012304.1)Azoarcus sp._seq2

AP012304.1)Azoarcus sp._seq1

FR850745.1)Rhizobium leguminosarum

381376528)Rubrivivax gelatinosus

AY196462.1)Uncultured nitrogen-fixing bacterium

SRSnifH120609CT15

FJ593866.1)Klebsiella sp.



AY544164.1)Delftia tsuruhatensis

HQ404304.1)Klebsiella pneumoniae

AJ563957.1)Methylobacter luteus

DQ481036.1)Uncultured bacterium

FJ822995.1)Agrobacterium tumefaciens

JN648883.1)Uncultured bacterium

CP001157.1)Azotobacter vinelandii

AM406670.1)Azoarcus sp.JX154793.1)Uncultured nitrogen-fixing bacterium

JX154830.1)Uncultured nitrogen-fixing bacterium

JX154844.1)Uncultured Dechloromonas sp.

AJ563286.1)Dechloromonas sp.

GU121498.1)Uncultured bacterium

SRSnifH120609CT14


AY768421.1)Tolypothrix sp.

JN162465.1)Uncultured bacterium

CP003548.1)Nostoc sp._seq1

CP003548.1)Nostoc sp._seq2

GU111829.1)Uncultured soil bacterium

DQ142699.1)Uncultured bacterium

EF408200.1)Uncultured cyanobacterium

HQ836199.1)Anabaena sp.

SRSnifH120609CT29

CP002364.1)Desulfobulbus propionicusSRSnifH120609CT57

HQ190142.1)Uncultured bacterium

CP001998.1)Coraliomargarita akajimensis

EU978427.1)Uncultured microorganism

JX545230.1)Stenotrophomonas maltophilia

CP002298.1)Desulfovibrio vulgaris

AY787578.1)Uncultured bacterium

JX268325.1)Uncultured bacterium


SRSnifH120

609CT17

JX2682

43.1)Un

cultured

bacteriu

m

JX2682

54.1)Un

cultured

bacteriu

m

GQ289

580.1)B

radyrhiz

obium ja

ponicum

DQ5203

42.1)Un

cultured

bacteriu

m

DQ7763

42.1)Un

cultured

soil ba

cterium

AY6010

51.1)Un

cultured

bacteriu

m

CP0011

24.1)Ge

obacter

bemidjie

nsis

CP00166

1.1)Geob

acter sp

.

CP002479

.1)Geob

acter sp

.


CP000769.1)Anaeromyxobacter sp

.

CP001390.1)Geobacter daltonii

AE017180.2)Geobacter sulfurreducens

SRSnifH120609CT34

SRSnifH120609CT38

GQ289581.1)Bradyrhizobium japonicum



GQ289574.1)Bradyrhizobium japonicum

FJ381622.1)Uncultured bacterium

FJ381624.1)Uncultured bacterium


SRSnifH120609CT20

SRSnifH120609CT27

SRSnifH120609CT24


SRSnifH120609CT62

SRSnifH120609CT45

SRSnifH120609CT23SRSnifH120609CT30

SRSnifH120609CT18

SRSnifH120609CT28

SRSnifH120609CT50

SRSnifH120609CT43

SRSnifH120609CT49

SRSnifH120609CT32

SRSnifH120609CT37

SRSnifH120609CT59

SRSnifH120609CT6

SRSnifH120609CT19

SRSnifH120609CT42

SRSnifH120609CT47

SRSnifH120609CT53

SRSnifH120609CT22

SRSnifH120609CT40

SRSnifH120609CT31

CP000482.1)Pelobacter propionicus


HQ586727.1)Uncultured alpha proteobacterium

0.1

Sequence alignment and

phylogenetic tree construction

Use high-throughput sequencing and

bioinformatics to analyze the diversity of the

functional diazotrophs and to determine the

major functional diazotrophs in sugarcane;

Isolate the major functional diazotrophs from

sugarcane plants or select ones from the

available culture collection for diazotrophs in

China;

Use the major functional diazotrophs to

inoculate the sugarcane cultivars shown high

nifH expression activity;

Use qRT-PCR and 15N isotope dilution methods

to determine the efficient sugarcane N2-fixing

systems;

Use in situ hybridization assay to find the N2-

fixing location in sugarcane plants.

