IRRI Rice Seminar Series

Roslen Anacleto
Current position: Senior Associate Scientist Education and training
2009, Projects in Controlled Environments (PRINCE2), Singapore 1997, MS in Computer Science, University of the Philippines, Los Baos, Laguna 1991, BS in Computer Science, University of the Philippines, Los Baos, Laguna

Work experience
2009 2005 2003 2000 1998 1991 present, Senior Associate Scientist, Grain Quality and Nutrition Center, IRRI 2009, Programmer, Experiment Station, IRRI 2005, Assistant Professor and Head, MIS Unit, University of the Philippines Open University 2003, IT Consultant, various local and international clients 2000, Academic Head, Systems Technology Institute, Cagayan De Oro City 1998, Assistant Professor, Central Mindanao University, Musuan, Bukidnon

Research highlights
Keyless data entry for grain quality evaluation Implemented barcoding for sample labeling and tracking at the quality evaluation laboratory Currently working on a LIMS implementation for GQNC Member of the IRRI Experiment Station ISO 14001:2004 certification working group Conversion of various databases at the Experiment Station from MS Access silos to a true relational database

Improving our knowledge of rice quality

Roslen Anacleto, Rosario Jimenez, Adoracion Resurreccion, Jeanaflor Crystal Concepcion, Venea Dara Daygon, Melissa Fitzgerald

Tools to measure quality do not give breeders accurate enough data about eating quality.
Current tools to measure amylose, gel temp and gel consistency are not globally standardised. Consumers do not have consistent adjectives to describe the quality of rice they like. New analytical technologies facilitate a surge on new understanding of sensory quality. Improved information and communication technologies make global collaboration routine rather than a challenge. We are in an era where genotyping is becoming routine. Serious investment into new, accurate phenotyping tools would greatly help genotyping work.
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The International Network for Quality Rice

Understanding quality needs collaboration

4

The INQR
80 members from almost every rice quality evaluation program. NARES

Researchers who work on rice quality.

PS

ARI

Companies who develop instruments for measuring traits of rice quality.

5

First INQR meeting 2007

Survey undertaken to identify priorities for INQR collaboration. Top priority was to fix the method to measure amylose by
Standardising the method amongst all the rice quality labs Bringing new science to move from apparent to actual amylose to increase accuracy of amylose measurement.

The amylose project then began with 30 labs, and by the time it was concluded in 2010, there were 45 labs and the International Standards Organisation involved.

Organisational levels of starch, and where amylose fits

Semi-crystalline zone Amorphous zone Crystalline lamella Amorphous lamella

Amylose Molecule Amylopectin Molecule Debranched Amylopectin Molecule Blocklets

Compound granules
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Improving the global measurement of amylose content

Singing from the same songsheet

There are many examples of different amylose contents reported for the same variety. Rice germplasms are exchanged all over the rice world together with its passport data --- quality data. Quality data gives an end-user/breeder expectations. If the sending center measures amylose one way and the receiving center another, then the expectations of the end-user are not met. It is important that everyone gets the same values for quality data from the same set of samples.

Standardising the measurement of amylose

Round 1 (Determine baseline) 17 samples 30 laboratories, INQR each lab used their own method amylose values and details of methods collected 8 methods to test variability calibrated standards and values provided 17% of values were outliers 3 out of 44 labs had no outliers 1 method which will be the ISO standard 5 standards calibrated by 6 labs (incl. Japan) 44 laboratories, INQR 10 18 samples

Round 2 (Choose best method)

Round 3 (Precision and proficiency tests)

Round 1 Determining the baseline

Enormous variability. Sample 3: 4 40% Waxies, which have no amylose: 0 12% IR64 is sample 11: 15 30% KDML is sample 13: 10 25% Several different methods in operation
45 40

Goami 2

Amylose Content (%)

35 30 25 20 15 10 5 0 -5 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

IR 24

IR 64

Sample Number
11

Round 1: Major source of variability

The current ISO standard specifies two ways of making a standard curve Potato amylose Calibrated rice varieties 15 labs used potato and 15 used calibrated rice. Variability is much higher for labs that used potato.
40 35

