Acknowledgments - ACIAR | Australian Centre for...

Final reportSmall research and development activity

project Proof on concept: verification of chain of custody of teak in Indonesia using DNA markers

project number FST/2014/028

date published 7/12/2015

prepared by Andrew Lowe, University of Adelaide

co-authors/ contributors/ collaborators

Eleanor Dormontt, University of AdelaideAnto Rimbawanto, Centre for Forest Biotechnology and Tree Improvement, Forestry Research and Development Agency

approved by Tony Bartlett, Forestry Research Program Manager

final report number FR2015-15

ISBN 978-1-925436-12-9

published by ACIARGPO Box 1571Canberra ACT 2601Australia

This publication is published by ACIAR ABN 34 864 955 427. Care is taken to ensure the accuracy of the information contained in this publication. However ACIAR cannot accept responsibility for the accuracy or completeness of the information or opinions contained in the publication. You should make your own enquiries before making decisions concerning your interests.

© Australian Centre for International Agricultural Research (ACIAR) 2015 - This work is copyright. Apart from any use as permitted under the Copyright Act 1968, no part may be reproduced by any process without prior written permission from ACIAR, GPO Box 1571, Canberra ACT 2601, Australia, [email protected].

Final report: Proof on concept: verification of chain of custody of teak in Indonesia using DNA markers

Contents

1 Acknowledgments.......................................................................................3

2 Executive summary.....................................................................................4

3 Introduction..................................................................................................6

4 Objectives and Activities............................................................................7

5 Technical Feasibility....................................................................................95.1 Molecular methods..............................................................................................................9

6 Political Feasibility.....................................................................................126.1 Workshop..........................................................................................................................12

6.2 On-ground Operations Visit...............................................................................................19

6.3 Overall comment on political feasibility..............................................................................21

7 Conclusions and recommendations........................................................227.1 Conclusions....................................................................................................................... 22

7.2 Recommendations............................................................................................................23

8 References..................................................................................................248.1 References cited in report.................................................................................................24

8.2 List of publications produced by project............................................................................24

9 Appendixes.................................................................................................259.1 Appendix 1: Minutes from project coordination meeting – Yogyakarta 30th October

2014................................................................................................................................. 25

9.2 Appendix 2: Table of representative 100 SNPs from candidate loci..................................27

Page


1 AcknowledgmentsThe authors would like to thank the ACIAR for its generous support of this project; staff and students at the University of Adelaide and FORDA for their hard work and enthusiasm; the participants of the workshop for their engagement with the project; and the staff of Perhutani for their on-ground hospitality and cooperation.

Page


2 Executive summaryAustralia is committed to working in partnerships with supplier countries to reduce the trade in illegal timber into Australia for the economic benefit of supplier countries and to improve environmental outcomes globally.

In this proof-of-concept project, an international team from Australia and Indonesia undertook research to demonstrate the viability of using DNA markers to verify the chain-of-custody of high value teak timber. The project provided the ground work for future research and development aiming to create a toolkit, processes and legal framework for verifying the chain-of-custody of high value teak timber that is sourced from Indonesia and potentially other regional producer countries.

The project focussed on demonstrating the technical feasibility of using DNA markers to prove provenance of teak timber from Indonesia, and explored the process by which the use of DNA markers could be used to verify legality of Indonesian teak timber products, grown by smallholder farmers.

The project also considered the political feasibility of deploying this methodology to verify chain-of-custody of teak exports from Indonesia and other countries.

The successfully completed activities of the project included:

Development of a bank of 249 single nucleotide polymorphism (SNP) DNA markers for teak that can be used for individual tree or concession/plantation level source verification;

The SNP markers were used to screen DNA (extracted from leaf material) from a range of plantation sourced teak trees (including Indonesia, India, Myanmar and Thailand) and from trees from natural populations sampled in Myanmar.

In addition the SNP markers could be applied to DNA extracted directly from teak timber;

The hosting of a workshop with small-holder timber growers in Indonesia to discuss opportunities for the application of DNA markers to their supply chains;

Submission (now accepted) of an additional ACIAR project proposal – ‘Developing a DNA chain of custody method to verify legally sourced teak in Indonesia and Myanmar’ - FST/2015/007

The outcomes of the project also helped to inform the development a concept note for a large integrated project ‘Development and Applications of DNA Markers for Timber Legality Verification and Good Forest Governance in Cambodia, Indonesia, and Myanmar’ which was submitted to the Japan-ASEAN Integration Fund (request $3 Million; for cosupport from ASEAN – Korea, request $100,000; and ASEAN Australia, request $1 Million). We have now been invited to submit a full proposal. During the progress of the project we have also been in close communication with The Nature Conservancy (TNC) and the Australian Government about the development and potential for integration with the new RAFT3 program.

The project has successfully demonstrated the technical feasibility of utilising DNA marker technology to support the Indonesian teak trade and facilitate compliance with new SVLK certification requirements. In addition, through direct engagement with Indonesian smallholder teak growers and government representatives, the project has mapped out future opportunities for the application of DNA marker technology in teak supply chains, and provides a route to verify small holder teak plantation sources. By doing so, growers can verify the legality of their forest resources and support existing certification schemes, and gain access to the increasing number of high-value markets sensitive to legality issues, including the EU, US, Australia and others.

Page


The close collaboration between Australia and Indonesia on this project has helped to strengthen relationships on a number of strategically important issues relating to forestry such as capacity building; legal requirements; systems that assure legality of timber and wood products but remove barriers to smallholders participating in international timber markets; enhanced forest law enforcement and governance; and the sourcing timber from legal and sustainable forest practices.

Page


3 IntroductionAustralia is committed to working in partnerships with supplier countries to reduce the trade in illegal timber into Australia for the economic benefit of supplier countries and to improve environmental outcomes globally.

DNA markers are being used for other timber species to distinguish between species, between populations and between individuals of trees and timber products.