Detect the effects of soil pH, nitrogen and

phosphorous levels, and microbes on nitrogen

fixation efficiency in sugarcane.

Gene cloning and transformation

1. Cloning and function analyses of different

families of SPS gene in sugarcane

2. Transformation of insect resistance gene

(Bt) into sugarcane

3. Transformation of cold resistance gene

into sugarcane

Cloning of SPS genes

Full length of cDNA sequences of 4 families of

SPS genes in sugarcane have been cloned:

SofSPSA (HM854011)

SofSPSB (JN584485)

SofSPSDⅢ (HQ117935)

SofSPSDⅣ

Infection, co-culture and screening Differentiation and propagation

Transformation of insect resistance gene (Bt

gene) through Bar screening

Hardening and rooting Root growth of Bt transgenic plants

3/13/201

4

Greenhouse culture of transgenic plants using

phosphomannose isomerase (PMI) as label for screening

Identification of transgenic plant resistance

to Chilo infuscatellus

Three larva of Chilo infuscatellus were inoculated into one

transgenic plants for experiment.

68

Proteomics related to sugarcane resistance

to adverse stresses

Materials Protein extraction

2-D eletrophoresis Scanning

Protocol for proteomic analyses

Software analysis Differential proteins

MS identification

Bioinformatics

Drought resistance related proteomics

greenhouse

Pot culture

Aquiculture

2-DE profiles of PDQuest analyses

ROC22

ROC16

CK PEG PEG+Si

ROC22

CK PEG PEG+Si

ROC16

Profiles of parts of enlarged differential protein

spots

MALDI-TOF-TOF/MS identification

1st profile

2nd profile

MALDI-TOF-TOF/MS profile for spot 25

Genome research

Although sugarcane genomes are very complex

and the genome research progresses very

slowly, it is important to sequence the whole

genome.

After years of preparation, Sugarcane Research

Center, CAAS/ GXAAS decided to begin the

whole sugarcane genome sequencing program

in China: F13TSFSCKF1043.

This is being conducted with a local collection of

Saccharum spontaneum, GXS87-16 (2n=64)

with the cooperation of Beijing Genome Institute

(BGI) –Shenzhen.

• The first term is planning to be completed in 3

years since July 2013. In the first term of the

project, 100 X (100 Gb) of WGS sequencing will

be done, 10 X BAC clones will be constructed

and 1 X (about 10,000 clones) will be selected

for BAC library construction and sequencing.

These data will be used to estimate complexity

of sugarcane genome, and the primary

sequence map will be obtained.

The program has completed small fragments

sequencing, and the data are under analyses.

BAC library has been completed, and will be

used for sequencing.

Acknowledgement

• China 863 Program (2013AA102604), China

International Cooperation Program

(2013DFA31600), National Natural Science

Foundation of China (31171504, 31101122,

31240056); Natural Science Foundation of

Guangxi Provence (2011GXNSFF018002,

2011GXNSFA018076, 2013NXNSFAA019073,

2013NXNSFAA019082), and Guangxi Key R &

D Program (GKC1123008-1, GKG1222009-1B).

5th IAPSIT International

Conference

(IS-2014)

Green Technologies for Sustainable

Growth of Sugar & Integrated

Industries in Developing Countries

November 25-28, 2014

Nanning, P.R. China

•

Guilin - Grand place with

wonderful landscape

Wonderful Seashores in Hainan province

3/13/201

4

021614 yang-rui li--research and development priorities for sugar industry of china

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Transcript of 021614 yang-rui li--research and development priorities for sugar industry of china