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Rice LabChip Analysis - Q01108

Adaptation Of DNA Analysis Techniques for the Analysis of Varieties, Adulterant Varieties and other Fragrant Rice Varieties for use on the Agilent 2100 BioAnalyzer

Final Technical Report

October 2007

12 June 2006 – 20 June 2007

Katherine Steele and Rob Ogden

Page 1 of 27 Table of Contents

1. Executive Summary 3

2. Glossary 5

3. Aims and Objectives of the Investigation 6 3.1 Why is enforcement needed for basmati rice? 6 3.2 Existing basmati rice tests with SSR markers 7 3.3 Alternative marker systems for rice 7 3.4 Aims and Objectives 8

4. Experimental Procedures 9 4.1. Sourcing of standard varieties and DNA extraction 9 4.2. Testing INDEL markers in different rice genotypes 10 4.3. Testing Rim2/Hipa and ISSR markers in different rice genotypes 10 4.4. Optimizing multiplex PCRs for INDELS 10 4.5. Developing a SOP for variety analysis of bulk extracts using the LabChip system 10 4.6. Optimizing existing SSRs for LabChip analysis 11 4.7. Evaluating INDEL markers for quantitative testing 11

5. Results and Discussion 12 5.1 Results with INDEL markers 12 5.2 Results with Rim2/Hipa and ISSR markers 12 5.3 Database of markers 14 5.4 Development of INDEL markers for variety testing 16 5.5 Quantitative analysis 16 5.6 Problems encountered when adapting the tests for the Agilent Bioanalyzer 17

6. Acknowledgements 17

7. References 18

Appendices 20

Page 2 of 27 1. Executive Summary

Aromatic basmati rice is sold at a premium price on the world market. The Code of Practice for Rice (The Rice Association, 2005) only allows the term „Basmati rice‟ to be applied to certain long grain varieties grown in India and Pakistan. It states that the maximum adulterant levels in basmati rice varieties can not exceed 7%.

Procedures for the detection of adulterant rice varieties and the identification of named basmati varieties have been developed and validated previously by the FSA. These methods rely on (SSR) markers and require separation of fragments on a DNA sequencer. DNA sequencers are not generally available to most public analysts. A more suitable platform is the Agilent 2100 BioAnalyzer, or LabChip system, which is simpler, more adaptable and cheaper than a DNA sequencer. However, the Agilent 2100 BioAnalyzer does not allow clear separation of all SSR fragments and cannot resolve fragments that differ in size by <4 bp. In this project, alternative rice markers were tested in order to identify a suitable set for testing basmati varieties using the Agilent 2100 BioAnalyzer.

Three novel PCR-based marker systems (ISSR, and INDELs, and Rim2/Hipa) were tested on a set of rice standard varieties. Of these systems, INDELs were found to be the most reliable and informative. INDELs gave fragment sizes that were sufficiently different for easy separation using the Agilent 2100 BioAnalyser. A set of INDEL PCR markers were optimized for use with the series II LabChip 1000 system, on the BioAnalyser. Panels of INDEL markers that can be run together in the same PCR (multiplex) were identified that can be used to determine rice varieties present in samples.

As with SSR markers, there was no ideal set of markers for identification of all known potential adulterants or separation of all basmati varieties from each other. We did not identify any INDEL markers that could distinguish Yamini from permitted basmatis. Two main criteria were used to select INDELs for variety testing: i) they should detect as many non-permitted varieties as possible and ii) the test should require a minimum of two PCR reactions. For these reasons, additional markers that could distinguish between permitted varieties (e.g Basmati 198 from other basmatis) were not included in the test. A set of 8 INDELs that can be run in two panels was able to detect the non-permitted varieties: Pak 386, Sherbati, Supra, Pusa Sugandha, Superfine, Mugad Sugandha, Basmati 2000, Shaheen Basmati and Pusa 1121. A simplified test using only one multiplex PCR (4 INDELs) was suitable for detecting all of these except Basmati 2000, Shaheen Basmati, and Pusa 1121. This test is highly cost effective and can be used in conjunction with a second PCR to detect these adulterants. The 4 INDEL variety test was validated and shown to give good, clear and reproducible results on the BioAnalyser, and it is highly suited for use by PAs for the detection of rice mixtures containing non-permitted varieties.

Page 3 of 27 The existing SSR marker (RM201) was shown to be effective for quantification using the BioAnalyser. In addition, we tested three INDEL markers that could be used for quantitative analysis. One of these (B8) was chosen for development into a second quantitative test because it is highly reproducible and gives clear PCR products on the BioAnalyser (but this test does not detect as many adulterant varieties as the RM201 test). Both quantitative tests were tested in two laboratories and used to compare a range of adulterant mixtures. Both were shown to be useable by PAs because they can give a reasonable estimation of the level of an adulterant present. However, we observed that the levels of accuracy and reproducibility of the tests when carried out on the BioAnalyser were not as high as when the same tests are carried out on a DNA sequencer.

The outcomes of this project were met:

1. An existing SSR marker (RM201) was adapted for the quantification of non- permitted rice varieties as adulterants in approved basmati rice using the Agilent 2100 BioAnalyzer (SOP 1). This test can detect and quantify levels of the adulterant varieties Sherbati, Pak 386, Mugad Sugandha and Superfine.

2. A novel INDEL marker (B8) was identified for the quantification of non- permitted rice varieties as adulterants in approved basmati rice and it was optimized for the Agilent 2100 BioAnalyzer (SOP 2). This test can detect and quantify levels of the adulterant varieties Sherbati and Pak 386.

3. A simple 4 INDEL (one-PCR) method for the identification of non-permitted rice varieties and common adulterants in approved basmati rice was developed for the Agilent 2100 BioAnalyzer (SOP 3). This method can be augmented with two additional markers to gain further information about varieties present.

4. All three methods were validated by the developer, Food DNA Services (FDNAS), and by an independent public analyst laboratory, Worcestershire Scientific Services (WSS). They were written up as three separate Standard Operating Procedures (SOPs) for presentation to the FSA.

An additional output has been the following publication: Steele K.A., Ogden R., McEwing R. Briggs H. Gorham J.R. (2007) INDEL markers distinguish Basmatis from other fragrant rice varieties. Field Crops Research. DOI: 10.1016/j.fcr.2007.08.001

Page 4 of 27 2. Glossary

Adulterant Rice variety not listed as an approved basmati

Base pair (bp) Unit of DNA sequence with 1 bp representing one nucleotide

Microsatellite Simple sequence repeat

INDEL Insertion/deletion. Sequence of DNA that differs between varieties. some varieties contain an extra insertion, others do not contain this, and are said to contain a deletion

ISSR Inter simple sequence repeat. PCR primers are based on the repeat sequence itself and multiple fragments are amplified, representing locations of the repeat in different regions throughout the .

Multiplex PCR where more than one region is amplified simultaneously to give multiple fragments. It requires a combination of primer pairs to be mixed into the reaction and each pair targets one region.

PCR Polymerase chain reaction

Polymorphism Heritable variants that are present in a population. Usually polymorphic DNA fragments are fixed within a rice variety, so they can be used as markers for that variety.

Rim2/Hipa This is a type of repeat sequence known as a transposon sequence. The primers are specific to the transposon which is present in multiple locations throughout the genome. The length of DNA sequence between the primers varies in different locations, and between different varieties, leading to polymorphism.

Simple Repetitive DNA sequence or microsatellite; usually a di- sequence nucleotide, e.g. (AT)n or tri-nucleotide (e.g. (GTC)n repeat, repeat (SSR) where n is the number of repeats. In any one variety n is fixed, but it is different in different varieties. The PCR primers used to amplify SSRs are designed from non-repetitive flanking sequences that are unique, so they only target one region of the genome at a time.

Standard Approved sample of a named variety from an approved source. Variety standards must be 100% pure. Quantitative standards are made from mixing two variety standards in known proportions by weight.