The cost of this technology is dropping dramatically and is already cost-effective. The technology is suitable for verifying existing chain-of-custody compliance

checks, and has already been proven to be effective for other species in other countries.

In Indonesia, much of the planted teak is grown by smallholder farmers who have difficulty in meeting international requirements related to legality verification and/or forest certification so mechanisms to support such farmers to reach external markets and improve livelihoods need to be explored.

Removing doubt about provenance creates certainty for industry and consumers, opens markets for timber and increases taxation revenue for governments in developing countries. It also provides a mechanism for community forestry suppliers to demonstrate sustainability to the global market.

Australia introduced illegal logging regulation on 30 November 2014. To support this new regulation, Australia wants to encourage and support increased legal trade between Indonesia and Australia.

An improved system of chain-of-custody verification is required to sustain forest resources and enable access to the increasing number of high-value markets sensitive to legality issues, including the EU, US, Australia and others.

DNA markers are a promising group of methods that can be applied to identify the origin of timber wood, from species identification, through region and concession source verification, down to tracking individual logs or timber products. In many cases, these DNA markers can be used in conjunction with or instead of non-genetic methods.Importantly, these methods can be applied at the point of consumer country importation,and can be used to overrule questionable certification documentation that may havebeen introduced along the supply chain. In addition, with the advancement of genetictechnologies, large-scale screening of DNA sequence variation can be done cheaply,routinely, quickly and with a statistical certainty that can be used in a court of law (Lowe and Cross 2011).

Whilst different levels of source can be identified using DNA markers, including species identification (DNA barcoding), verification of source, both at the regional(phylogeography) and concession scales (population genetic assignment), and fortracking individual logs or timber products (DNA fingerprinting), it is this latter application, using DNA fingerprinting methods, that have so far been used to support chain-of-custody (Lowe and Cross 2011), and have been selected as the most appropriate for the current project.

The ACIAR Forestry Program aims to contribute to poverty alleviation and natural resource conservation and rehabilitation through scientific support for the establishment, management and sustainable utilisation of forests, providing optimum social, economic and environmental benefits to partner countries and Australia. This project contributes to the sustainable management of forests, and efficient and sustainable forest industries.

Page


4 Objectives and ActivitiesThis project aimed to demonstrate the viability of a larger program that would create a toolkit, processes and legal framework for verifying the chain-of-custody of high value teak timber that is sourced from Indonesia and potentially other regional producer countries.

The project is a proof of conept to demonstrate the technical feasibility of using DNA fingerprinting markers to identify the individual tree and plantation/population of origin of teak timber from Indonesia, and to explore the political feasibility of deploying this methodology to verify chain-of-custody of teak exports. It is recognised that this is the first stage of a larger body of research and development that will be needed to achieve adoption of this technology in Indonesia and the possible expansion of its use to cover planted and natural occurring teak in other Asian countries.Through the close collaboration of the Australian and Indonesian partners, the project aimed to help to strengthen relationships between the Australian and Indonesian governments on issues relating to:

Capacity building Legal requirements Systems that assure legality of timber and wood products but remove barriers to

smallholders participating in international timber markets Enhanced forest law enforcement and governance Sourcing timber from legal and sustainable forest practices

The activities supported by this proof-of-concept project are split into two components, technical and political feasibility, with the following components:

Technical feasibilityo Development of initial genetic markers to work with teak that could be used

to track supply chains and identify provenance, even in processed timber;o Test the variability of these markers on a selection of teak DNA sourced

from leaf material from different natural and plantation populations o Test the application of these markers to DNA extracted from teak timber;o Outline a process by which these DNA markers could be used to verify the

legality of Indonesian teak timber products, such as furniture produced in Java from teak grown by smallholder farmers.

Political feasibilityo Host a workshop to scope a project proposal to develop a broader regional

DNA teak source verification tracking detailed DNA map of teak across Indonesia and Myanmar, and to establish a legal framework for the integration of this technology into a chain-of-custody system for both countries;

Prepare a report that outlines the initial research undertaken on DNA markers for planted teak from Indonesia, the prospects for utilising this technology to enhance Indonesian smallholder farmer access to international timber markets; any additional research that is needed to enable adoption of this technology; and an indication of the political support for implementing this technology in Indonesia and other countries in the region.

Minutes from the project coordination meeting can be found at Appendix 1.

Once the use of DNA tools for verifying the chain of custody for teak have been established and shown to be viable practically and politically in Indonesia, the aim was to prepare a larger proposal to develop more comprehensive maps of DNA variation in teak populations and to work with regional producer and consumer governments to implement a legal framework for verifying the chain of custody of teak exports.

Page


Such a system can support both efficient value-chains for teak products and a robust chain of custody system. The implementation of systems that can verify provenance to local areas would also support the ability of community forestry cooperatives to attract a price premium in the international market.

Page


5 Technical Feasibility

5.1 Molecular methodsThe aim of this component of work was to develop a set of molecular markers (single nucleotide polymorphisms, or SNPs) for use in teak chain of custody tracking. The project aimed to develop a test bank of 300 SNPs which could be further optimised in the future for individualisation tests and examined for potential utility in regional assignment. Previous work has highlighted that strong genetic structure exists across the range of teak, but that natural populations are highly variable genetically (Madan et al 2005; Volkaert et al 2007; Fofana et al 2009; Verhaegen et al 2010; Lowe & Volkaert 2013; Widyatmoko et al 2013; Win et al 2015).

Available marker development methods have progressed very quickly recently, and whilst still involving considerable laboratory work, we are now able to develop molecular markers for non-model organsiations relatively quickly and for non-prohibitive budgets (e.g. Jardine et al 2015). Once developed the markers need to be transferred onto a screening platform (a MassArray system) to be fully operational.

