ACP PROJECT 1.2
Increasing sugar productivity through the
development of high sucrose and early
ripening genotypes
Mid-Term Review
1 October 2012
MSIRI
Réduit
Mauritius
Dr Goolam Badaloo
Dr Asha Dookun-Saumtally
Increasing sugar productivity through the development
of high sucrose and early ripening genotypes
Implementing Institution: Mauritius Sugarcane Industry
Research Institute
Countries targeted:
ACP sugar producing countries
Duration of project:
> 4 years
Cost of project:
€ 935,650, excl. NIR €155000
Importance of breeding high
sucrose/early ripening varieties

As a result of centralization of sugar mills, the milling
period has extended & harvest is starting earlier

Lack of high performing varieties for early stage

Increase in sugar content throughout the harvest season

Increase in sugar productivity
Sucrose accumulation in sugarcane



Sucrose accumulation patterns differ among varieties.
Early-ripening, ER, varieties produce more
sucrose/tonne of cane at the start of the ripening
season compared to late varieties.
However, ER varieties accumulate less sucrose at
middle and late-season
Pol % cane of different variety types at early, mid and late season
(%)
18
M 2343/77
(High Sucrose)
16
14
M 937/77
(Low Sucrose)
12
CP 721210
10 (Early)
8
6
4
2
R 570
(Late)
0
H1
(mid-May)
H2
(mid-Aug)
H3
(3rd wk Oct)
4 different
variety types
identified with
very distinct
sucrose
accumulation
patterns and
the early
variety
significantly in
advance
Juice purity of different variety types at early, mid and late season
(%)
100
M 2343/77 (High Sucrose)
90
80
M 937/77 (Low Sucrose)
CP 721210
(Early)
70
60
50
40
R 570
(Late)
H1
(mid-May)
H2
(mid-Aug)
H3
(3rd wk Oct)
Maturity differing
between the 4
variety types with
the early one
harvestable at
nearly 80% juice
purity in mid-May
well ahead of the
start of the harvest
season
Overall objective
Increase sugar productivity/unit area through the development of
early-ripening & high sucrose varieties (ER/HS), to ensure the
sustainability and the competitiveness of sugar industries in ACP
countries
Activity 1
Develop/validate a methodology for characterisation of
ER/HS genotypes & characterise 400 parent varieties for
ER/HS
Activity 2
Identify molecular markers linked to earliness of ripening
and high sucrose as a tool for marker-assisted selection
Activity 3
Develop ER/HS sugar cane genotypes for use in the
breeding and selection programmes and for commercial
exploitation
Activity 1: Progress
Activity 1
2011
2012
2013
2014
1.1 Establish replicated trials with sub set of 10
parents in three environments and evaluate for
sucrose accumulation pattern1
1.2 Screening of 400 parents for sucrose
accumulation2
1.3 Develop and update databases3
1
2
3
- Completed with 8 parents and is being followed up.
- 200 varieties planted in 2010 and 200 in 2011 are being followed up
All data are quality controlled and stored in Excel worksheets for future database development once a first full set will be available
Activity 1