Page 5 of 27 3. Aims and Objectives of the Investigation

3.1 Why is enforcement needed for basmati rice?

A code of practice was issued in July 2005 by The Rice Association, 2005. It lists the varieties that are currently approved by the Indian and Pakistani authorities that can use the description „basmati‟, and lays down the minimum specifications and labelling requirements for basmati rice sold in the UK. The maximum limit for non-basmati rice is 7%. This code of practice replaces the FSA‟s previous guidance on the use of the term „basmati‟ and it came into practice for all products labelled after January 1st 2006.

There are import duties on rice for entry into Europe as specified by the European Commission Regulation 1549/2004. For premium basmati varieties from India and Pakistan there was complete exemption from EU duty payments in 2004, representing a saving of at least €65 per tonne. The reduction in duty is decreasing, and currently stands at 50%. However, by 2009 there will be no exemption from duty for premium basmati rice. EU regulations currently define premium varieties as only the following: Basmati 217; Basmati 370; Basmati 386; Kernel (Basmati); Pusa Basmati; Ranbir Basmati; Super Basmati; Taraori Basmati (HBC-19); Type-3 (Dehradun). Although the duty reduction is a driver for fraud, the market price for these premium varieties can be up to 3 times that of non-basmati rice. Therefore variety mixtures made using lower quality basmati varieties are likely to continue to remain a hazard for consumers. Such practice results in the consumer being sold a lower quality product in the guise of a superior one. The main adulterants detected in 2003 with SSR markers were Sherbati and Pak 386 (FSA Basmati Rice survey, 2004), but newer varieties such as Pusa Sugandha and Supra are now appearing in the U.K.

Enforcement of labeling regulations is necessary in order to ensure that basmati rice is correctly described and labelled so that consumers are accurately informed. Public Analyst (PA) laboratories need appropriate and validated methods to test basmati rice and detect levels of adulteration with non-permitted varieties. It is important to ensure that such methods of analysis are used correctly and, if necessary, be defensible in the courts.

The FSA has identified the Agilent 2100 BioAnalyser as a suitable platform for use by PAs. It is smaller, cheaper and easier to use than a DNA sequencer. A major advantage of the LabChip system compared with horizontal agarose gels is that it avoids the use of the highly carcinogenic intercalating dye ethidium bromide. So even when PCR products can be separated on agarose, the LabChip system makes a major contribution to laboratory and environmental safety.

Page 6 of 27 3.2 Existing basmati rice tests with SSR markers

The FSA has funded work to produce robust methodology that can identify rice cultivars. PCR methods for the detection of non-basmati rice in basmati rice using DNA polymorphism analysis have already been developed that use Simple Sequence Repeats (SSRs or ). Two SOPs were previously developed and validated by the FSA in association with J Gorham and K.A. Steele, Bangor University. These were (i) Microsatellite method for the identification of certain basmati rice varieties (2005) and (ii) Protocol for the quantitative analysis of adulteration of basmati rice with Sherbati, Mugad Sugandha, Pak 386 or Superfine (2006). In this report, these two procedures will be referred to as the variety test and the quantitative test, respectively.

For the variety test with twelve SSR markers, the recommended method for electrophoretic separation of fragments was a DNA sequencer (either an ABI 377 or CEQ8000 platform) that gives resolution of ± 1 bp. Resolution of an Agilent 2100 BioAnalyser is not sufficient for all of these SSR markers. Therefore, in order to adapt the variety test protocol for the BioAnalyser we sought alternative PCR markers where polymorphic fragments had differences of > 4 bp. Such alternative DNA marker techniques have previously been developed and tested for use in rice, but had not to our knowledge been tested on basmati and known adulterant varieties.

For the quantitative test, the microsatellite RM201 has been well tested and validated in FSA studies using DNA sequencers for fragment separation and estimation of peak area. With this marker the polymorphic fragments for basmati and non-basmati varieties differ by 16 bp. This is sufficient for separation on the Agilent 2100 BioAnalyser, and so we elected to adapt this method directly for use with the LabChip system.

3.3 Alternative marker systems for rice

We identified three alternative PCR marker systems that have been developed in rice and that we considered to be potentially suitable for separation on the BioAnalyser. These are ISSR (Saini et al., 2004; Nagaraju et al., 2002), INDELs (Shen et al., 2004) and transposon display methods based on a unique rice element known as „Rim2/Hipa‟ (Kwon et al., 2005).

The public availability of rice genomic sequences allows segments of indica and japonica genomic sequence to be compared. Shen et al. (2004) have produced a genome-wide polymorphism database that contains four types of polymorphism including Insertion-Deletion (INDEL) sites. This is available at the following web site: http://shenghuan.shnu.edu.cn/ricemarker. Size differences of less than 100 base pairs (bp) are attributed to INDELs, of which there are more than 400,000 in the database, representing one INDEL every 953 bp. Eight PCR-based markers for INDELs from this database were developed by Dr. A.H. Price of Aberdeen

Page 7 of 27 University (personal communication) on 4 . Fifty PCR-based markers distributed across all 12 chomosomes are given by Shen et al. (2004). One additional marker situated in the for betaine aldehyde dehydrogenase 2, which is a key component in the aroma biosynthetic pathway, has been published by Bradbury et al. (2006). The PCR products reveal polymorphic fragments that differ by 25-50 bp. Hence INDELs are ideal markers for multiplex use with the Agilent 2100 Bioanalyzer and the products range in size from 185 to 330 bp. Another advantage of INDELs is that they do not produce the stutter characteristic of SSRs and so they are much easier to score than SSRs.

Inter Simple Sequence Repeat (ISSR) markers were developed from rice DNA sequences that contain simple sequence repeats (SSRs) and their flanking regions. ISSR is a fingerprinting technique, like AFLPs and RAPDs, and the are likely to give several polymorphic bands per PCR. They have been used to differentiate taxa at all levels (Parsons et al., 1997; Blair et al., 1999; Joshi et al., 2000; Virk et al., 2000), and particularly to distinguish basmati and non- basmati varieties (Nagaraju, et al., 2002; Saini et al., 2004). One feature of ISSRs is that the polymorphic PCR products can be sequenced and the information used to design Sequence Tagged Site (STS) primers, although such development was beyond the scope of this project. In contrast to SSR primers which anneal to the flanking sequences (forward and reverse primer) of simple sequence repeats, the ISSR primers amplify the regions between SSRs with the same repeated sequence. Only one PCR primer is used to generate many fragments. Greater specificity is achieved by adding a flanking sequence („anchor‟) to either the 5‟- or 3‟- end of the ISSR primer. Fragments are amplified that range in size from about 100 to 1500 bp. Although the maximum number of clearly defined bands can only be seen on large polyacrylamide gels, there are still enough bands resolved on the Agilent 2100 BioAnalyzer to make this a useful technique.

Over 34% of the rice genome is transposon sequences and of these nearly 3% are Class II CACTA-type elements (International Rice Genome Sequencing Project, 2005). Wang et al. (2003) confirmed that Rim2/Hipa elements are unique to the rice genome and that they show a high level of variation within their internal sequences. Hence polymorphic bands are amplified when primers from their sequences are used. It has been estimated that there are total of 600–700 Rim2/Hipa elements present in the whole rice genome and they occur non- randomly on the chromosomes, as visualized by fluorescence in situ hybridization (Wang et al., 2003).

3.4 Aims and Objectives

In this project we adapted existing rice marker systems for basmati authentication on the Agilent 2100 BioAnalyser. The different marker systems were tested on a range of varieties and suitable ones identified. We aimed to produce a database of alleles with the novel markers, and identify a set of

Page 8 of 27 markers for variety testing in multiplex PCR. The database of varieties was extended to include non-basmati fragrant varieties such as Thai fragrant rice KDML-15 and US fragrant rice (). We also included „new‟ adulterant varieties Pusa Sugandha (three separate samples) Superfine and Supra. Two separate samples of Sherbati were also compared.