5.1.1 SNP developmentSNP discovery was made following standard procedures (as outlined in Jardine et al 2015), using two sets of restriction enzymes to isolate restriction site associated DNA (RAD) markers (a method termed ddRAD-Seq). Traditional RAD uses one restriction enzyme and random shearing to generate fragments from genomic DNA, it is a broad spectrum sequencing of the genome but generates substantial losses where a reference genome is not available. In ddRAD-Seq genomic DNA is digested with two restriction enzymes, creating a smaller, more specific pool of loci for sequencing. This complexity reduction approach creates a greater representation of those loci across individuals and a more repeatable methodology.

Twenty three samples were selected for SNP marker development in teak (Table 1). The samples represented wood, cambium and leaf/bud tissue, which was due to a combination of availability (leaf vs cambium samples) and to allow the early stage assessment of the quality of wood-extracted DNA. DNA extraction methods varying as appropriate for the tissue. Leaf/bud tissue (samples 1-14 were extracted using a CTAB protocol by FORDA in Indonesia and shipped to Adelaide as dry DNA; wood tissue (samples 15-18) were extracted at University of Adelaide using the BOTAB protocol; and cambium tissue (samples 19-24) were extracted at AGRF (Australian Genome Research Facility) in Adelaide).

Table 1. Samples used to develop and screen SNP loci

Page

Sample numbers Country Tissue Source of material Number of

samples1-3 Myanmar Leaf/bud Indonesian provenance trial 34-6 India Leaf/bud Indonesian provenance trial 35-12 Indonesia Leaf/bud Indonesian provenance trial 613-14 Thailand Leaf/bud Indonesian provenance trial 215-18 Unknown Wood Indonesian plantation (FORDA) 419-24 Myanmar Cambium Field collection in Myanamar 6

Total 24


All samples were quantified on the QuantiFluor® dsDNA System (Promega). Samples were diluted to 100 ng in 20 µL and restriction enzyme (RE) digested using EcoRI and MseI. Adapters were ligated to either end of the digested fragments, and then roughly size selected using a double AMPure purification. This removed all small and large DNA fragments, leaving only those in the range of ~150-1000 bp. Quantiative PCR (qPCR) was undertaken to increase the concentration of the library and to add sample-identifying end tags, ‘indices’. The cycle number was controlled manually based on the products rate of amplification. The run was terminated when all samples reached exponential amplification ensuring PCR artefacts were at a minimum (15-19 cycles). Identifier tags ‘barcodes’ (at the front of the sequence) and indices (at the end of the sequence) were all in place by this point, the ‘barcodes’ during adapter ligation and the ‘indices’ embedded in the qPCR primers.

Samples were pooled (each bioinformatically distinguishable) and the library quantified using the QuantiFluor® dsDNA System (Promega) and TapeStation (Agilent). Further size selection was undertaken on the Pippin Prep (Sage Sciences), with a target range of 400-600 bp, and this product was quantified using the Bioanalyzer (Agilent) for the peak size, and the QuantiFluor® dsDNA System (Promega), for the mass in ng/uL. Together these results informed molarity calculations for sequencing. Sequencing was carried out at the South Australian Health and Medical Research Institute (SAHMRI) on the Illumina MiSeq using MiSeq Reagent Kit v3 300 bp, paired end (PE) chemistry.

Over 20 million reads were generated by the sequencing stage. These were de-multiplexed, trimmed and de novo assembled to create a reference sequence library. Individual sample reads were mapped back onto this de novo reference library. The consensus sequences of all the sample mappings were then reassembled to compare the loci across samples. In this way the loci were screened for SNP marker candidates, with non-variable loci and paralogous loci removed manually.

5.1.2 SNP marker choice A total of 294 loci were selected as candidate SNP markers, which were identified during the bioinformatics stage (outlined above), and these loci exhibited variation across the reference samples used for maker development; had a single SNP per locus, had no more than 2 base pair variation at SNPs, and had sufficient flanking sequence either side of the SNP to design genotyping primers (Table 1). Of these 294 loci, 100 representative SNPs are presented in Appendix 2.

5.1.3 DNA quality from leaf, timber and cambium sourcesDNA extracted from leaf material (supplied by the Indonesian laboratory) were highest quality, followed by wood samples extracted at the University of Adelaide. Samples that performed least well were cambium samples extracted by AGRF. Both the DNA quality and quantity could be contributory factors, and these are influenced by the method of extraction. Different extraction methods perform differently in fluorometry based quantification systems such as the QuantiFluor® dsDNA System. DNA quantity may be overestimated due to other residual components of the BOTAB extraction (Jardine, pers. comm.), in turn influencing the outcome of the restriction enzyme digest and down-stream procedures. However our tests here indicate that DNA can reliably be extracted from teak timber and that the resulting DNA is of sufficient quality and quantity to be sequenced and SNP genotyped.

5.1.4 SNP potential future applicationPreliminary data analysis based on development sequences will be strongly influenced by missing data (see Appendix 2) and so is inappropriate at this stage. Robust analyses must wait for the next phase of this project, where more samples from populations across the distribution of the species can be screened on an appropriate genotyping platform. We

Page


recommend genotyping the 204 SNPs (or a subset of these) on the Agena Biosciences MassARRAY system. Previous work at the University of Adelaide (e.g. Jardine et al 2015) has shown excellent results on this platform with DNA extracted from leaf/bud, cambium and wood, with conversion rates (number of loci successfully working on the platform) ranging from 40-85%. Assuming the most conservative conversion rate (40%) the number of candidate SNPs identified here would still likely generate somewhere in the region of 120 usable SNPs. Assuming that the two alleles at a locus have equal frequency (i.e.e 0.5 each, the most conservative situation), 120 SNPs would be capable of determining a genetic fingerprint of individual teak genotypes that would have less than a 1 in 1036 (termed an “undecillion”) chance of being being duplicated by another teak individual.