Develop and validate a methodology for
characterisation of early-ripening/high sucrose
genotypes
&
characterise 400 parent varieties available in
the germplasm for early-ripening ability and
high sucrose content
Activity 1.1 - Establish replicated trials with ten
parent varieties in three contrasting environments
for evaluation of sucrose evolution
Three trials ongoing, one planted in 2009 in the
superhumid zone (Esperance), two in 2010 in the
humid & subhumid zones (Etoile & St. Antoine)
Eight (8) parent varieties being followed
Trials were sampled at the scheduled dates for
early season (mid-May), mid-season (end-August)
& late-season (Nov./Dec.)
Harvest was at the same age of 12 months in 1st
ratoon for the 2010 trial whilst the one planted in
2009 was in 2nd ratoon
Activity 1.2 - Screening of 400 parent varieties for
sucrose accumulation
 The 1st batch of 200 varieties, planted in
2010, was sampled and harvested at the
scheduled dates - mid-May, mid-August & midNovember.
2nd batch of 200 varieties followed in plant
cane crop in 2011 at Réduit E.S. under irrigation.
 Samples were analyzed for cane quality
characters - Brix, Pol & fibre % cane, juice purity,
dry matter in cane
Activity 1.3 - Develop a database for use by all ACP
sugar-producing countries
 Agronomy & crop management data collected in PC
and 1R crops.
 Data keyed and stored in Excel
 Development of the database will start after a
complete dataset is available (H3 harvest in November
2012).
User-friendly applications, easy access and querying
of database, downloading and export for in depth
statistical and other analyses.
Activity 1: Future works
Activity 1
2011
2012
2013
2014
Remarks
Establish replicated trials with sub
set of 10 parents in three
environments and evaluate for
sucrose accumulation pattern1
8 parent varieties planted in
3 environments
•Replicated trials
•Sampling for pol% cane &
purity
Screening of 400 parents for
sucrose accumulation2
Done in 2 batches of 200
parents varieties planted in
2010/2011 in replicated
trials; for sampling for pol%
cane and purity at 3 dates
Develop and update databases3
Database will be created in
2013
1
2
3
- Completed with 8 parents and is being followed up.
- 200 varieties planted in 2010 and 200 in 2011 are being followed up
All data are quality controlled and stored in Excel worksheets for future database development once a first full set will be available
ACTIVITY 2
Develop genetic maps and
identify molecular markers for
use in marker-assisted
selection
Will be dealt by Dr A Dookun-Saumtally
Activity 3: Progress
Activity 3.1 Field evaluation and selection
2011
2012
Series 1
(15000)
Series 2
(15000)
through a seedling stage and successive clonal
stages
3.1.1 Seedling stage1
Raised beds, 1st ratoon sampling, pol % cane,
Sample weight and visual grade (family)
3.1.2 1st clonal stage
5-m plot size
3.1.3 2nd clonal stage
2 x 5-m plots, replicated
1
Crosses for series 1 done in 2010
and crosses for series 2 done in 2011
Series 1
2013
2014
Activity 3
Production of an array of improved high
sucrose and early-ripening sugar cane
genotypes
• Crossing of parents based on pre-evaluation data
(2007-2010)
• Production of 15000 seedlings from true sexual
seeds.
• Evaluation and selection across three selection
stages, namely
• Seedling (stage 1)
• 1st clonal (stage 2)
• 2nd clonal (stage 3)
Activity 3.1.1
Seedling stage
Series 1
 14 500 seedlings produced from crosses made in
2010 and planted in 2011 in replicated trials (FUEL)

28 families planted in 3 replicates (60 seedlings per
replicate)
◦ Remaining seedlings planted family-wise in an adjacent field
for practical reasons

Population was stubble-shaved in August 2011 to
simulate a 1st ratoon for selection in 2012
Activity 3.1.1
Seedling stage - Series 1

Sampling - April 2012

Fifteen millable stalks per family per replicate
◦ Pol and fibre on fresh and dry weight basis, purity

Field characters:
◦ Family visual grade (1 poor to 5 excellent)
◦ Sample weight of 15 millable stalks
◦ Stalk diameter, number, height, growth habit
Activity 3.1.1
Seedling stage - Series 1

10 families had at least one parent classified as
precocious high/high sucrose content.

Families were ranked according to pol % cane,
sample weight and visual grade

Combined selection (family and individual)
◦ Differential selection rates selection
 More genotypes selected from the best families
Activity 3.1.1
Seedling stage - Series 1

Highly significant differences between families for
most characters (1%).

Purity as high as 0.82,

Precocious/early ripening families displayed rapid
growth..

Families with low sucrose parents had considerably
lower pol % (both fresh and dry basis), dry matter %
cane and purity
Results – Activity 3.1.1 – Seedling stage – Series 1
Sample
weight (kg)
Family
Visual
grade
Selection
Rate (%)
Combs
Female
Type
Male
Pol %
cane
773/10
W 681049
Early high
CP 62258
10.5
16.7
4.5
35
484/10
L 6025
Precocious high
Polycross
10.6
12.8
4.3
33
462/07
CP 67412
Precocious high
CP 44101
8.6
17.3
3.8
24
668/10
TUC 692
Polycross
9.2
16.5
3.1
11
865/10
CP 67412
Polycross
8.0
16.3
3.0
11
662/10
J 593
Polycross
9.2
15.8
2.7
5
High
Precocious high
High
Results – Activity 3.1.1 – Seedling stage – Series 1
Sample
weight (kg)
Family
Visual
grade
Selection
Rate (%)
Combs
Female
Type
Male
Pol %
cane
673/10
CP 67412
Precocious high
Polycross
10.8
10.4
4.0
30
1149/10
CP 67412
H 493533
10.5
10.0
3.4
18
1316/10
CP 67412
Polycross
10.3
10.0
3.0
10
Polycross
9.3
9.7
3.5
8
Precocious high
Precocious high
Precocious high
1187/10
CP 67412
Activity 3.1.2
1st Clonal stage – arising from selection of
seedlings
Selection
 Best genotypes selected (397) and planted at the
1st clonal stage on 1 x 5-m plots, without replication