Amylose Content (%)

30 25 20 15 10 5 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Sample Number
12

Amylose content using 2 brands of potato amylose, done in one laboratory, by one person
40 35

Amylose Content (%)

30 25 20 15 10 5 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 13 17

Brand 1 Brand 2

Sample Number

Round 2 Choosing the best method

44 participating labs 8 methods were used to test variability due to wavelengths, to standing time, and to methods of calibrating the standards Two wavelengths were compared: 620 nm and 720 nm Two standing time were compared: 0 minutes and 30 minutes. Two methods of calibrating the standards were compared: by iodine (using ISO 6647-1:2007(E)) and also by Size Exclusion Chromatography (by 5 INQR members). Eliminated potato amylose and used 4 types of rice varieties according to known amylose contents: waxy, low, intermediate, and high

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The standard: iodine or SEC

Iodine Iodine is easy for every lab to do Iodine needs to be calibrated against something reference standards, known rice varieties, potato amylose It is not a direct measure of amylose We also know that iodine binds to amylopectin. SEC SEC is a direct method to quantify the chains that belong to amylose vs the chains that belong to amylopectin. It is not a routine method In a network such as ours, it is possible to calibrate standards by SEC and distribute them as reference material. 15

Moving from iodine to SEC calibration values

Using the standard curve made from iodine values led to higher values for all samples. The average difference is not proportional Low: 3.5% Inter: 5.1% High: 6.3% Amylopectin contribution increases with increasing amylose.
45 40

Values from iodine curve Values from SEC curve

Amylose Content (%)

35 30 25 20 15 10 5 0 -5 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Sample number

16

Testing the relationship between amylose content by SEC and by iodine using both calibration methods
Calibration values by iodine
35
30

Calibration values by SEC

Amylose content % by iodine

30

25

25

20

20
15

15
10

10
5

5
0

0
-5.0 0.0 -5 5.0 10.0 15.0 20.0 25.0 30.0

0 -5

10

15

20

25

30

35

Amylose content % by SEC

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Round 1 vs Round 2 (620-0, 620-30, 720-0, 720-30)

Goami 2
40

30

Amylose content (%)

20

10

IR 24
0
0 1 2 3 4 5 6 7 8 9 10

IR 64
11 12 13 14 15 16 17

Sample No. -10

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Other difference is wavelength

Spectrophotometer scan showing 620nm still has lots of AP while at 720nm AP is almost invisible By using the higher wavelength, we are significantly reducing the contribution from amylopectin Amylose = AM Amylopectin = AP
0.5

AM
0.4

Absorbance

0.3

90% AP
0.2

70% AP
0.1 0 480 520 560 600 640 680 720 760 800

Wavelength (nm)
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Selecting the best method

Chain-length dist of DB starch

Technically, the best method should be the one that more accurately reports amylose. This means calibration by SEC and making the AP/iodine complex invisible, so a wavelength of 720 nm. AM-I and AP-I signatures fade through time, thus a standing time of 0 min was chosen.

0.7

Normalized DRI

0.3

0.4

0.5

High Intermediate Low Waxy

0.6

0.1

0.2

AM

AP

10000 10000

1000 1000

100 100

10 10

11
20

Chain-Length

Performance
SEC calibrated standards measured at different wavelengths at 0 min
Goami 2 Goami 2

IR 24

IR 64

IR 24

IR 64

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Round 3 Precision and proficiency tests

Rigid test for the 720nm_0_ SEC method Five standards were used from each category, each with allele of the Waxy gene: waxy, very low, low, intermediate, and high Six INQR labs with SEC calibrated the standards Note that SEC measures true AC so lower values are shown in the chart
Amylose measurements done by six INQR labs based on SEC calibrated values
30 25

Amylose Content (%)

20 15 10 5 0 0 1 2 3 4 5
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Standard

Repeatability and reproducibility conditions

Within-Lab Repeatability conditions Between-Labs Reproducibility conditions

Using the same test method Measuring on identical material At the same laboratory By the same operator Using the same equipment At different laboratories By different operators Using different equipment

Within a short interval of time

Tang Luping and Bjrn Schouenborg, 2000, Methodology of Inter-comparison Tests and Statistical Analysis of Test Results Nordtest project No. 1483-99, p8.