Based on our experience with other projects, the SNP markers developed in this proof-of-concept phase will be sufficient for determining individual identity of timber products along supply chains and also for application to determine the region of origin of timber.

Page


6 Political Feasibility

6.1 WorkshopTo establish the political support and feasibility of verifying teak origin using DNA methods, a workshop was organsied to bring together project scientists, FORDA, SVLK and ministry staff and teak smallhodler and planation managers to discuss the issues. A group of 35 people attended a workshop on ‘Application of genetic information for the verification of teak’ held at the FORDA meeting rooms at Jogjakarta on 5th May 2015.

6.1.1 BackgroundIn an effort to suppress illegal timber trade, the Ministry has implemented a Sistem Verifikasi Legalitas Kayu (Timber Legality Verification System) or SVLK, which serves to ensure wood products and their raw materials are obtained or derived from sources whose origin and management meet the legal requirements. Wood is certified as legal when the origin of the timber, logging licenses, systems and procedures for harvesting, transporting, processing, and trading or transfer of ownership can be demonstrated to comply with laws and regulations.

Genetic information can be used to verify the legality of timber by means of:

a) Determining the identity of the type of wood being traded (DNA barcoding);b) Verifying the source or origin of wood, both at the regional level (phylogeography)

or at the level of the concession (population genetics)c) Track the source of logs or wood products (DNA fingerprinting)

This workshop aimed to gather information about the potential use of genetic information for the verification of the legality of teak. This workshop discussed the technical aspects required to facilitate the development of DNA identity verification of the legality of teak.

6.1.2 Speakers1. Dr. Bambang Sukmananto, Director of Processing and Marketing of Forest

Products, SVLK 2. Prof. Andrew Lowe, University of Adelaide 3. Dr. Anto Rimbawanto, Research Institute of Forest Biotechnology and Plant

Breeding

6.1.3 Attendees

No. Organization/Farmer Cooperative Contact Persons

1 Kelompok Tani Kumpul Makaryo Warsana and Wagiran

2 Kelompok Tani Sedyo Rukun I Widarno and Susilo

3 Kelompok Tani Wukir Mulyo Wasito and Subandi

4 Kelompok Tani Sumber Makmur Budi Atmono and Marjono

5 KHTR Jati Pendowo Sutari and Anang

Page


6 PTHR Ngudi Lestari Sukimin and Sugeng S

7 HTR Wono Martani-Koperasi Akur Suripta and Handek

8 Kelompok Tani Margo Mulyo Paija and Sutri Yanto

9.Directorate of Marketing and Processing of Forest Product, SVLK

Dr. Bambang Sukmananto

10 Perhutani Research and Development Centre

Suwarno

11 District Forestry Service of Gunung Kidul

Bambang WS, Taufik, Benny, Sumardanto

12 CFBTI Management and Researcher

Dr Mahfudz, Purnamila Sulistyawati, Vivi Yuskianti, Dana Apriyanto, Istiana Prihatini, Hamdan, Sigit, Anton Widyatmoko, Anto Rimbawanto, Eva Sinaga, Liliana Baskorowati, Anis Fauzi, Nurdin

13 University of Adelaide, Australia Prof Andrew Lowe

14 Double Helix Tracking Technologies

Bart van Assen

6.1.4 Location Maps

1. Overview of Java

Page


2. Location of teak small holder plantations represented at meeting

3. Location of Perhutani R&D Centre – large scale teak plantation in Java

Page


6.1.5 Workshop Photographs

1. Welcoming delegates

2. Dr Rimbawanto introduces meeting and opening given by Dr Mahfudz, CFBTI Director

3. Talks given by Director of SVLK, Dr. Bambang Sukmananto, and Prof Andrew Lowe

Page


4. Question and answer session

5. Question and answer session continued

6. Talks given by Dr Rimbawanto and Mr Suwarno, Perhutani R&D Centre

Page


7. Expert panel discussion with Bart van Assen, and questions

8. More questions and panel discussion with Bart van Assen and Dr Rimbawanto

9. Group photo at CFBTI – May 5th 2015

Page


6.1.6 Outcomes of Group Discussions1. Perhutani

a. There are 7 Forest Management Units of Perhutani which have received FSC certificate. Having FSC certificate add around 10% to the cost. But this is compensated by the fact that most of the teak are exported to Europe, so they can get premium price.

b. Another way to recoup the cost of certification is to increase their plantation productivity by using genetically improved materials (seeds/clones).

c. Perhutani is getting the benefit of certification (FSC or SVLK) because they operate from plantation to industry.

2. Kelompok Tani Kumpul Makaryo (Farmer Group “Kumpul Makaryo”).

a. Practice mix planting of teak and cash crops, no monoculture of teak planting.

b. Certification is something new for us, but we would be interested if it gives us benefit. Cost of certification is one of important issues to consider when small group such as farmer’s cooperative has to make a decision on joining any certification scheme.

c. We are interested to replace our teak stand with genetically improved seeds/seedlings, and would like to receive assistance in establishing demonstration plot.

3. District Forest Service of Gunung Kidul

a. Community forest/smallholder plantation is very important for the district, currently there are about 43,000 ha of established plantation, majority is teak (up to 80%), potentially it can have 50,000 ha.

b. Recognizing the importance of teak, the local government is keen to see that small holder teak plantations use improved teak seeds/seedlings.

c. One important issue with certification, is how the DNA marker will be implemented in small holder teak plantation.

4. Koperasi Hutan Wana Manunggal

a. Received sustainable forest management of PHBML or LEI (Lembaga Ecolabelling Indonesia), managing about 815 ha. The problem they had was selling the timber to overseas market, as they did not have access. In the process they form an agreement with a furniture industry who agreed to get timber from the cooperative, but this only went on for 2 years. In the end, because of lack of funding to maintain the certificate, the certificate was revoked. In summary, having certification was a burden for the cooperative.