We also aimed to identify markers that can be used for quantification of different possible adulterants in order to extend the range on adulterant varieties that could be detected. We selected RM201 and one INDEL marker and for quantitative analysis on the Agilent 2100 BioAnalyser.

The adapted quantitative and a variety tests were validated in conjunction with an independent Public Analyst. The main outputs were three new SOPs, two for quantification of adulterants and one for detection of varieties or mixtures.

4. Experimental Procedures

Full details of all optimized procedures are given in SOPs 1, 2 and 3. All methods for marker systems were adapted from the procedures given in publications cited in the previous section: “Alternative marker systems for rice”. The DNA extraction method remains the same as it was described in the previous SOPs.

4.1. Sourcing of standard varieties and DNA extraction

Authenticated rice samples were obtained from the FSA, Tilda Riceland and IRRI to supplement the existing rice reference collection held at Bangor University. DNA was extracted from de-husked or milled samples of a core set of 24 standards of rice (Appendix 1). This set contains at least one representative sample of all groups of rice varieties that can be separated with the current set of SSR markers.

DNA extractions from 1 g of each powdered sample were be carried out using the modified Nucleon Phytopure DNA extraction kit (Tepnel, U.K.) procedure. For standards, where only a few grains were available, the extraction procedure was adapted to extract from 200 mg of powdered sample. All DNA samples were stored in a -20°C freezer. The DNA concentration of samples was determined, either using the Nanodrop or the Pico Green method (Molecular Probes, Netherlands), and the concentration adjusted to 10 ng l-1.

„Test‟ samples were chosen from commercial samples recently tested by FDNAS, according to the results obtained with the SSR variety and quantitative tests. For these DNA was used that had been extracted for the commercial tests, following the existing SOP.

For quantitative standards, DNA was extracted from mixtures of two standard varieties that were mixed together in known proportions by weight before grinding.

Page 9 of 27

4.2. Testing INDEL markers in different rice genotypes

We obtained previously developed PCR primers designed to amplify INDEL sequences. These were tested on a range of basmati and non-basmati long grain varieties. Reaction conditions were optimized from those given by Shen et al., 2004 (parameters for PCR optimization included: MgCl2 concentration, temperature profile and times, reagent kits). Initial screening of fragments used high resolution agarose gel electrophoresis to confirm polymorphism. Thirteen primer pairs that revealed good size separation and polymorphism between basmati, other fragrant and non-basmati standard varieties were repeated and fragments were separated with the Agilent 2100 BioAnalyser. Multiplex pools were developed for variety tests, and primers adjusted to prevent fragment overlap.

4.3. Testing Rim2/Hipa and ISSR markers in different rice genotypes

In our laboratory we had previously optimized the PCR methodology for transposon display. In this project we tested 5 Rim2/Hipa primer pairs (A, B, C, D and E) on a set of rice reference material and separated fragments using high resolution agarose gel electrophoresis. The fragments were scored as either present or absent. The PCRs were repeated in order to check for repeatability. For full details see Steele et al. (2007).

Anchored ISSR primers from sequences given by Blair et al. (1999) were used to test a range of standard varieties.

4.4. Optimizing multiplex PCRs for INDELS

We used the QIAGEN Multiplex Master Mix to optimize the PCR for three multiplex panels, each containing different combinations of primers for four INDELs. Reaction conditions were adjusted to give even amplification of all fragments. Two of the panels were designed to be run simultaneously for a test that could detect the presence of the nine adulterants. These were tested with a range of varieties to check for clear fragment separation on the BioAnalyser. Two forward primers (for I13 and A5) were adjusted so that the PCR amplified larger fragments that did not overlap with other fragments in the same panel. The panels were used for analysis of different „test‟ samples that were chosen because they represented a range of known varieties and variety mixtures. Marker profiles were compared with the expected profiles of standard varieties.

4.5. Developing a SOP for variety analysis of bulk extracts using the LabChip system

Two criteria were used to select INDELs for variety testing: i) they should detect as many non-permitted varieties as possible; ii) the test should require a minimum of two PCR reactions. These limitations meant that additional markers

Page 10 of 27 that could distinguish between permitted varieties (e.g Basmati 198 from other Basmati varieties) could not be included in the test, because it would increase the number of PCRs required to at least three.

A simple one panel (4 INDEL) test was developed that required only one multiplex PCR and it was suitable for detecting seven common adulterants. Two other markers can be used subsequently to identify the additional adulterants. The SOP was validated with standard and test varieties by Worcestershire Scientific Services.

4.6. Optimizing existing SSRs for LabChip analysis

We adapted the existing procedure for SSR marker RM201 for quantitative detection by comparison of peak areas separated with the Agilent 2100 BioAnalyzer software. Instead of using a labelled forward primer, necessary for florescence detection on the DNA Sequencer, we used unmodified primers that gave clearer separation on the Agilent 2100. Quantitative standards were made up as known mixtures (% by weight) of Taraori and Sherbati (0, 5, 10, 15, 20, 30, 40 and 50% of adulterant in an approved basmati variety). PCR products were run with the Lab Chip 1000 kit and Agilent 2100 BioAnalyser software data was used to give peak areas for the relevant bands from which ratios were calculated. The peak areas are used to create standard curves using Microsoft Excel software according to the results from known standards. The ratio of the non- basmati to basmati total areas are calculated and plotted against the non- basmati percentages of the standards. The percentages of non-basmati in the unknown samples were calculated from the regression equation of the standard curve.

The method was validated by Worcestershire Scientific Services, using two sets of standards and four test samples. The results of both laboratories were compared. The data were assessed to examine the precision, accuracy and reproducibility of results.

4.7. Evaluating INDEL markers for quantitative testing

We considered that a second test was necessary in order to detect and measure levels of adulteration with „new‟ adulterant varieties that cannot be detected using RM201. Three INDELs were used to test standard mixtures of 10% and 30%. The clarity of bands revealed on the BioAnalyser was used to identify the most suitable marker for validation as a quantitative test. The test with B8 was validated with WSS, using two sets of standards (the same ones used for the RM201 test) and four test samples. The results of both laboratories were compared. The data were assessed to examine the precision, accuracy and reproducibility of results.

Page 11 of 27 5. Results and Discussion

5.1 Results with INDEL markers

A total of 52 INDEL primer sets were tested on the 24 rice standards (Appendix 2). Only one gave consistently no amplification. Most INDELS revealed two distinct alleles although a few amplified more than two alleles. The difference in size of alleles is due to an insertion/deletion polymorphism. The sizes of insertion/deletions ranged from approximately 15 to 70 bp. A total of 103 alleles were detected. The amplified products were scored independently by two workers for presence or absence, and the differences highlighted which alleles were difficult to score.

The full-set of results were summarized as a data matrix for multivariate analysis. The data were used to find Jaccard‟s similarity coefficient for each pair of standards and UPGMA (Unweighted Pair Group Method with Arithmetic mean) clustering was carried out to summarise the clusters as a dendrogram (Figure 1a). The most informative 13 markers were: B8, I13, I12, A5, I10, I6, I5, I22, B5, A4, B4, I11 and F4. The smallest sub-set of INDEL markers that could give useful groupings was identified as nine (Figure 1b)

5.2 Results with Rim2/Hipa and ISSR markers

The ISSR PCR products could be detected with the BioAnalyser (Figure 2) but the band intensity was variable and polymorphisms were considered to be very difficult to score in comparison to the other methods. Therefore this method was not tested further.

The Rim2/Hipa primer sets gave multiple polymorphic alleles, with up to 15 fragments amplified (Appendix 3). The A primer-set was the most reliable while the B primer set revealed the most fragments. Of 41 possible fragments detected with five primer pairs, approximately 50% were repeatable across runs. Unique profiles were obtained for 16 varieties, but the basmati varieties could not be readily separated from non-basmati varieties.