Site - Deep River Beau Champ on 11th May 2012
(i) Design - Augmented Latin Square
◦ Two standard commercial varieties
◦ M 52/78 and M 1400/86

(ii) Design - Line and Column
◦ One standard commercial control M 1400/86
Activity 3.1.1
Production of an array of improved high sucrose
and early-ripening sugar cane genotypes
Series 2
 New set of 15000 seedlings planted in April
2012.
 Replicated trials, RCBD with 3 blocks of 56
seedlings per replicate
 Raised beds (0.75-m x 0.75-m)
 10 control varieties with different ripening
pattern
◦ precocious high, early high, middle high and late stable
high
Constraint – Activities 1.1 & 1.2
Methodology for characterisation of ER/HS
genotypes & characterise 400 parent varieties
The unavailability of the Infracana equipment for
handling of large number of samples for analysis of
laboratory quality characters such as pol % cane, Brix
% cane and juice purity constitutes a handicap
Activity 3: Future works
Activity 3.1 Field evaluation
2011 2012 2013 2014 Remarks
and selection through a
seedling stage and successive
clonal stages
Seedling stage
Intermating of genotypes
from precocious/early
ripening families.
1st clonal stage
Possibility of intermating
parent genotypes
2nd clonal stage
ACTIVITY 2
Develop genetic maps and
identify molecular markers for
use in marker-assisted
selection
Dr A Dookun Saumtally
Breeding for early ripening sugarcane cultivars





Early ripening trait: poorly understood in sugarcane
Unknowns: number of genes involved/pathways/switch?
Early ripening varieties currently selected by measuring
sucrose accumulation at different intervals in the season
Ripening influenced by the environment
Therefore, there is room for improvement to bring
classical selection more accurate, less costly, & much
faster
Improvement For Selection of Early Ripening
Introduce marker assisted selection(MAS)
as an additional tool:
 to enhance the efficiency of the selection
programme
 to select for markers tightly linked to
early ripening trait
QTL Mapping For Early Ripening In Sugarcane
How to get there?
 Construction of a linkage map for an early ripening
sugarcane cultivar
 Association of phenotypic trait of a mapping population
to molecular markers to identify markers linked to early
sucrose ripening gene (s) -Quantitative Trait Loci-QTL
Construction of a linkage map of late ripening/low sucrose cultivar
& identification of markers linked to genes contributing to the
suppression of sucrose accumulation
Linkage Map Construction
Based on recombination (crossing over) during meiosis
Requirements:
 Genetically distant parental lines with diverging traits
 Mapping Population of at least 200 individuals derived
from selected parents

Activity 2: Develop genetic map & identify
molecular markers for marker assisted selection
Activity 2
2.1.1 Establish one segregating population of
HS/ER x LS/LR
2.1.2 Establish 1st clonal stage
2.1.3 Establish replicated trials in two
environments
2.1.4 Field evaluation of 200-250 progeny for
sucrose accumulation
2.2
Identification of markers
2011
2012
2013
2014
Selection of Distant Parents


Most divergent parents selected after screening with
more than 100 SSR markers and diversity analysis
CP 67412: Precocious ripening, high sucrose
M 245/76 : Late ripening, low sucrose
Mapping population derived bi-parental cross produced
CP 67412 x M 245/76
HS/ER
LS/LR
477 progeny

Population planted in the field in March 2012
Choice of Marker System(s)?




