Intensifying legume/cereal cropping systems in Malawi
021614 yang-rui li--research and development priorities for sugar industry of china
1. 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
2. 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
⑧⑨
Major Sugarcane
Growing Area in
Mainland China
3. 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.
7. 1. More than 80% of sugarcane is grown in
rain-fed upland fields, and easily affected by
drought.
8. 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.
9. 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
14. Sugarcane germplasm innovation
• 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.
16. E. arundinaceus - S. spontaneum complex (AS complex) creating
E. arundinaceus × S. spontaneum
15 crosses, 8000 seedlings
Progeny detection : Phenotype + molecular (SSR-PCRSRAP-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
17. 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
Germplasm innovation with S. spontaneum and E. arundinaceus
25. 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
Sugarcane germplasm innovation
26. 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
Sugarcane germplasm innovation
28. 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:
Sugarcane germplasm innovation
29. 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.
30. 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.
31. 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
32. 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)
New sugarcane varieties
33. Released in 2011
Cross: ZZ92-126×CP72-2086
Mid maturity, high yield, good
ratoon ability, good drought
tolerance
GT37 (GT03-2357)
New sugarcane varieties
34. 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)
New sugarcane varieties
35. 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)
New sugarcane varieties
39. • 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.
52. (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.
53.
54. 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.
56. 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
57. 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
58. 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
59. Proteomic analysis of interaction between N-fixing bacterium
and sugarcane
GT21, Control
pH4-7,loaded 300μg/gel,silver stained
11
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)
60. AB188122.1)Azohydromonaslata
EU048175.1)Unculturedbacterium
EU542578.1)Ideonelladechloratans
AB188121.1)Azohydromonasaustralica
SRSnifH120609CT51SRSnifH120609CT33
EU048169.1)Unculturedbacterium
AJ505315.1)Sinorhizobiumsp.
JX081993.1)Ensiferadhaerens
EU586055.1)Unculturedbacterium
AB188120.1)Pelomonassaccharophila
EU544203.1)Unculturedbacterium
FJ008185.1)Uncultured
soilbacterium
AP012304.1)Azoarcus
sp._seq2
A
P
012304.1)A
zoarcus
sp._seq1
F
R
850745.1)R
hizobium
legum
inosarum
381376528)R
ubrivivax
gelatinosus
AY196462.1)Uncultured nitrogen-fixing bacterium
SRSnifH120609CT15
FJ593866.1)Klebsiella sp.
EU048149.1)Uncultured bacterium
EU048059.1)Uncultured bacterium
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
AY231551.1)Uncultured nitrogen-fixing bacterium
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 propionicus
S
R
S
nifH
120609C
T
57
H
Q
190142.1)U
ncultured
bacterium
CP001998.1)Coraliom
argarita
akajim
ensis
EU978427.1)Unculturedmicroorganism
JX545230.1)Stenotrophomonasmaltophilia
CP002298.1)Desulfovibriovulgaris
AY787578.1)Unculturedbacterium
JX268325.1)Unculturedbacterium
AY196392.1)Unculturednitrogen-fi
SRSnifH120609CT17
JX268243.1)Unculturedbacterium
JX268254.1)Unculturedbacterium
GQ289580.1)Bradyrhizobiumjaponicum
DQ520342.1)Unculturedbacterium
DQ776342.1)Unculturedsoilbacterium
AY601051.1)Unculturedbacterium
CP001124.1)Geobacterbemidjiensis
CP001661.1)Geobactersp.
CP002479.1)Geobactersp.
EU912951.1)Unculturedbacterium
CP000769.1)Anaeromyxobactersp.
CP001390.1)Geobacterdaltonii
AE017180.2)Geobactersulfurreducens
SRSnifH120609CT34
S
R
S
nifH
120609C
T38
G
Q
289581.1)B
radyrhizobium
japonicum
JX
2
6
8
2
6
8
.1
)U
n
cu
ltu
re
d
b
a
cte
riu
m
JX268291.1)Uncultured bacterium
GQ289574.1)Bradyrhizobium japonicum
FJ381622.1)Uncultured bacterium
FJ381624.1)Uncultured bacterium
JX268505.1)Uncultured bacterium
SRSnifH120609CT20
SRSnifH120609CT27
SRSnifH120609CT24
GU117592.1)Uncultured bacterium
SRSnifH120609CT62
SRSnifH120609CT45
SRSnifH120609CT23SRSnifH120609CT30
SRSnifH120609CT18
SRSnifH120609CT28
SRSnifH120609CT50
SRSnifH120609CT43
SRSnifH120609CT49
SRSnifH120609CT32
SRSnifH120609CT37
SRSnifH120609CT59
S
R
S
nifH
120609C
T
6
S
R
S
nifH
120609C
T19
SRSnifH120609CT42
SRSnifH120609CT47
SRSnifH120609CT53
SRSnifH120609CT22
SRSnifH120609CT40
SRSnifH120609CT31
0.1
Sequence alignment and
phylogenetic tree construction
61. 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;
62. 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.
63. 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
64. 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
65. 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
67. Identification of transgenic plant resistance
to Chilo infuscatellus
Three larva of Chilo infuscatellus were inoculated into one
transgenic plants for experiment.
68. 68
Proteomics related to sugarcane resistance
to adverse stresses
Materials
Protein extraction
2-D eletrophoresis
Scanning
Protocol for proteomic analyses
Software analysisDifferential proteins
MS identification
Bioinformatics
73. 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.
74. • 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.
75. The program has completed small fragments
sequencing, and the data are under analyses.
BAC library has been completed, and will be
used for sequencing.
76. 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).