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Repeatability and reproducibility conditions

Within-Lab Between-Labs

Procedure
1 5 SEC calibrated standards, 720 nm wavelength, 0 min standing time 18 samples from the same source distributed to 44 participating labs Agreed time frame for conducting the experiment Each lab assigned the same technician and equipment
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Repeatability conditions

Reproducibility conditions

Using the same test method Measuring on identical material At the same laboratory By the same operator Using the same equipment At different laboratories By different operators Using different equipment

Within a short interval of time

Boxplot of results

Amylose Content (%)

IR 74 IR 64 Seraup 27

High Intermediate Low Very low Waxy

RC 18

Sample

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Use ISO standard for interlaboratory ring tests

ISO 5725-2:1994, Accuracy (trueness and precision) of measurement methods and results Part 2: Basic method for the determination of repeatability and reproducibility of a standard measurement method

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Statistical analysis flowchart

1 Collate results Form A

ISO 5725-2:1994(E)
Excel worksheets

2 Discard discordant data 3

Compute replication means and std. dev.

Form B, Form C 5

Discard or correct outliers Check the results for consistency and outliers**
Outliers found?

Yes 6 Recalculate replication means and std. dev.

No **ISO 5725 suggests two ways of checking for consistency and outliers: 1. Graphical Mandels k and h statistics 2. Numerical Cochrans and Grubbs tests 7

Compute the general mean, repeatability std. dev., and reproducibility std. dev., etc.

Formulas are given in the standard Statistical Analysis Report

8 Report the results

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Using graphical technique in detecting outliers (Mandels k and h Tests)

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Within-laboratory consistency statistic

According to ISO 5725-2:1994(E)
5.00

4.50

4.00

1% significance 5% significance

Sample 1 Sample 2 Sample 3 Sample 4

Mandels k-statistic

3.50

k=2.10
3.00

Sample 5 Sample 6 Sample 7 Sample 8 Sample 9 Sample 10 Sample 11

k=1.72
2.50

2.00

Sample 12 Sample 13 Sample 14 Sample 15 Sample 16 Sample 17 Sample 18

1.50

1.00

0.50

0.00 lab1 lab2 lab4 lab6 lab7 lab10 lab11 lab12 lab13 lab15 lab17 lab18 lab21 lab22 lab23 lab24 lab25

Laboratory

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Within-laboratory consistency statistic (continuation)

According to ISO 5725-2:1994(E)
5.00

4.50

4.00

1% significance 5% significance

Sample 1 Sample 2 Sample 3

Mandels k-statistic

3.50

Sample 4

k=2.10 2.10
3.00
1.72 k=1.72

Sample 5 Sample 6 Sample 7 Sample 8 Sample 9 Sample 10 Sample 11

2.50

2.00

Sample 12 Sample 13 Sample 14 Sample 15 Sample 16 Sample 17 Sample 18

1.50

1.00

0.50

0.00 lab26 lab27 lab28 lab29 lab30 lab32 lab33 lab36 lab37 lab38 lab41 lab42 lab46 lab47 lab54 lab64 lab65

Laboratory

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Within-laboratory consistency statistic (outliers)

According to ISO 5725-2:1994(E)
5.00

4.50

4.00

1% significance 5% significance

Sample 1 Sample 2 Sample 3

Mandels k-statistic

3.50

Sample 4

k=2.10
3.00

Sample 5 Sample 6 Sample 7 Sample 8 Sample 9 Sample 10 Sample 11

k=1.72
2.50

2.00

Sample 12 Sample 13 Sample 14 Sample 15 Sample 16 Sample 17 Sample 18

1.50

1.00

0.50

0.00 lab4 lab6 lab10 lab13 lab26 lab28 lab30 lab47 lab54 lab65

Laboratory

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Between-laboratory consistency statistic