5. Farmers Group Petani Jati Pendowo Nglipar

a. Received FSC certificate since 2012/2013. Experiencing a booming sale where they fetched a price of up to Rp. 3 million of teak per m3, a significant jump from Rp. 200,000 they normally received. However, since the crisis in Yunani, the demand has significantly dropped. Become much more aware of the price of teak timber, and they can have a better say with the buyer on deciding the price.

b. One of the positive points of having certificate, is farmers become much aware of the price of teak timber, and they can have a better say with the buyer on deciding the price. Farmers also adopting better recording system since plantation to harvesting.

Page


6.2 On-ground Operations Visit

6.2.1 Trip SummaryIn order to understand some of the current chain of custody (CoC) systems employed in teak production, Dr Anto Rimbawanto visited Cepu to learn about the CoC system of teak managed by Perhutani. the trip was made from 23-24 June 2015. The information was obtained from 3 offices:

Forest Management Unit/FMU of Cepu; Deputy Head/Mr. Teguh Waluyo Independent Business Unit for Timber Marketing: Manager/Mr. Gembong Nurjoko Teak Processing Plant of Cepu: Manager/Mr. Henki

Documents collected: Standard Operating Procedure for CoC, published by Perhutani.

The forest management unit (FMU) of Cepu obtained FSC certification in 2012. Other FMUs with FSC certification are Randublatung, Kendal, Kebonharjo, Mantingan, Lawu and Madiun. FMU Cepu manages some 33,019 ha, dominated by teak, annual timber production is between 20,000 and 30,000 m3.

The benefits of having a FSC certificate are:

Corporate image Surcharge of 7.5% for all timber sold (domestic and export market)

It’s not possible to calculate whether having the additional 7.5% is sufficient to cover the cost of obtaining and maintaining the certificate, as the certification requires compliance to technical, environment and social components.

All trees in a logging compartment are mapped based on GPS coordinates. Hence, if a stump is missing, the location of the stump is still on record. The teak stump and its root are a commodities for furniture or art works. The company policy states that a stump must not be removed within the first two years after logging. However, for reasons of social cohesion, a district manager would not stop farmers from unearthing the stump soon after logging.

Random sampling to check compliance to the standard operating procedure (SOP) for CoC is performed monthly. So far, all checks have revealed compliance with procedures.

In order to get first-hand experience of how CoC is applied, Dr Rimbawanto visited the Pasar Sore log yard, some 20 km from the town of Cepu. Pasar Sore is the biggest log yard of FMU Cepu, with a capacity of up to 10,000 m3. In the log yard, the Receiving Foreman will record information on the logs from the Logging Foreman, and if he is in agreement regarding the volume and quality of the logs, he will paint his mark on the log and the form.

To test the accuracy of the record, Dr Rimbawanto picked up a random log and checked the document and origin. In this case, a log from tree no 384 was assessed and verified with its stump in the logging compartment.

Once a log has left the log yard, documentation on its origin is the responsibility of the operator and maybe monitored by the local forestry service. Similarly, in the teak processing plant, which makes furniture products, tracking the timber origin of a product based on the documentation is not observed.

In this trip, verbal support has been given by FMU Cepu to take samples from any point of the CoC, when the on-ground project is operational.

Page


6.2.2 Trip Photographs

1. General view of the Pasar Sore log yard, the biggest log yards managed by FMU Cepu

2. Marking of the log consists of tree number, cutting number, length of the log, volume and forest district. The log is from tree no. 384

Page


3. The stump of tree 384

3. Marking is in transition from painting to stamp hammer

6.3 Overall comment on political feasibilityThe need to obtain certification (SVLK and/or FSC) is clearly recognised by teak growers (small-holder farmers to large scale plantation oweners) to access high-value international markets, and is a situation supported by Indonesian Ministry of Environment and Forestrystaff. However the cost of gaining international certification standards (FSC), appears to be a significant financial barrier, particularly for small holder growers.

Teak supply chains have relatively high transparency and using the cheaper SVLK certification system as a framework, it would be possible to use DNA methods to verify the source origin of teak along the early stages of supply chains (e.g. between saw mill and plantation/farm). This system is already used in Indonesia to verify supply chains of merbau using DNA integrated into the certification standard Certisource (Lowe et al 2010).

Page


7 Conclusions and recommendations

7.1 ConclusionsThis project has successfully used DNA samples originating from teak trees across, Java, Sulawesi, Myanmar, India, and Thailand to generate a set of ~300 candidate SNP loci suitable for individualisation of teak timber along supply chains. The project has also demonstrated the utility of the wood DNA extraction protocol for successfully genotyping timber material without the need for more DNA rich tissue sources such as leaf/bud and cambium. The ability to successfully utilise DNA directly from timber is essential for utility of these tests in the timber industry. We predict that further development of this technology will allow individual matching with unprecedented certainty, facilitating robust and independent verification of teak supply chains.

The stakeholder meeting and discussion clearly highlighted the perceived value of certification (mainly SVLK, but also FSC for international markets) and there was tremendous interest in the ability to provide verification through independent DNA tests to bolster the value of exported teak, although these tests are very price sensitive.

Following disucssions with FORDA, ministry, SVLK staff and plantation managers (Perhutani), it was agreed to undertake a trial DNA chain of custody verification test using the molecular makers developed as part of this project from some Perhutani plantations. These samples are being collected and will be screened as part of the follow up ACIAR project.

The project has demonstrated that there is the technical ability and political interest in utilising DNA marker technology to support the Indonesian teak trade and facilitate compliance with new SVLK certification requirements. Through direct engagement with Indonesian teak plantation owners, the project has been able to identify where DNA marker technology can be applied to teak supply chains (between plantation and saw mills, by tracking individual sawn timber back to cut stumps if required). By doing so, growers have the option to provide independent verification of the legality of their forest resources, which should help gain access to the increasing number of high-value markets sensitive to legality issues, including the EU, US, Australia and others.