5.3 Database of markers

A database has been produced for two alternative marker systems, INDELs (Appendix 2) and Rim2/Hipa (Appendix 3) transposon display. A summary of the total number of alleles detected and varieties tested with each system is given in Table 1.

Table 1. Summary of data sets for two novel rice marker systems used to test rice varieties. Marker type Number of varieties Number of alleles Rim 2/Hipa transposon 23 38 INDEL 23 103

Page 12 of 27 Table 2. Fragment sizes (bp) detected in the two panel variety test using INDEL multiplex panels (IMPX 1 and IMPX2). Adulterant varieties that can be detected are denoted by *. IMPX1 IMPX2 B8 I13 I12 A5 I10 I6 I5 I22 Basmati 370 273 213 200 230 168 578 205 317 Dehra Dun (type 3) 273 213 200 230 168 578 205 317 Basmati 217 273 213 200 230 168 578 205 317 Ranbir 273 213 200 230 168 578 205 317 Taraori 273 213 200 230 168 578 205 317 Kernel 273 213 200 230 168 578 205 317 Pusa Basmati 273 213 200 185 168 538 152 317 Super Basmati 273 213 200 230 134 578 205 317

Basmati 198 273 213 200 230 168 578 205 317 Basmati 385 273 213 200 185 168 578 205 317 Kasturi 273 213 200 230 134 538 205 268 (152) Mahi Sugandha 273 213 200 185 134 538 151 268 Haryana Basmati 273 213 200 230 134 538 205 317 Punjab Basmati 273 213 200 230 168 578 205 317 (185) (134) (153)

Pak 386 * 354 213 167 185 134 538 152 317 Sherbati 1 * 354 213 167 185 134 538 152 268 Sherbati 2 * 354 241 200 185 134 538 152 268 Sherbati (awns) * 354 213 167 185 134 538 152 268 Supra * 273 213 167 185 134 538 152 268 =348 Pusa Sugandha 1 * 273 213 200 185 134 538 152 268 (317) Pusa Sugandha 2 * 273 213 200 185 168 538 152 317 Pusa Sugandha 3 * 273 213 200 185 168 538 152 317 Superfine * 273 213 200 185 134 538 152 268 (167) Mugad Sugandha 273 241 200 185 134 538 152 268 (India) (213) (167) Basmati 2000 * 273 213 200 185 134 578 205 317 Shaheen Basmati * 273 213 200 185 134 578 205 317 Yamini * 273 213 200 230 168 578 205 317 Pusa 1121 * 273 213 200 185 168 538 205 317 Sugandha-1 (Nepal) 273 213 200 185 134 538 152 268 Azucena (Philippines) 273 241 200 230 168 578 205 317 KDML 105 (Thai) 273 213 200 185 134 538 152 268 Jasmati (USA) 273 241 200 230 168 578 152 317 (134) (205) IR64 354 213 200 185 134 538 152 268 Kalinga III (India) 354 213 200 185 134 538 152 268

Page 13 of 27

5.4 Development of INDEL markers for variety testing

Eight INDEL markers were identified that can distinguish most of the main adulterants from permitted basmati varieties. They were grouped into 2 panels, each consisting of four markers for multiplex PCR (Table 2). The primers for two markers (I13 and A5) were re-designed to give products with clear separation on the Agilent Bioanalyzer 2100 that do not overlap other fragments in the same panel.

Panel 1 (IMPX1) contains a common control fragment amplified by B8 (>650 bp) that should be amplified in all rice samples. Absence of this fragment suggests that the DNA extract is not suitable for the test. The panels were tested on a range of genotypes (Figure 3) and some mixed samples of different genotypes. The two optimized pools and PCR conditions have been incorporated into one SOP but they were not validated because the alternative one-panel method was considered to be more cost-effective (see below).

An alternative single panel of four INDEL markers (B8, A4, A5 and I11) was developed and optimized for the Bioanalyzer. This method requires only one PCR and can identify Pak 386, Sherbati 1, Sherbati 2, Sherbati (awns), Supra, Pusa Sugandha 1, Pusa Sugandha 2, Pusa Sugandha 3, Superfine and Mugad Sugandha. This panel when used alone can not be used to identify individual varieties, but it can readily detect the presence of common adulterants in one test. One of the markers used in the test is also used in SOP 2. Compared to the microsatellite method it cannot detect as many individual varieties, nor distinguish Basmati 198 from other basmatis. Two additional markers, I5 and I10, can be used to augment this test (Table 3). INDEL I5 separates Pusa Basmati 1 from Pusa 1121. INDEL I10 separates Basmati 385 from Basmati 2000 and Shaheen Basmati.

A SOP for this one-panel (4 INDEL) test has been developed and validated. The interpretation for nine “unknown” commercial samples matched that given by the microsatellite test. This is the most cost-effective method available for screening basmati samples and we recommended its use by PAs for detecting for the presence of non-permitted varieties and mixtures. This is an ideal first step to identify which samples contain non-permitted varieties that can then be tested with a more sensitive quantification test.

Detailed data for the variety validation study (SOP 3) is provided in the excel file, „Validation of variety SOP‟.

Page 14 of 27 Table 3. Fragment sizes (bp) detected in the 4 INDEL marker test. Fragment sizes are also given for two optional markers that can detect Pusa 1121, Basmati 2000 and Shaheen Basmati in conjunction with the test.

B8 I11 A5 A4 options I5 I10 Approved Basmati 198 273 214 230 142 205 168 Listed Basmati 370 273 214 230 142 205 168 Listed Dehra Dun (type 3) 273 214 230 142 205 168 Listed Basmati 217 273 214 230 142 205 168 Listed Ranbir 273 214 230 142 205 168 Listed Taraori 273 214 230 142 205 168 Listed Kernel 273 214 230 142 205 168 Listed Super Basmati 273 214 230 142 205 134 non-permitted Yamini 273 214 230 142 Can't separate from Basmatis 205 168 Listed Pusa Basmati 273 214 192 174 test with I5 152 168 non-permitted Pusa 1121 273 214 192 174 test with I5 205 168 Approved Basmati 385 273 214 192 142 test with I10 205 168 Approved Punjab Basmati 273 214 230 +192 142 test with I10 205 + 152 168 + 134 non-permitted Basmati 2000 273 214 192 142 test with I10 205 134 non-permitted Shaheen Basmati 273 214 192 142 test with I10 205 134 Approved Kasturi 273 256 230 142 205 + 152 134 Approved Haryana Basmati 273 256 230 174 152 134 Approved Mahi Sugandha 273 256 192 142 205 134 non-permitted Pak 386 354 214 192 174 152 134 non-permitted Sherbati 1 354 214 192 174 152 134 non-permitted Sherbati 2 354 256 192 174 152 134 non-permitted Sherbati (awns) 354 256 192 174 152 134 non-permitted Pusa Sugandha 1 273 256 192 174 152 134 non-permitted Pusa Sugandha 2 273 256 192 174 152 168 non-permitted Pusa Sugandha 3 273 256 192 174 152 168 non-permitted Superfine 273 256 192 174 152 134 non-permitted Mugad Sugandha 273 256 192 174 152 134 non-permitted IR64 354 256 192 174 152 134 non-permitted Kalinga III 354 256 192 141 152 134 non-permitted Supra 273 + 348 256 192 142 152 134

Page 15 of 27 5.5 Quantitative analysis

The quantitative test for non-permitted basmatis using microsatellite RM201 (previously on the CEQ8000) has been adapted for use on the Agilent 2100 BioAnalyser (SOP1). There was some „noise‟ for the non-basmati peak, but this was minimized by optimizing the integration parameters on the BioAnalyser software to determine peak area. There was a large range of variation in results between runs on the BioAnalyser and large standard errors for standard mixtures containing ≥40% non-basmati. The estimates given for non basmati content were slightly higher than the equivalent results using the CEQ8000, but they followed the same pattern (Figure 8). The test could be used by PAs to gain an indication of the level of adulteration. However, we recommend that the CEQ8000 method should be used for enforcement purposes.