According to ISO 5725-2:1994(E)
6.00

5.00

h=2.45 k=1.91 1% significance 5% significance

Sample 1 Sample 2 Sample 3

4.00

3.00

Mandels h-statistic

2.00

Sample 4 Sample 5

1.00

Sample 6 Sample 7 Sample 8

0.00

Sample 9 Sample 10

-1.00

Sample 11

lab1

lab2

lab4

lab6

lab7

lab10

lab11

lab12

lab13

lab15

lab17

lab18

lab21

lab22

lab23

lab24

lab25

Sample 12 Sample 13

-2.00

Sample 14 Sample 15

-3.00

k=-1.91 h=-2.45

Sample 2 Sample 3 Sample 4 Sample 5

Sample 16 Sample 17 Sample 18

-4.00

-5.00

-6.00

Laboratory

32

Between-laboratory consistency statistic (continuation)

According to ISO 5725-2:1994(E)
6.00

5.00

h=2.45 k=1.91 1% significance 5% significance

Sample 1 Sample 2 Sample 3

4.00

3.00

Mandels h-statistic

2.00

Sample 4 Sample 5

1.00

Sample 6 Sample 7 Sample 8

0.00

Sample 9 Sample 10

-1.00

Sample 11

lab26

lab27

lab28

lab29

lab30

lab32

lab33

lab36

lab37

lab38

lab41

lab42

lab46

lab47

lab54

lab64

lab65

Sample 12 Sample 13

-2.00

Sample 14 Sample 15 Sample 16

-3.00

k=-1.91 h=-2.45

Sample 17 Sample 18

-4.00

-5.00

-6.00

Laboratory

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Between-laboratory consistency statistic (outliers)

According to ISO 5725-2:1994(E)
6.00 5.00 4.00 3.00

h=2.45 k=1.91

1% significance 5% significance
Sample 1 Sample 2 Sample 3 Sample 4

Mandels h-statistic

2.00 1.00 0.00 -1.00 lab12 -2.00 -3.00 -4.00 lab26 lab30 lab54 lab65

Sample 5 Sample 6 Sample 7 Sample 8 Sample 9 Sample 10 Sample 11 Sample 12 Sample 13 Sample 14 Sample 15

k=-1.91 h=-2.45

Sample 16 Sample 17 Sample 18

-5.00 -6.00

Laboratory

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Summary of outliers using Mandels k and h statistics

Lab designations Sample 1 2 3 4 5 6 7 8 9 4 30, 47 28 26 13, 65 26, 30, 65 6, 10 4, 26 26, 28, 65 Within-Lab Between-Lab 26, 54 12, 30, 54 12 12, 54 12, 30 30 30 30 30 Sample 10 11 12 13 14 15 16 17 18 Lab designations Within-Lab 28, 30, 47 28, 54 47 26 26, 65 30 30, 65 26, 47, 65 30 Between-Lab 30 12, 65 54, 65 30 30 30 30 30 30

17 14 Total within-lab outliers - 33 (10 unique labs) Total between-lab outliers - 25 (5 unique labs) Total outliers - 58

16

11

4 labs in common 26,30, 54, 65

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Using numerical technique in detecting outliers (Cochrans and Grubbs Tests)

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Repeatability/Reproducibility Statistics (samples 1-9)

Sample Number of valid laboratories General mean Repeatability std. dev. (sr) Reproducibility std. dev. (sR) Repeatability covariance (%) Reproducibility covariance (%) Repeatability limit Reproducibility limit Number of excluded outliers Outlier/Reasons 1 34 3.560 0.605 2.549 16.986 71.607 1.693 7.138 0 2 28 0.809 1.240 5.592 8.576 2.264 3.472 6 12g, 26g, 30g, 47c, 54g, 65g 3 27 0.782 2.272 2.817 8.182 2.190 6.362 7 4c, 12g, 18c, 26g, 28c, 30g, 54g 4 29 0.870 1.952 3.128 7.023 2.435 5.467 5 4c, 12g, 26c, 54g, 65c 5 30 0.570 1.179 5.250 10.855 1.596 3.300 4 12g, 13c, 30c, 65c 6 30 0.568 1.438 3.217 8.150 1.590 4.027 4 12c, 26c, 30c, 65c 7 32 -1.795 0.437 1.260 -24.362 -70.189 1.224 3.527 2 12g, 30g 8 31 0.920 1.282 3.891 5.425 2.575 3.590 3 4c, 26c, 30g 9 30 1.021 2.347 3.427 7.879 2.859 6.573 4 26c, 28c, 30g, 65c