The outcomes of the project also helped to inform the development a concept note for a large integrated project ‘Development and Applications of DNA Markers for Timber Legality Verification and Good Forest Governance in Cambodia, Indonesia, and Myanmar’ which was submitted to the Japan-ASEAN Integration Fund (request $3 Million; for cosupport from ASEAN – Korea, request $100,000; and ASEAN Australia, request $1 Million). We have now been invited to submit a full proposal. During the progress of the project we have also been in close communication with The Nature Conservancy (TNC) and the Australian Government about the development and potential for integration with the new RAFT3 program.

The close collaboration between Australia and Indonesia on this project has helped to strengthen relationships on a number of strategically important issues relating to forestry such as capacity building; legal requirements; systems that assure legality of timber and wood products but remove barriers to smallholders participating in international timber markets; enhanced forest law enforcement and governance; and the sourcing timber from legal and sustainable forest practices.

Page


7.2 RecommendationsThe recommendations of this project form the basis of a subsequent ACIAR project entitled ‘Developing a DNA chain of custody method to verify legally sourced teak in Indonesia and Myanmar’ - FST/2015/007 – conducting research activities on:

Developed molecular markers should be optimised for the MassArray genotyping system to facilitate rapid and inexpensive DNA typing of teak material.

Markers suitable for geographic provenancing and individualisation should be validated through screening of additional reference material from across the range of teak and within specific concessions.

Pilot implementation of DNA verification of CoC should be undertaken in partnership with teak plantations/concessions to demonstrate functionality of the system.

Implementation of DNA verification of teak CoC should proceed in close collaboration with SVLK to ensure that the system is compatible with the Indonesian national certification system.

Consideration of teak small holder circumstances should inform implementation plans to ensure that future adoption of DNA verification of teak CoC results in improved market access for these farmers.

Page


8 References

8.1 References cited in reportFofana, I. J., D. Ofori, M. Poitel, and D. Verhaegen. 2009. Diversity and genetic structure of teak (Tectona grandis L) in its natural range using DNA microsatellite markers. New forests 37:175-195.

Hirao, T., A. Watanabe, and S. Goto. 2015. Current genetic structure of teak (Tectona grandis) in Myanmar based on newly developed chloroplast single nucleotide polymorphism and nuclear simple sequence repeat markers. Tropical Conservation Science 8.

Jardine, D., E. E. Dormontt, K. J. van Dijk, R. R. M. Dixon, B. Dunker, A. J. Lowe. 2015. A set of 204 SNP and INDEL markers for Bigleaf maple (Acer macrophyllum Pursch). Conservation Genetics Resources. Published online. doi: 10.1007/s12686-015-0486-7

Lowe, A.J., K.N. Wong, Y.-S. Tiong, S. Iyerh, F.-T. Chew. 2010. A DNA Method to Verify The Integrity of Timber Supply Chains; Confirming The Legal Sourcing of Merbau Timber From Logging Concession to Sawmill. Silvae Genetica 59:263-268.

Lowe, A. J. and H. B. Cross. 2011. The Application of DNA to Timber Tracking and Origin Verification. Journal of the International Association of Wood Anatomists 32:251-262.

Lowe, A. J., and H. Volkaert. 2013. The evolutionary and plantation origin of teak. Pages 23-25 in Y.-M. Balasingamchow, editor. Jalan Jati (teak road). The migrant Ecologies Project. Royal Botanic Gardens, Edinburgh.

Shrestha, M. K., H. Volkaert, and D. V. D. Straeten. 2005. Assessment of genetic diversity in Tectona grandis using amplified fragment length polymorphism markers. Canadian Journal of Forest Research 35:1017-1022.

Verhaegen, D., I. J. Fofana, Z. A. Logossa, and D. Ofori. 2010. What is the genetic origin of teak (Tectona grandis L.) introduced in Africa and in Indonesia? Tree genetics & genomes 6:717-733.

Volkaert, H., A. Lowe, S. Davies, S. Cavers, I. Puthanveettil, S. Sudarsono, A. Vanavichit, D. Van Der Straeten, and H. Wellendorf. 2008. Developing know-how for the improvement and sustainable management of teak genetic resources. Final Report for TEAKDIV.

Widyatmoko AYPBC, R. A, and C. AR. 2013. Genetic relationship among teak (Tectona grandis L.) populations based on RAPD (Random Amplified Polymorphic DNA) markers. Jurnal Pemuliaan Tanaman Hutan 7:151-166.

8.2 List of publications produced by projectThe project has produced preliminary data for a range of scientific publications on teak genetic markers. Additional data will be collected and publications finalised as part of the follow on ACIAR project ‘Developing a DNA chain of custody method to verify legally sourced teak in Indonesia and Myanmar’ - FST/2015/007.

Page


9 Appendixes

9.1 Appendix 1: Minutes from project coordination meeting – Yogyakarta 30th October 2014

PresentAnto Rimbawanto (CFBTI)

Anton Widyatmoko (CFBTI)

Purnamila Sulistyowati (CFBTI)

Andrew Lowe (University of Adelaide)

Mark Saunders (University of Adelaide, Skype)

Eleanor Dormontt (University of Adelaide)

Darren Thomas (DoubleHelix)

Progress against project activities

1. Development of initial genetic markers to work with teak that could be used to track supply chains and identify provenance, even in processed timber; Test the application of these markers on teak timber from IndonesiaFORDA to screen microsatellites on Indonesian provenance material (from natural and plantation populations), plus DNA extracted from wood supplied by Adelaide lab - approx. $2.5k for collection costs and $7.5 consumables for microsatellite screening

Adelaide to develop bank of single nucleotide polymorphism markers for teak screening. 24 individuals to be included in marker development run, including:

4 wood samples (from FORDA – and supplied to Andy)