In addition to the microsatellite RM201, we tested three INDEL markers (B8, I11, and I12) for use in quantitative analysis of Sherbati in Taraori (Figure 5). I12 was not very reliable so it was dropped. The „non-basmati‟ allele for I11 was also amplified in three basmati varieties, so for this reason I12 was not developed as a quantitative test. However, this marker could be used as a follow-on test to quantify Pusa Sugandha in mixtures.

The INDEL marker B8 was chosen to be developed for a test on the BioAnalyser (SOP2) because it readily distinguishes between aromatic and non-aromatic varieties, including the most commonly detected adulterants from the 2003 survey. However, it does not detect more adulterants than the RM201 test. The procedure was adapted from the RM201 quantitative test and quantification is based on the ratio of the non-basmati peak area and the basmati peak area. This test uses four PCR primers for marker B8 and gives three possible fragments (Figure 7 a and b). One of these is present in all rice samples and so can be treated as a control for successful amplification.

Both quantitative tests (microsatellite RM201 and INDEL B8) were validated in two laboratories with standards (prepared at FDNAS) and four commercial samples known to contain adulterants. (Appendix 4).

The INDEL B8 results were relatively consistent between laboratories. However, there was a difference in relative proportion of non-permitted variety detected with this method compared to the RM201/CEQ method (Figure 8). The pattern of estimated non-basmati present was different to that given for the same samples with RM201. This discrepancy is probably due to the fact that some non- permitted varieties contain the “Basmati” allele of B8 and thus they evade detection with this marker. The proportion of non-basmati that was determined was much smaller where samples contained adulterants that were not identified as Sherbati or Pak 386. This test misses some of the less common but potential adulterants (Mugad Sugandha and Superfine), For example, in sample #3101 (likely to be Super contaminated with a variety similar to Mugad Sugandha) the level of non-basmati detected was three times lower than that detected with

Page 16 of 27 RM201 on the CEQ8000 in the same sample. Therefore, we cannot recommend the INDEL B8 quantitative test for use on its own, without a variety test analysis to identify rice varieties likely to be present. The INDEL B8 quantitative test is best used as a follow-up test to the 4 INDEL test (SOP3) if the B8 fragment is detected.

5.6 Problems encountered when adapting the tests for the Agilent Bioanalyzer

The two quantitative SOPs were compared with the results from RM201 on the CEQ8000 (Figure 1). Across different runs and chips the Agilent Bioanalyzer gave a range of sizes for each fragment consequently it was more difficult to select the “Area” values for the relevant fragments from the Agilent output than it is on the CEQ8000. The results must be extracted from the Agilent output (.csv file) manually (by copying and pasting), whereas with the CEQ8000 they can be exported directly into Excel. Therefore, there is a higher chance of operator error during data handling with the Agilent method.

The default threshold for height is 20 on the Agilent 2100 Bioanalyzer software. This causes problems for quantitative analysis because there is no optimum level to which to set the height threshold manually (Figure 6). We have chosen a value of 2 as a standard for quantitative analysis because lower values give more spurious calls. It must be remembered that very low levels of adulteration where the non-basmati peaks are <2 will be reported as having zero values for the non- basmati peak.

The four INDEL variety test (Figure 3) cannot distinguish between the following varieties: Taraori, Basmati 370, Dehradun (Type 3) Basmati 217, Ranbir, Kernel, Super, Yamini and Basmati 198. Of these, only Yamini is non-permitted, but it is currently not thought to be a problem. The test cannot separate Pusa Basmati from Pusa 1121 and it cannot separate Basmati 385 from Pujab Basmati, Basmati 2000 and Shaheen basmati. Additional INDELS or microsatellite markers can be used to distinguish most of these varieties if more precise analysis is needed.

6. Acknowledgements

We thank the following for their input to this project: Mark Woolfe and Sandy Primrose (FSA) John Gorham, Ross McEwing, Helen Briggs and Liz Heap (FDNAS) Carol Stevens and Carole Lincoln (Worcestershire Scientific Services) Gwen Edwards (CAZS Natural Resources, Bangor University) Nandini Sheshadri (Tilda Riceland, India)

Page 17 of 27 7. References

Blair MW, Panaud O, McCouch SR. 1999. Inter-simple sequence repeat (ISSR) amplification for the analysis of microsatellite motif frequency and fingerprinting in rice ( L.). Theoretical and Applied Genetics 98: 780:792. Bradbury LMT, Henry RJ, Jin Q, Reinke RF, and Waters DLE. 2006. A perfect marker for fragrance genotyping in rice. Molecular Breeding 16: 279–283 European Commission Regulation (EC) No 1549/2004 of 30 August 2004 derogating from Council Regulation (EC) No 1785/2003 as regards the arrangements for importing rice and laying down separate transitional rules for imports of basmati rice. http://europa.eu.int/eur- lex/pri/en/oj/dat/2004/l_280/l_28020040831en00130022.pdf Food Standards Agency Basmati Rice Survey. 2004 http://www.food.gov.uk/multimedia/pdfs/fsis4704basmati.pdf International Rice Genome Sequencing Project. 2005. The map-based sequence of the rice genome 436, 793-800 (11 August 2005) [doi: 10.1038/nature03895] Joshi SP, Gupta VS, Aggarwal RK, Ranjekar PK, Brar DS. 2000. Genetic diversity and phylogenetic relationship as revealed by inter simple sequence repeat (ISSR) polymorphism in the genus Oryza. Theoretical and Applied Genetics 100: 1311-1320. McCouch S.R. Teytelman L, Xu Y, Lobos KB, Clare K, Walton M, Fu B, Maghirang R, Li Z, Xing Y, Zhang Q, Kono I, Yano M, Fjellstrom R, DeClerck G, Schneider D, Cartinhour S, Ware D and Stein L. 2002. Development and mapping of 2240 new SSR markers for rice (Oryza sativa L.). DNA Research 9, 199-207. Kwon SJ, Park KC, Kim JH, Lee JK, Kim NS. 2005. Rim 2/Hipa CACTA transposon display; A new genetic marker technique in Oryza . BMC Genetics, 6:15. Nagaraju J, Kathirvel M, Kumar RR, Siddiq EA, Hasnain SE. 2002. Genetic analysis of traditional and evolved Basmati and non-Basmati rice varieties by using fluorescence-based ISSR-PCR and SSR markers. Proceedings of the National Academy of Science U.S.A. 99: 5836-5841. Parsons BJ, Newbury, HJ, Jackson MT, Ford-Lloyd BV. 1997. Contrasting genetic diversity relationships are revealed in rice (Oryza sativa L.) using different marker types. Molecular Breeding 3: 115-125. Saini N, Jain N, Jain S, Jain RK. 2004. Assessment of genetic diversity within and among Basmati and non-Basmati rice varieties using AFLP, ISSR and SSR markers. Euphytica 140: 133-146. Scheuke M. 2000. An economic method for the fluorescent labelling of PCR fragments. Nature Biotechnology, 18: 233-234. Shen YJ, Jiang H, Jin JP, Zhang ZB, He YY, Wang G, Wang C, Qian L, Li X, Yu QB, Liu HJ., Chen DH, Gao JH, Huang H, Shi TL., Yang ZN. 2004

Page 18 of 27 Development of genome-wide DNA polymorphism database for map-based cloning of rice . Plant Physiology 135: 1198-1205. Singh, R.K., Singh, U.S., Khush, G.S., 2000. Aromatic , Science Publishers, Inc. Plymoth, UK. Steele K.A., Ogden R., McEwing R. Briggs H. Gorham J.R. (2007) INDEL markers distinguish Basmatis from other fragrant rice varieties. Field Crops Research. DOI: 10.1016/j.fcr.2007.08.001 The Rice Association, 2005. Code of Practice for basmati Rice. July 2005 [http://www.riceassociation.org.uk/]. Virk PS, Zhu J, Newbury HJ, Bryan GJ, Jackson MT, Ford-Lloyd BV, 2000. Effectiveness if different classes of molecular marker for classifying and revealing variation in rice (Oryza sativa) germplasm. Euphytica 112: 275-284. Wang et al. 2003. Genomic characterization of Rim2/Hipa elements reveals a CACTA-like transposon superfamily with unique features in the rice genome. Molecular Genetics and Genomics 270, 234 – 242.