RFLP : low throughput X
Genomic SSR and AFLP: low/medium throughput but
no information on sequence data X
EST-SSR: targets genes, enables comparative mapping
with related crop species
√
RADs - Restriction Site Associated DNA sequencing √
Application of EST-SSR to linkage mapping
of CP 67412
• 4,500 sugarcane EST-SSR primers available
• 600 sorghum EST-SSR primers evenly distributed amongst the
gramineae genome also available
• About 650 EST-SSR screened by PCR for polymorphism between
mapping parents CP 67412 & M 245/76
Method
 Label reverse primer with radioactive P33
 Carry out PCR on genomic DNA of mapping parents (in duplicate)
 Denature PCR products and run on polyacrylamide sequencing gels
 Expose gels to X-ray films and develop films in 2-3 days
 Score for polymorphism
Results





More than 55% primers (out of 650) polymorphic for the early
ripening clone CP 67412
i.e present in CP 67412 & absent in M 245/76
75% polymorphism if present in M245/76 & absent in CP67412
also considered
Average level of polymorphism between mapping parents = 2
(useful markers need to segregate in 1:1 ratio)
Mapping population to be genotyped & mapped on both
parents
RADseq

Also known as Genotyping By Sequencing (GBS)

Most high throughput genotyping system available so far



Based on Next Generation Sequencing (NGS) technology &
identification of Single Nucleotide Polymorphism (SNP)
markers - single base substitution or deletion/insertion
Make use of Illumina sequencing : that can provide several
hundred million reads from a sequencing library (1 lane) in
1week at a lower cost compared to capillary sequencing
Mapping population can be pooled for sequencing
Parent 1
Single Nucleotide Polymorphism (SNP)
Parent 2
Illumina
sequencer
RADseq applied to sugarcane population
DNA extracted from 360 individuals & two parents
 Digestion of DNA with two restriction enzymes MseI and NsiI
 Ligation with adaptors containing barcodes 1-48 & index 1-12
(combination of barcode and index determine the individual)
 Amplification of ligated product using adaptor directed primers
 Quantitate amplicon concentration from each individual
 Create sequencing library by pooling equal amounts of
template DNA from each individual
 Sequence library using Illumina HiSeq (Univ of Illinois, Urbana
Champaign)

RADseq
250 samples currently being processed at University of IllinoisUrbana Champaign, USA
 Samples divided into three lanes
 Lane1produced >144 million reads (sequence fragments)
 Sequence Quality / Quality score : Excellent
 Awaiting sequence data from lanes 2 and 3
 Expected number of markers : More than 5,000
(compared with SSR: 1 per day, AFLP 10 per day)
Quality of data will depend on:
1.
Genome coverage (proportion of genome sequenced)
2.
and sequencing depth (representation of each sequence fragment
among the total number of reads)

Use of Bioinformatics
Sequence data analysis requires use of supercomputers

Align sequences of mapping parents to a reference
genome (sorghum) and determine the level of SNPs

Screen mapping population for SNPs distribution

Score SNP markers segregating 1:1 ratio

Construct linkage map using Joinmap
Activity 2: Develop genetic map and identify
molecular markers for marker assisted selection
Activity
2011 2012 2013 2014 Remarks
Establish one segregating
population of HS/ER x LS/LR
Parents chosen following
screening with SSR markers
Cross HS/ER x LS/LR
performed
Establish 1st clonal stage
Planted in April 2012
Establish replicated trials in two
environments
Field evaluation of 200-250
progeny for sucrose accumulation
Evaluation will need to be
carried out beyond 2014
Identification of markers
Application of RAD Seq
technology in progress
Future work
Laboratory: Combine marker data & construct high density map using Joinmap
Major limitation of RADseq: missing data due to incomplete sequence
depth
How to remedy?
1. Re-sequencing of the library
2. Construct additional library based on new restriction enzyme
combination for better genome coverage
Field:
Establishing trials with mapping population: 2 environments, 3 replications
& 3 harvest dates
Phenotypic traits scoring in trials: ER/LR
Associating laboratory and field data
Collaborators in the project
Dr A Dookun-Saumtally
Dr Kishore Ramdoyal
Mr Razack Nayamuth
Dr Goolam Badaloo
Mr Yogesh Parmessur
Mr Satish Koonjah
Mr Harrydas Mungur
Ms Manesha Sukhoo
Ms Lovena Nowbut
Acknowledgements