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ACP P ROJECT 1.2 Increasing sugar productivity through the development of high sucrose and early ripening genotypes Mid-Term Review 1 October 2012 MSIRI.

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Presentation on theme: "ACP P ROJECT 1.2 Increasing sugar productivity through the development of high sucrose and early ripening genotypes Mid-Term Review 1 October 2012 MSIRI."— Presentation transcript:

1 ACP P ROJECT 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

2 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

3 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

4 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

5 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 CP (Early) M 2343/77 (High Sucrose) M 937/77 (Low Sucrose) R 570 (Late) (%) H1 (mid-May) H2 (mid-Aug) H3 (3 rd wk Oct)

6 Juice purity of different variety types at early, mid and late season CP (Early) M 2343/77 (High Sucrose) M 937/77 (Low Sucrose) R 570 (Late) (%) H1 (mid-May) H2 (mid-Aug) H3 (3 rd 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

7 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 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 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

8 Activity 1: Progress Activity Establish replicated trials with sub set of 10 parents in three environments and evaluate for sucrose accumulation pattern Screening of 400 parents for sucrose accumulation Develop and update databases Completed with 8 parents and is being followed up varieties planted in 2010 and 200 in 2011 are being followed up 3 All data are quality controlled and stored in Excel worksheets for future database development once a first full set will be available

9 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

10 Activity 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 1 st ratoon for the 2010 trial whilst the one planted in 2009 was in 2 nd ratoon

11 Activity Screening of 400 parent varieties for sucrose accumulation The 1 st batch of 200 varieties, planted in 2010, was sampled and harvested at the scheduled dates - mid-May, mid-August & mid-November. 2 nd 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

12 Activity 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.

13 Activity 1: Future works Activity Remarks Establish replicated trials with sub set of 10 parents in three environments and evaluate for sucrose accumulation pattern 1 8 parent varieties planted in 3 environments Replicated trials Sampling for pol% cane & purity Screening of 400 parents for sucrose accumulation 2 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 databases 3 Database will be created in Completed with 8 parents and is being followed up varieties planted in 2010 and 200 in 2011 are being followed up 3 All data are quality controlled and stored in Excel worksheets for future database development once a first full set will be available

14 Develop genetic maps and identify molecular markers for use in marker-assisted selection ACTIVITY 2 Will be dealt by Dr A Dookun-Saumtally

15 Activity 3: Progress Activity 3.1 Field evaluation and selection through a seedling stage and successive clonal stages Seedling stage 1 Raised beds, 1 st ratoon sampling, pol % cane, Sample weight and visual grade (family) Series 1 (15000) Series 2 (15000) st clonal stage 5-m plot size Series nd clonal stage 2 x 5-m plots, replicated 1 Crosses for series 1 done in 2010 and crosses for series 2 done in 2011

16 Activity 3 Production of an array of improved high sucrose and early-ripening sugar cane genotypes Crossing of parents based on pre-evaluation data ( ) Production of seedlings from true sexual seeds. Evaluation and selection across three selection stages, namely Seedling (stage 1) 1 st clonal (stage 2) 2 nd clonal (stage 3)

17 Activity Seedling stage Series 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 1 st ratoon for selection in 2012

18 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 Seedling stage - Series 1

19 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 Seedling stage - Series 1

20 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 Seedling stage - Series 1

21 Results – Activity – Seedling stage – Series 1 CombsFemaleTypeMale Pol % cane Sample weight (kg) Family Visual grade Selection Rate (%) 773/10 W Early highCP /10 L 6025Precocious highPolycross /07 CP Precocious high CP /10 TUC 692 High Polycross /10 CP Precocious high Polycross /10 J 593 High Polycross

22 CombsFemaleTypeMale Pol % cane Sample weight (kg) Family Visual grade Selection Rate (%) 673/10CP 67412Precocious highPolycross /10CP Precocious high H /10CP 67412Precocious highPolycross /10CP Precocious high Polycross Results – Activity – Seedling stage – Series 1

23 Activity st Clonal stage – arising from selection of seedlings Selection Best genotypes selected (397) and planted at the 1 st clonal stage on 1 x 5-m plots, without replication Site - Deep River Beau Champ on 11 th 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

24 Activity Production of an array of improved high sucrose and early-ripening sugar cane genotypes Series 2 New set of seedlings planted in April 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

25 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

26 Activity 3: Future works Activity 3.1 Field evaluation and selection through a seedling stage and successive clonal stages Remarks Seedling stageIntermating of genotypes from precocious/early ripening families. 1 st clonal stagePossibility of intermating parent genotypes 2 nd clonal stage

27 Develop genetic maps and identify molecular markers for use in marker-assisted selection ACTIVITY 2 Dr A Dookun Saumtally

28 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

29 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

30 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

31 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

32 Activity 2: Develop genetic map & identify molecular markers for marker assisted selection Activity Establish one segregating population of HS/ER x LS/LR Establish 1 st clonal stage Establish replicated trials in two environments Field evaluation of progeny for sucrose accumulation 2.2 Identification of markers

33 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 Population planted in the field in March 2012 CP x M 245/76 HS/ER LS/LR 477 progeny

34 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

35 Application of EST-SSR to linkage mapping of CP Method Label reverse primer with radioactive P 33 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 Method Label reverse primer with radioactive P 33 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 & M 245/76

36 Results More than 55% primers (out of 650) polymorphic for the early ripening clone CP i.e present in CP & 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

37 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

38 Parent 1 Parent 2 Single Nucleotide Polymorphism (SNP) Illumina sequencer

39 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)

40 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)

41 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

42 Activity 2: Develop genetic map and identify molecular markers for marker assisted selection Activity 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 1 st clonal stagePlanted in April 2012 Establish replicated trials in two environments Field evaluation of progeny for sucrose accumulation Evaluation will need to be carried out beyond 2014 Identification of markersApplication of RAD Seq technology in progress

43 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

44 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

45 Acknowledgements


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