Page 19 of 27 Appendix 1. Authenticated rice samples sourced by the FSA or CAZS-NR, indicating varieties approved in the EU for labelling as Basmati (Rice Association, 2005) Origin Noteworthy parents Other details EU listed Basmati varieties Basmati 370 India Selection from landrace Dehra Dun (Type 3) India Selection from landrace Basmati 217 (IRGC 53637) India Ranbir India Basmati 370 Early Basmati Anantnag Taraori (HBC 19, Karnal India Selection from landrace local) Kernel (Pakistan Basmati) Pakistan CM7/Basmati 370 Pusa Basmati 1 (IET 10364) India (IARI) IR8 and Basmati 370a Semi-dwarf, high yielding Super Basmati Pakistan (RRI-KSK) Basmati 320/IR661 Shabnam in India

Other approved Basmati varieties Basmati 198 Pakistan (RRI-KSK) Basmati 385 Pakistan (RRI-KSK) T(N)1/Basmati 370 Kasturi (IET 8580) India (DRR, Hyderabad) Basmati 370/CR88-17-1-5 High-yielding Haryana Basmati (IET 10367) India (RRS, Kaul) Sona/Basmati 370 Dwarf Mahi Sugandha India BK79/Basmati 370 Punjab Basmati (Bauni India (RRS Kapurthala) Sona/Basmati 370 Basmati)

Non-approved fragrant varieties Basmati 2000 India Shaheen Basmati Pakistan Salt tolerant Mugad Sugandha India Superfine Pusa Sugandha 1 India (IARI) Potential adulterant Pusa Sugandha 2 India (IARI) Potential adulterant

Page 20 of 27 Pusa Sugandha 3 India (IARI) Potential adulterant Yamini (CSR 30) India Possibly Pusa Basmati 1 Semi dwarf, salt tolerant Supra Pusa 1121 (Pusa sugandha India (IARI) Sister lines of Pusa Basmati 1 Semi-dwarf 4) Sugandha-1 Nepal (LI-BIRD/CAZS-NR/NARC) Pusa Basmati 1 Azucena Phillipines Selection from landrace Upland tropical japonica KDML 105 Thailand Khao Dawk Mali (landrace) Jasmati RiceTec (USA) Possibly Della?

Non-fragrant varieties Sherbati 1 India Potential adulterant Sherbati 2 India Potential adulterant Sherbati (awns) India Potential adulterant Pak 386 Pakistan Potential adulterant IR64 Philippines (IRRI) Multiple lines High yielding semi-dwarf Kalinga III (CR 237-1) India AC 540 and Ratna Upland rice, early a See page 87 in Singh et al. (2000) for full ancestry of Pusa Basmati 1 DDR, Directorate of Rice Research; IARI, Indian Agricultural Research Institute; IRRI, International Rice Research Institute, RRI-KSK, Rice Research Institute, Kala Shaha Kaku ;RRS, Rice Research Station.

Page 21 of 27 Appendix 2. INDEL results database

B B B D D D D B B E E L L I I I I I I K K G G C C F F C C F F G G 8 8 8 0 0 0 0 0 0 1 1 1 1 5 5 1 1 2 2 2 2 1 1 1 1 1 1 2 2 2 2 2 2 - - - 4 4 7 7 4 4 1 1 1 1 - - 3 3 2 2 3 3 ------1 2 3 ------2 1 ------1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 Basmati 198 0 0 1 1 0 1 0 1 0 1 0 1 0 0 1 1 0 0 1 1 0 1 0 1 0 1 0 0 1 1 0 1 0 Mahi Sugandha 0 0 1 0 1 0 1 0 1 0 1 1 1 1 0 1 0 1 0 0 0 1 0 1 0 1 0 0 1 0 1 0 1 Basmati 370 1 0 1 1 0 1 0 1 0 1 0 1 0 0 1 1 0 0 1 1 0 1 0 1 0 0 1 0 1 1 0 0 0 Basmati 385F 1 0 1 1 0 1 0 1 0 1 0 1 0 0 1 1 0 0 1 1 0 1 0 1 0 1 0 0 1 1 0 0 0 Pusa Basmati 0 0 1 1 0 1 0 1 0 1 0 1 0 0 1 1 0 0 1 1 0 1 0 1 0 0 1 0 1 1 0 1 0 Ranbir 1 0 1 1 0 1 0 1 0 1 0 1 0 0 1 1 0 0 1 1 0 1 0 1 0 0 1 0 1 1 0 1 0 Super Basmati 0 0 1 1 0 1 0 1 0 1 0 1 0 0 1 1 0 1 0 1 0 1 0 1 0 0 1 0 1 1 0 1 0 Taraori 1 0 1 1 0 1 0 1 0 1 0 1 0 0 1 1 0 0 1 1 0 1 0 1 0 0 1 0 1 1 0 1 0 Basmati 2000 1 0 1 1 0 1 0 1 0 1 0 1 0 0 1 1 0 0 1 1 0 1 0 1 0 0 1 0 1 1 0 0 0 Pak 386 0 1 0 1 0 1 1 1 0 0 1 0 1 0 1 1 0 1 0 0 1 1 0 1 0 1 0 0 1 0 1 1 0 Pusa 1121 1 0 1 0 0 1 0 1 0 1 0 1 0 0 1 1 0 0 1 1 0 1 0 1 0 0 1 0 1 1 0 1 0 Pusa Sugandha 1 0 0 1 1 1 0 1 1 1 0 1 1 0 1 0 1 0 0 1 0 1 1 1 1 0 1 0 0 1 0 1 1 1 Pusa Sugandha 2 1 0 1 1 0 0 1 1 0 0 1 1 0 0 1 1 0 1 0 0 1 1 0 1 0 1 0 0 1 0 1 1 0 Pusa Sugandha 3 0 0 1 1 0 0 1 1 0 0 1 1 0 0 1 1 0 1 0 0 1 1 0 1 0 1 0 0 1 0 1 1 0 Shaheen Basmati 1 0 1 1 0 1 0 1 0 1 0 1 0 0 1 1 0 0 1 1 0 1 0 1 0 1 0 0 1 1 0 1 0 Sherbati 1 0 0 0 0 1 0 1 0 1 0 1 0 1 1 0 0 1 1 0 0 1 1 0 1 0 0 1 0 1 0 1 0 1 Supra 0 0 0 0 1 1 0 0 1 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 0 1 1 0 1 1 Sherbati 2 0 0 0 0 1 0 1 0 1 0 1 1 1 1 0 0 1 1 0 0 1 1 0 1 0 0 1 0 1 0 1 0 1 Azucena 1 0 1 1 0 1 0 1 0 1 0 1 0 0 1 0 1 0 1 1 0 0 1 0 1 0 1 1 0 1 0 0 0 IR64 0 1 0 0 1 0 1 0 1 0 1 0 1 1 0 1 0 1 0 0 1 1 0 1 0 1 0 0 1 0 1 0 1 Kalinga III 0 0 0 0 1 0 1 0 1 0 1 1 1 1 0 1 0 1 0 0 1 1 0 1 0 1 0 0 1 0 1 0 1 Sugandha-1 0 0 1 1 0 0 1 0 1 0 1 1 1 1 0 1 0 0 1 0 0 1 0 1 0 1 0 0 1 0 1 0 1 KDML 105 1 0 1 1 0 0 1 0 1 0 1 1 1 1 0 1 0 1 0 0 0 1 0 1 0 1 0 0 1 0 1 0 1