14 individuals from FORDA provenance trials (including material from India, Myanmar, Thailand, Java and Sulawesi, - provenances: 1, 4, 9, 10, 11, 12, 13, 15, 17, 18, 23, 26, 29) – to be collected as young leaf material and send to University of Adelaide by end of November

6 individuals from Myanmar populations (3 individuals from each of 2 populations, although collections of 20-30 individuals will be made from each population) – extra cost of $3500 for consultant to sample material

2. Outline a process by which the use of DNA markers could be used to verify legality of Indonesian teak timber products, such as furniture produced in Java from teak grown by smallholder farmersFORDA is leading this work on certification of smallholders within existing institute resources, and will provide an understanding (as part of the final report) of what chain of custody is required in small holder teak market in Indonesia. Some additional resources for travel ($1000) are required particularly to help link up this project with an ACIAR funded project lead by FORDA on small holder teak production (FST/2006/117 "Improving added value and small medium enterprises capacity in the utilisation of plantation timber for furniture production in Jepara region” which has been completed and had final review just recently).

Page


3. Host a workshop to scope a project proposal to develop a broader regional DNA teak source verification tracking detailed DNA map of teak across Indonesia and Myanmar, and to establish a legal framework for the integration of this technology into a chain-of-custody system for both countriesSeveral discussions have been held by Skype and phone with Andrew Ingles and Allison Lewin from The Nature Conservancy (TNC). There is a significant opportunity to coordinate teak verification frameworks with the activities of the proposed RAFT3 project.

We plan to hold the workshop to scope a project proposal to develop a broader regional DNA teak source verification system to coincide with the regional development meeting set for Myanmar in Jan 2015. Many of the potential project partners will be at this meeting, which will also allow some budget to be saved and reallocated to other unforeseen project costs.

In July Andy met with Mehm Ko Ko Chi (formerly employed and now consulting to Myanmar Forestry Department) and John Leake (Myanmar consultant) to outline the plan for the project proposal and wokshop, which was warmly received.

During this visit Andy also had discussions with Thiha, a private sector auditor (DoubleHelix, and also ex-forestry department) who are developing a chain of custody certification system which includes DNA verification with MTE (Myanmar Timber Enterprise).

Contact has been made from potential future project science partners from Thailand (Hugo Volkaert, Centre for Agricultural Biotechnology, Kasetsart University) and India (Dr. Indira Edakkeppurath Puthanveettil – Kerala Forest Research Institute) have been made and they have been notified of the potential planned proposal meeting.

4. Preparation of a report that outlines the initial research undertaken on DNA markers for planted teak from Indonesia, the prospects for utilising this technology to enhance Indonesian smallholder farmer access to international timber markets; any additional research that is needed to enable adoption of this technology; and an indication of the political support for implementing this technology in Indonesia and other countries in the region.The technical results describing genetic variation within and between teak provenances, ability to distinguish between regions, concessions and individuals and application of markers to teak wood will be described in a report prepared by University of Adelaide in consultation and with input from FORDA. It is intended that this will be used as a basis for a joint publication on the state of knowledge of teak genetic diversity within Indonesia, which will include description of previously published work.

FORDA will lead the section of the report outlining the application of DNA methods to help with certification of smallholders and the political context for this in Indonesia. FORDA will work with DoubleHelix to identify the chain of custody application of DNA markers to government forestry activities in Myanmar.

Page


9.2 Appendix 2: Table of representative 100 SNPs from candidate loci

Genotypes for the teak samples used in marker development for the top 100 SNPs. Standard IUPAC nucleotide code describe the genotypes (http://www.bioinformatics.org/sms/iupac.html). Blanks represent missing data.

