ACP PROJECT 1.2

Increasing sugar productivity through the development of high sucrose and early

ripening genotypes

Mid-Term Review1 October 2012

MSIRIRéduit

Mauritius

Dr Goolam BadalooDr 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

4 different variety types identified with very distinct sucrose accumulation patterns and the early variety significantly in advance

Series10

2

4

6

8

10

12

14

16

18

CP 721210 (Early)

M 2343/77 (High Sucrose)

M 937/77(Low Sucrose)

R 570(Late)

(%)

H1(mid-May)

H2(mid-Aug)

H3(3rd wk Oct)

Juice purity of different variety types at early, mid and late season

Series140

50

60

70

80

90

100

CP 721210 (Early)

M 2343/77 (High Sucrose)

M 937/77 (Low Sucrose)

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

Activity 1Develop/validate a methodology for characterisation of ER/HS genotypes & characterise 400 parent varieties for ER/HS

Activity 2Identify molecular markers linked to earliness of ripening and high sucrose as a tool for marker-assisted selection

Activity 3Develop ER/HS sugar cane genotypes for use in the breeding and selection programmes and for commercial exploitation

Overall objectiveIncrease 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: 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 - Completed with 8 parents and is being followed up.2 - 200 varieties planted in 2010 and 200 in 2011 are being followed up3 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 & mid-November.

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 - Completed with 8 parents and is being followed up.2 - 200 varieties planted in 2010 and 200 in 2011 are being followed up3 All data are quality controlled and stored in Excel worksheets for future database development once a first full set will be available

Develop genetic maps and identify molecular markers for

use in marker-assisted selection

ACTIVITY 2

Will be dealt by Dr A Dookun-Saumtally

Activity 3: ProgressActivity 3.1 Field evaluation and selection through a seedling stage and successive clonal stages

2011 2012 2013 2014

3.1.1 Seedling stage1

Raised beds, 1st ratoon sampling, pol % cane,Sample weight and visual grade (family)

Series 1(15000)

Series 2(15000)

3.1.2 1st clonal stage5-m plot size

Series 1

3.1.3 2nd clonal stage2 x 5-m plots, replicated

1 Crosses for series 1 done in 2010 and crosses for series 2 done in 2011

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 3.1.1Seedling stage

Series 114 500 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

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 3.1.1Seedling 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 3.1.1Seedling 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

Activity 3.1.1Seedling stage - Series 1

Results – Activity 3.1.1 – Seedling stage – Series 1

Combs Female Type MalePol %cane

Sample weight (kg)

Family Visual grade

Selection Rate (%)

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 692High

Polycross 9.2 16.5 3.1 11

865/10 CP 67412Precocious high

Polycross 8.0 16.3 3.0 11

662/10 J 593High

Polycross 9.2 15.8 2.7 5

Combs Female Type MalePol %cane

Sample weight (kg)

Family Visual grade

Selection Rate (%)

673/10 CP 67412 Precocious high Polycross 10.8 10.4 4.0 30

1149/10 CP 67412Precocious high

H 493533 10.5 10.0 3.4 18

1316/10 CP 67412 Precocious high Polycross 10.3 10.0 3.0 10

1187/10 CP 67412Precocious high

Polycross 9.3 9.7 3.5 8

Results – Activity 3.1.1 – Seedling stage – Series 1

Activity 3.1.21st Clonal stage – arising from selection of

seedlings

SelectionBest 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 3.1.1Production of an array of improved high sucrose

and early-ripening sugar cane genotypesSeries 2New set of 15000 seedlings planted in April

2012.Replicated trials, RCBD with 3 blocks of 56

seedlings per replicateRaised 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 and selection through a seedling stage and successive clonal stages

2011 2012 2013 2014 Remarks

Seedling stage Intermating of genotypes from precocious/early ripening families.

1st clonal stage Possibility of intermating parent genotypes

2nd clonal stage

Develop genetic maps and identify molecular markers for

use in marker-assisted selection

ACTIVITY 2

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 meiosisRequirements: 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 2011 2012 2013 2014

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

Selection of Distant Parents

Most divergent parents selected after screening with more than 100 SSR markers and diversity analysisCP 67412: Precocious ripening, high sucrose

M 245/76 : Late ripening, low sucrose Mapping population derived bi-parental cross produced

Population planted in the field in March 2012

CP 67412 x M 245/76 HS/ER LS/LR

477 progeny

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

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

• 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

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

Parent 2

Single Nucleotide Polymorphism (SNP)

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 Illinois-Urbana 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 markersCross 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 coverageField: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-SaumtallyDr Kishore RamdoyalMr Razack NayamuthDr Goolam BadalooMr Yogesh ParmessurMr Satish KoonjahMr Harrydas MungurMs Manesha SukhooMs Lovena Nowbut

Acknowledgements