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F F G G H H C C F F G G B B A A B B C C A A F F I I C C F F C C G G G 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 5 6 6 6 6 6 6 7 7 7 7 8 ------1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 2 1 1 2 1 2 1 2 1 2 1 2 1 2 1 Basmati 198 1 0 1 0 0 1 1 0 1 0 1 0 1 0 1 0 1 0 0 1 0 1 1 0 0 1 1 0 0 0 0 1 0 0 1 Mahi Sugandha 0 1 0 1 1 0 0 1 0 1 0 1 1 0 1 0 0 1 1 0 1 0 1 0 1 0 1 0 1 0 0 1 1 0 0 Basmati 370 1 0 1 0 0 1 1 0 1 0 1 0 1 0 1 0 1 0 0 1 0 1 1 0 0 1 1 0 0 0 0 1 1 0 1 Basmati 385F 0 1 1 0 0 0 0 1 0 1 1 0 1 0 1 0 1 0 0 1 1 0 1 0 0 1 1 0 0 1 0 1 1 0 0 Pusa Basmati 0 1 0 1 1 0 0 1 1 0 1 0 0 1 0 1 0 1 1 0 1 0 1 0 1 0 1 0 0 1 0 1 1 0 0 Ranbir 1 0 1 0 0 1 0 0 1 0 1 0 1 0 1 0 1 0 0 1 0 0 1 0 0 1 1 0 0 1 0 1 1 0 1 Super Basmati 1 0 1 0 0 1 1 0 1 0 1 1 1 0 1 0 1 0 0 1 0 1 1 0 0 1 1 0 0 1 0 1 1 0 1 Taraori 1 0 1 0 0 1 0 1 1 0 0 1 1 0 1 0 1 0 0 1 0 1 1 0 0 1 1 0 0 1 0 1 1 0 1 Basmati 2000 0 0 1 0 0 1 1 0 0 1 1 0 1 0 1 0 1 0 0 1 1 0 1 0 0 1 1 0 0 1 0 1 1 0 0 Pak 386 0 1 0 0 0 0 1 0 1 0 1 1 0 1 0 1 1 0 0 1 1 0 1 0 1 0 1 0 0 1 0 1 1 0 0 Pusa 1121 1 0 1 1 1 1 0 1 1 0 0 1 1 1 0 1 0 1 0 1 1 0 1 0 1 0 1 0 0 1 0 1 1 0 1 Pusa Sugandha 1 1 1 1 0 0 0 0 1 0 1 1 1 1 0 0 1 1 1 1 1 1 0 1 0 1 0 1 0 1 0 0 0 1 0 0 Pusa Sugandha 2 1 0 1 0 1 0 0 1 0 1 0 1 0 1 0 1 1 0 0 1 1 0 1 0 1 0 1 0 1 0 0 1 1 0 0 Pusa Sugandha 3 1 0 1 0 1 0 0 1 0 1 0 1 0 1 0 1 1 0 0 1 1 0 1 0 1 0 1 0 1 0 0 1 1 0 0 Shaheen Basmati 1 1 1 0 0 1 1 0 1 0 1 0 1 0 1 0 1 0 0 1 1 0 1 0 0 1 1 0 0 1 0 1 1 0 1 Sherbati 1 0 1 1 0 1 0 0 1 0 1 0 1 0 1 0 1 0 1 0 1 1 0 1 0 1 0 1 0 1 0 0 1 1 0 0 Supra 0 1 1 1 1 1 0 1 0 1 1 0 1 0 1 0 0 1 0 1 1 0 1 0 1 0 1 0 1 0 0 1 1 0 0 Sherbati 2 0 1 0 1 1 0 0 1 0 1 0 1 0 1 0 1 0 1 0 1 1 0 1 0 1 0 1 0 1 0 0 1 1 0 0 Azucena 0 1 1 0 0 1 1 0 1 0 0 1 1 0 0 0 0 1 0 1 0 1 0 1 0 1 0 1 0 0 1 0 1 0 1 IR64 0 1 0 1 0 0 0 1 0 1 0 1 0 1 0 1 0 1 1 0 1 0 1 0 1 0 1 0 1 0 0 1 1 0 1 Kalinga III 0 1 0 1 1 0 0 1 0 1 1 0 0 1 0 1 0 1 1 0 1 0 1 0 1 0 1 0 1 0 0 1 1 0 0 Sugandha-1 0 1 0 0 1 0 1 0 0 1 0 1 0 1 0 1 0 1 1 0 1 0 1 0 1 0 1 0 1 0 0 1 1 0 0 KDML 105 0 1 1 0 0 0 0 1 0 1 0 1 0 1 1 0 0 1 1 0 1 0 1 0 1 0 1 0 0 0 0 1 1 0 0

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G H C C F F C C G G F F H H I I C C I I F C C I I F F C C I I F F G G 8 8 8 8 8 8 9 9 9 9 9 9 9 9 9 9 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ------0 0 0 0 0 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 1 1 2 1 2 1 2 1 2 1 2 1 2 1 2 ------1 2 1 2 1 1 2 1 2 1 2 1 2 2 1 1 2 1 2 Basmati 198 0 1 0 1 0 1 1 0 1 0 0 1 0 0 0 1 0 1 0 1 1 0 1 1 0 0 0 0 1 0 1 1 0 1 0 Mahi Sugandha 1 1 1 0 1 0 1 0 0 1 0 0 1 0 0 1 1 0 1 0 1 0 1 0 1 1 0 0 1 0 1 0 1 1 0 Basmati 370 0 1 0 1 0 1 0 0 1 0 0 1 0 0 0 1 0 1 0 1 1 0 1 1 0 0 1 0 1 0 1 0 0 1 0 Basmati 385F 1 1 0 1 0 1 1 0 1 0 0 1 0 0 0 1 0 1 0 1 1 0 1 1 0 0 0 0 1 0 1 0 0 1 0 Pusa Basmati 1 1 0 1 1 0 0 1 1 0 0 1 0 0 0 1 0 1 0 1 1 1 0 1 0 1 0 0 1 0 1 0 0 1 0 Ranbir 0 1 0 1 0 1 1 0 1 0 0 1 0 0 0 1 0 1 0 1 1 0 1 1 0 0 0 0 1 0 1 1 0 1 0 Super Basmati 0 1 0 1 1 1 1 0 1 0 0 1 0 0 0 1 0 1 1 1 1 1 0 1 0 1 0 0 1 0 1 0 0 1 0 Taraori 0 1 0 1 0 1 1 0 1 0 0 1 0 0 0 1 0 1 0 1 1 0 0 1 0 0 0 0 1 0 1 1 0 1 0 Basmati 2000 1 1 0 1 0 1 1 0 1 0 0 1 0 0 0 0 0 1 1 0 1 1 0 1 0 0 0 0 1 0 1 1 0 1 0 Pak 386 1 1 1 0 0 1 0 1 0 1 0 0 1 0 0 1 0 1 1 0 1 1 0 0 1 1 0 0 1 1 0 0 0 1 0 Pusa 1121 0 1 0 1 1 0 1 0 1 0 0 1 0 0 0 1 0 1 0 1 1 1 0 1 0 1 0 0 1 0 1 0 0 1 0 Pusa Sugandha 1 1 1 1 0 1 0 0 1 0 1 0 1 1 0 0 1 0 1 0 1 1 1 0 0 1 1 0 0 1 0 1 0 0 1 0 Pusa Sugandha 2 1 1 1 0 1 0 0 1 0 1 0 0 1 0 0 1 0 1 0 1 1 1 0 0 1 1 0 0 1 0 1 0 1 1 0 Pusa Sugandha 3 1 1 1 0 1 0 0 1 0 1 0 0 1 0 0 1 0 1 0 1 1 1 0 0 1 1 0 0 1 0 1 1 1 1 0 Shaheen Basmati 1 1 0 1 0 1 1 0 1 0 0 1 0 0 0 1 0 1 1 0 1 1 0 1 0 0 0 0 1 0 1 1 0 1 0 Sherbati 1 1 1 1 0 1 0 0 1 0 1 0 0 1 0 0 1 0 1 1 0 1 1 0 0 1 1 0 0 1 0 1 0 0 1 0 Supra 1 1 1 1 0 1 0 1 0 1 0 1 0 0 0 1 0 1 1 0 1 1 0 1 0 1 0 0 1 1 1 1 1 1 0 Sherbati 2 1 1 1 0 1 0 0 1 0 1 0 1 1 0 0 1 0 1 1 0 1 1 0 0 1 1 0 0 1 1 0 0 0 1 0 Azucena 0 1 0 1 0 1 1 0 0 1 1 0 0 0 0 0 0 1 0 1 1 0 1 0 1 0 1 1 1 0 1 1 0 0 1 IR64 0 1 1 0 1 0 0 1 0 0 0 1 0 0 0 1 0 1 1 0 1 1 0 0 1 1 0 0 1 0 1 1 0 1 0 Kalinga III 1 1 1 0 1 0 0 1 0 1 0 1 1 0 0 1 0 1 1 0 1 0 0 0 1 1 0 0 1 0 1 0 1 1 0 Sugandha-1 1 1 1 0 1 0 0 1 0 1 0 0 1 0 0 1 0 1 1 0 1 1 0 0 1 1 0 0 1 0 1 0 0 1 0 KDML 105 1 1 0 1 1 0 0 1 0 1 0 0 0 1 0 1 0 0 1 0 1 0 0 0 1 0 1 0 1 0 1 0 1 1 0