Loci►

Samples▼

_817 _836

_1150

_1253

_1262

_1634

_2339

_2563

_2794

_2810

_3505

_3820

_4208

_4347

_5767

_5939

_6421

1 T R Y A Y T S G G Y T T T A R Y

2 Y G C T T K

3 Y C T T T

4 C R T

5 T G T K T

6 Y T

7 C R T

8 C G T

9 C A T

10 C G T

11 T R C R Y C C G Y T T K T W A Y

12 T R C R T C C R T T K T K W R Y

13 T G C A T S T T T T K W A T

14 T R T A Y Y T T

15 T G C A T C C R T T K T W Y

16 T T T Y

17 T G C R T C C G G Y T T K T A A T

18 Y R C A Y C G Y K K K A R T

19 T R C A T Y S G G T T K T T W A Y

20 T G A Y Y S G T K T A A T

21 T G C A Y C C G T T K T K W R Y

22 C T

Page

http://www.bioinformatics.org/sms/iupac.html


23 Y G C R Y S G Y T T T A A Y

24 T G T R Y C G Y C T T K A T

Page


Loci►

Samples▼ Loci

_6598

_6665

_7456

_7511

_7999

_8201

_8761

_8880

_9299

_10320

_11733

_11880

_11934

_11981

_12062

_12346

_12464

1 TG-0-1 Y A K M T R S R A G T C G T T T

2 TG-0-3 Y Y T

3 TG-0-4 Y

4 TG-0-6 A

5 TG-1-31 T S C Y

6 TG-1-32

7 TG-1-33 A

8 TG-2-31

9 TG-2-32

10 TG-2-33

11 TG-3-1 T A K M K G S R R W G T C G T T T

12 TG-3-12 T A K M T R S A G A Y C R Y Y T

13 TG-3-2 Y A K A T G C T C R T Y T

14 TG-3-20 T A G C Y Y Y

15 TG-3-24 T C G A K G C A G G Y C R T Y T

16 TG-3-3 Y G S Y

17 TG-3-56 T A K M T G S A G A R Y C R T Y Y

18 TG-3-64 Y G A T R C A G W G Y Y G T T Y

19 TG-3-72 Y M K A K G S R G A R T C G T Y T

20 TG-3-73 T K A K G S A G A G Y C G T Y T

21 TG-3-89 Y K M T G C A G A G C G Y Y T

22 TG-3-9

23 TG-3-93 Y K M K G S A G W R T C G T Y T

24 TG-3-94 Y A K M K G S A R A G C G T Y Y

Page


Loci►

Samples▼ Loci

_12893

_12974

_13346

_13591

_14020

_14915

_15179

_15374

_16016

_16866

_17296

_18274

_19000

_20394

_20649

_21557

_22258

1 TG-0-1 G G S R G S Y Y C K G T A A T G

2 TG-0-3

3 TG-0-4

4 TG-0-6

5 TG-1-31 S

6 TG-1-32

7 TG-1-33

8 TG-2-31

9 TG-2-32

10 TG-2-33

11 TG-3-1 R K G R R R S T C Y K G Y R A Y R

12 TG-3-12 G K G R G C Y C C G G Y R A T R

13 TG-3-2 G G G R S Y C C G K Y R R

14 TG-3-20 G T G G

15 TG-3-24 G K G A G R T C C G K T R A T G

16 TG-3-3 R

17 TG-3-56 R K G A G R S T C Y G G Y R A Y

18 TG-3-64 G G G R R A S T Y Y K G T A R T G

19 TG-3-72 R G G R G R S Y C Y G G Y A R T G

20 TG-3-73 G K G R G R S Y C C K G Y A A T R

21 TG-3-89 R G G R G R S T C Y K G Y R A T G

22 TG-3-9

23 TG-3-93 R G S R R A C Y C C G G R R G

24 TG-3-94 R G G R G A C Y C Y G G T A R G

Page


Loci►

Samples▼ Loci

_22634

_22968

_25871

_27189

_29554

_31132

_31448

_32426

_35177

_37029

_41225

_43641

_44357

_46576

_46810

_48239

_50563

1 TG-0-1 R Y A Y A K R A A G Y R R Y G R

2 TG-0-3

3 TG-0-4

4 TG-0-6

5 TG-1-31

6 TG-1-32

7 TG-1-33

8 TG-2-31

9 TG-2-32

10 TG-2-33

11 TG-3-1 G T C R C R K G M M R T A R C R R

12 TG-3-12 Y C A C A K R A A G T A R Y G R

13 TG-3-2 G C Y A T G A G Y G G A

14 TG-3-20 K G

15 TG-3-24 Y S C A K G M R T R R C R A

16 TG-3-3

17 TG-3-56 R T S R C R K R M M G T R C R R

18 TG-3-64 G T C R C A T R A M R T A R Y G R

19 TG-3-72 G T C R C R T R M M R T A R Y G R

20 TG-3-73 G T C R C A K G A A R T A R Y R R

21 TG-3-89 G T C R Y A K R M M G T A G Y R R

22 TG-3-9

23 TG-3-93 G C A C A K M A G A G Y G R

24 TG-3-94 G T C R C A K G M M R T A R Y G A

Page


Loci►

Samples▼ Loci

_53534

_55372

_55533

_56472

_56564

_57653

_59183

_66074

_75119

_115438

_116342

_120019

_210175

_241092

_265039

_266271

_304282

1 TG-0-1 T R A W Y G A G G T Y T R

2 TG-0-3 A K

3 TG-0-4 Y T

4 TG-0-6 G

5 TG-1-31 C T

6 TG-1-32

7 TG-1-33 G

8 TG-2-31 A

9 TG-2-32 G

10 TG-2-33 A

11 TG-3-1 T G A A Y R G A G G G T Y K R

12 TG-3-12 T R R W T R G R G R G Y Y Y K G

13 TG-3-2 G A A Y G G G K Y C T T

14 TG-3-20 C K

15 TG-3-24 T R A W T G K R S R G T Y T T

16 TG-3-3 C K

17 TG-3-56 T G A A T R G A S R Y A Y T K R

18 TG-3-64 T R R W T R G A G R K Y A Y K G

19 TG-3-72 Y R A W Y G K A S R G Y Y T T R

20 TG-3-73 T G A W Y G K A G G G Y C T T G

21 TG-3-89 T G R W Y G K R G G G Y Y T T G

22 TG-3-9

23 TG-3-93 Y G R W T G G R C T T G

24 TG-3-94 T G A W Y R G A G K Y G C Y T G

Page


Loci►

Samples▼ Loci

_318463

_337863

_418500

_2920

_8480

_10419

_1664

_14672

_12005

_1027

_17083

_6050

_4486

_4283 _53

1 TG-0-1 T R R C Y R R C A C R W C T Y

2 TG-0-3 R C Y A R

3 TG-0-4 A C Y A

4 TG-0-6 A

5 TG-1-31 A C T R T Y

6 TG-1-32 C

7 TG-1-33

8 TG-2-31

9 TG-2-32

10 TG-2-33

11 TG-3-1 K A G C Y R R C A Y R W C Y Y

12 TG-3-12 K A G C Y R R C W Y A T Y Y Y

13 TG-3-2 K A G C Y A R C W Y R T C Y Y

14 TG-3-20 A C Y R C W Y W C

15 TG-3-24 T A G Y Y R R C A Y A W C Y Y

16 TG-3-3 A Y Y R A S A C A

17 TG-3-56 K A G C Y R R C A Y A T C Y T

18 TG-3-64 T R G C Y R R S A Y R W C Y T

19 TG-3-72 T A R C Y R R C A Y A W C Y Y

20 TG-3-73 K A R C T R R C A Y R W C Y Y

21 TG-3-89 T A R C Y R R C A Y A W C Y Y

22 TG-3-9

23 TG-3-93 K A G C Y R R C A Y A W C Y Y

24 TG-3-94 K A R C Y R R C A Y R W Y Y Y

Page

Acknowledgments - ACIAR | Australian Centre for...

Documents

Transcript of Acknowledgments - ACIAR | Australian Centre for...