14.459 27.769 27.802 10.858 17.646

23.635 29.792

Note on the suffixes: - c denotes rejection due to Cochrans statistic - g denotes rejection due to Grubbs statistic 37

Repeatability/Reproducibility Statistics (samples 10-18)

Sample Number of valid laboratories General mean Repeatability std. dev. (sr) Reproducibility std. dev. (sR) Repeatability covariance (%) Reproducibility covariance (%) Repeatability limit Reproducibility limit Number of excluded outliers Outlier/Reasons 10 31 13.458 0.697 1.737 5.180 12.904 1.952 4.862 11 31 6.589 0.627 1.132 9.512 17.178 1.755 3.169 12 31 6.516 0.697 1.014 10.694 15.566 1.951 2.840 13 32 1.114 1.785 6.593 10.568 3.119 4.999 14 31 0.972 1.747 4.725 8.492 2.722 4.892 15 33 0.946 2.792 3.569 10.532 2.649 7.818 1 30c 16 31 -1.305 0.375 0.929 -71.187 1.051 2.601 3 30c, 54g, 65c 17 29 -1.387 0.536 1.138 18 32 29.555 1.223 3.449 4.139 3.425 9.658

16.893 20.574 26.509

-28.766 -38.673 1.501 3.186

-82.071 11.670

3 3 3 2 3 28c, 12g, 54c, 47c, 54g, 26c, 30g 26c, 30c, 47c 65g 65g 30g, 65c

5 2 26c, 30g, 54g 30g, 47c, 54g, 65c

Note on the suffixes: - c denotes rejection due to Cochrans statistic - g denotes rejection due to Grubbs statistic

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Summarized results
Laboratories with repeatability problems -> lab 26, 28, 30, 47, 54, 65 Laboratories with reproducibility problems -> lab 12, 26, 30, 54, 65 Laboratories with both problems! -> lab 26, 30, 54, 65

4 out of 34

11.76%
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Analysis of lab proficiency

With repeatability issues : 26, 28, 30, 47, 54,65 With reproducibility issues : 12, 26, 30, 54, 65

IRRI GQNC

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The result of the amylose project

Completion of the 1st INQR standardisation project. New method and calibrated standards were launched by ISO at the INQR symposium at the IRRC28 in November 2010 in Hanoi. Providing calibrated standards and testing to achieve uniform measurement is only possible in a collaborative network. Each year, newly grown standards and calibration values (from 6 labs) will be distributed to INQR members. We are getting inquiries from companies who want to buy the standards...
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What this means for breeders

35 % Amylose by New Method 30 25 20 15 10 5 0 -5 -5 0 5 10 15 20 25 30 35

VL

VH

% Amylose by Old Method

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So then
Previously the categories were waxy, very low, low, intermediate, high. That will not change. Current work is done in establishing the relationship between the values obtained using both methods This relationship would be determined through rigorous computational methods on precise data sets Any immutable relationship will be used as correction factor should breeders/rice scientists refer to old values

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Timeline to integrate the new method

ISO has approved the method to be the new standard. At IRRI we are currently collecting data from both methods. We intend to switch fully to the new method by the end of this year. Other INQR labs are following this timeline.

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Conclusions
Rigorous analytical and computational procedures were done to move from apparent to actual amylose content A platform for collaborative work is in place --- INQR Current classifications for amylose content: waxy, very low, low, high, and very high will not change Work is in progress to determine the relationship between old and new amylose values A series of other collaborative work will be undertaken to improve existing tools for measuring quality Current research activities at the Grain Quality and Nutrition Laboratory progressively improve our understanding of rice quality
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Acknowledgement
The authors thank The INQR participating laboratories and other contributing members PBGB breeders and GRC for providing quality samples The GQNC technicians: Teodie, Johnny, Boy, Dennis, Leah, Ferdie, and Lucy for their usual outstanding support

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