Page 24 of 27 Appendix 3. Database of alleles detected with Rim2/Hipa markers

Variety A A A A A A A A A A B B B B B B B B B B B B B B B C C C C C C C C C C C C D ------1 2 3 4 5 6 7 8 9 1 1 2 3 4 5 6 7 8 9 1 1 1 1 1 1 1 2 3 4 5 6 7 8 9 1 1 1 1 0 0 1 2 3 4 5 0 1 2 Bas 370 0 0 0 0 0 0 0 0 0 0 1 1 1 1 0 0 1 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 1 1 0 0 1 Type 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Bas 217 1 1 0 1 0 0 0 0 1 1 1 0 1 1 0 0 1 0 0 0 0 0 1 0 0 1 1 0 0 0 0 0 0 1 1 0 0 1 Ranbir 1 1 0 1 0 0 0 0 1 1 1 0 1 1 0 0 1 0 0 0 0 0 1 0 0 1 1 0 0 0 0 0 0 1 1 0 0 1 Tarori 1 1 0 1 0 0 0 0 0 1 1 0 1 1 0 0 1 0 0 0 0 0 1 0 0 1 1 0 0 0 0 0 0 1 1 0 0 1 Bas 386 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Kernal 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 1 1 0 0 0 Pusa Bas 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Super 1 1 0 1 0 0 0 0 0 0 1 0 1 1 0 0 1 0 0 0 0 0 1 1 0 1 1 0 0 0 0 0 0 1 1 0 0 1 Bas 198 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Bas 385 1 1 0 1 1 1 0 0 1 1 1 0 1 1 0 0 1 0 0 0 0 0 1 1 0 1 1 0 0 1 0 0 0 1 0 0 0 1 Kastori 1 0 0 0 1 1 0 1 1 1 1 0 0 1 0 0 1 1 0 1 0 0 1 0 1 1 1 0 0 1 1 0 1 1 0 0 1 1 Haryana Bas 1 0 1 0 1 0 1 0 1 1 1 0 1 1 0 1 1 0 1 0 0 0 1 0 0 1 1 0 0 1 1 1 1 1 0 0 0 1 Mahi Sugandha 0 0 0 0 0 0 0 0 0 0 1 1 1 0 1 0 0 0 0 0 1 0 0 0 0 0 0 1 1 1 1 0 0 1 0 0 0 1 Punjab Basmati 1 1 0 1 0 0 0 0 1 1 1 0 1 1 0 0 1 0 0 0 1 1 1 0 0 1 1 0 0 1 0 1 0 1 0 1 1 1 Basmati 2000 0 0 0 0 0 1 0 0 0 1 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 1 0 0 0 1 1 0 0 1 Shaheen 0 0 0 0 0 0 0 0 0 0 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 1 0 0 0 1 Sherbati 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 Mughad 1 0 0 1 1 1 1 1 0 1 1 0 1 1 1 0 1 0 0 1 0 1 0 0 0 1 1 0 1 1 1 1 0 1 1 1 0 1 Sughandha Pak 386 1 0 0 0 1 0 0 0 0 0 1 0 1 1 1 0 0 0 0 1 0 0 0 0 0 1 1 0 1 1 0 0 0 1 0 0 0 1 Superfine 1 0 0 0 1 0 0 0 0 0 1 0 1 1 0 0 0 0 0 1 0 0 0 0 0 1 1 0 1 1 1 0 0 1 0 0 0 1 Pusa Sugandha 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1 1 0 0 1 1 0 0 1 Yamini 1 1 0 1 0 0 0 0 1 1 1 0 1 1 0 0 1 0 0 0 0 0 1 0 0 1 1 0 0 0 0 0 0 1 1 1 0 1 Azucena 1 0 0 0 1 0 1 0 0 0 1 IR64 0 0 0 0 1 1 1 0 0 0 0 Kalinga III 0 0 0 0 1 0 0 0 1 1 1 Sugandha 1 1 0 1 1 1 1 0 0 0 1 1

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Appendix 4. Summary of results from all electrophoresis runs for validation of Quantitative tests. Two different sets of standards (B and C) were used.

Lab FDNAS WSS WSS FDNAS WSS WSS FDNAS RM201 RM201 RM201 INDEL INDEL method RM201 B C C B C INDEL Platform CEQ Agilent Agilent Agilent Agilent Agilent Agilent Results form microsatellite Sample mean s.d. mean s.d mean s.d mean s.d mean s.d mean s.d mean s.d variety test A complex mixture that includes several permitted varieties (possibly Basmati 370, Basmati 217 and Ranbir) and 2861 1.7 1.61 7.2 0.72 9.9 1.43 9.2 3.68 7.4 2.79 7.2 2.48 7.8 1.26 non-permitted Contains a high proportion of Pak 386 3021 30.9 1.67 58.9 10.02 39.0 5.85 33.3 1.48 57.8 26.93 58.6 11.27 33.2 8.73 Mixture of Pusa and Super plus some non- 3028 2.0 1.88 5.2 0.68 4.0 0.12 5.4 0.49 1.8 1.23 3.0 1.42 1.6 0.61 basmati Super plus non- permitted variety similar to Mugad 3101 19.7 1.28 29.0 2.80 23.7 3.18 21.5 1.12 6.4 0.95 5.2 1.53 4.6 0.71 Sugandha

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Appendix 5. Standard operating procedures developed in this project.

SOP1 STANDARD OPERATING PROCEDURE FOR Word file THE QUANTITATIVE ANALYSIS OF ADULTERATION OF BASMATI RICE WITH THE VARIETIES SHERBATI, MUGAD SUGANDHA, PAK 386 OR SUPERFINE USING THE AGILENT 2100 BIONALYSER AND A MICROSATELLITE PCR MARKER

SOP2 STANDARD OPERATING PROCEDURE FOR Word file THE QUANTITATIVE ANALYSIS OF ADULTERATION OF BASMATI RICE WITH THE VARIETIES SHERBATI OR PAK 386 USING THE AGILENT 2100 BIONALYSER AND A PCR MARKER BASED ON AN INDEL SEQUENCE

SOP3 STANDARD OPERATING PROCEDURE FOR Word file THE DETECTION OF NON-PERMITTED RICE VARIETIES BY INDEL-PCR ANALYSIS USING THE AGILENT 2100 BIONALYSER

Appendix 6. Figures (see accompanying .pdf file)

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