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fragrant (Gulfmont) parents which were segregating for fragrance. The assay also .... A band of 257 bp indicates a variety or individual is homozygous fragrant.
New Markers for Australian Rice Improvement

A report for the Rural Industries Research and Development Corporation by Robert Henry and Daniel Waters

November 2006 RIRDC Publication No 06/123 RIRDC Project No USC-6A

© 2006 Rural Industries Research and Development Corporation. All rights reserved.

ISBN 1 74151 386 3 ISSN 1440-6845

New markers for Australian rice improvement Publication No. 06/123 Project No. USC-6A The information contained in this publication is intended for general use to assist public knowledge and discussion and to help improve the development of sustainable industries. The information should not be relied upon for the purpose of a particular matter. Specialist and/or appropriate legal advice should be obtained before any action or decision is taken on the basis of any material in this document. The Commonwealth of Australia, Rural Industries Research and Development Corporation, the authors or contributors do not assume liability of any kind whatsoever resulting from any person's use or reliance upon the content of this document. This publication is copyright. However, RIRDC encourages wide dissemination of its research, providing the Corporation is clearly acknowledged. For any other enquiries concerning reproduction, contact the Publications Manager on phone 02 6272 3186.

Researcher Contact Details Professor Robert Henry

Centre for Plant Conservation Genetics, Southern Cross University PO Box 157 LISMORE NSW 2480 Phone: (02) 6620 3010 Fax: (02) 6622 2080 [email protected]

In submitting this report, the researcher has agreed to RIRDC publishing this material in its edited form. RIRDC Contact Details Rural Industries Research and Development Corporation Level 2, 15 National Circuit BARTON ACT 2600 PO Box 4776 KINGSTON ACT 2604 Phone: Fax: Email: Web:

02 6272 4819 02 6272 5877 [email protected]. http://www.rirdc.gov.au

Published in November 2006 Printed on environmentally friendly paper by Canprint

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Foreword Molecular markers are pieces of DNA that flag the presence or absence of particular traits and allow selection to be undertaken on the basis of a simple laboratory test of a small tissue sample, rather than direct measurement of the character itself. These tests offer several advantages including being free from the confounding affects of the environment, a single platform test for multiple traits and early generation testing, often before the trait can be measured by other means. The objectives of this project were to identify, adapt and evaluate molecular markers for use in the Australian rice-breeding program. The work reported here involved the evaluation of germplasm and development of markers for the major fragrance gene (fgr), rice starch gelatinisation temperature, blast disease resistance and hybrid rice. This project was funded from industry revenue which is matched by funds provided by the Federal Government. This report, an addition to RIRDC’s diverse range of over 1500 research publications, forms part of our Rice R&D program, which aims to improve the profitability and sustainability of the Australian rice industry. Most of our publications are available for viewing, downloading or purchasing online through our website: • •

downloads at www.rirdc.gov.au/fullreports/index.html purchases at www.rirdc.gov.au/eshop

Peter O’Brien Managing Director Rural Industries Research and Development Corporation

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Acknowledgments The Centre for Plant Conservation Genetics, Southern Cross University acknowledges the contribution of Prof. Qingsheng Jin of Crop Research Institute, ZAAS, Hangzhou, China for assistance with the development of the molecular marker for rice fragrance. Dr Russell Reinke, Dr Melissa Fitzgerald and the rice breeding team of the Yanco Agricultural Institute were integral to the completion all work reported here.

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Abbreviations IRRI; International Rice Research Institute PCR; Polymerase Chain Reaction SNP; Single Nucleotide Polymorphism SSR; Simple Sequence Repeat 2AP; 2-acetyl-1-pyrroline BAD2; betaine aldehyde dehydrogenase 2 GT; Gelatinisation temperature SSIIa; Starch synthase IIa SD; Standard deviation M; Mean

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Contents Foreword ..................................................................................................................................iii Acknowledgments.................................................................................................................... iv Abbreviations............................................................................................................................ v Executive Summary ............................................................................................................... vii 1.

A perfect molecular marker for fragrance ..................................................................... 1 1.1 Background....................................................................................................................... 1 1.2 Approach and outcomes ................................................................................................... 2 1.3 Significance and utility of outcomes ................................................................................ 4

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Molecular markers for rice starch gelatinisation temperature .................................... 5 2.1 Background....................................................................................................................... 5 2.2 Approach and outcomes ................................................................................................... 5 2.3 Significance and utility outcomes................................................................................... 11

3. Markers for disease resistance .......................................................................................... 12 3.1 Background..................................................................................................................... 12 3.2 Approach and practical significance of outcomes .......................................................... 12 4. Molecular markers for hybrid rice................................................................................... 14 References ............................................................................................................................... 16

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Executive Summary Molecular markers are pieces of DNA that flag the presence or absence of particular traits and allow trait selection to be undertaken on the basis of a simple laboratory test of a small tissue sample, rather than direct measurement of the character itself. These tests offer several advantages including being free from the confounding affects of the environment, a single platform test for multiple traits and early generation testing, often before the trait can be measured by other means. The objectives of this project were to identify, adapt and evaluate molecular markers for use in the Australian rice-breeding program. The flavour and fragrance of Basmati and Jasmine style rice have been associated with increased levels of the compound, 2-acetyl-1-pyrroline (2AP). Rice breeders require a simple, accurate and inexpensive method to distinguish fragrant from non-fragrant rice within a breeding program if they are to efficiently develop fragrant rice varieties and take advantage of the premium consumers are willing to pay for fragrant rice. A number of methods have been utilised to assist breeders in selecting fragrant rice but they have limitations when processing large numbers of samples. A perfect molecular marker is a marker that is within the gene that codes for a trait. Workers at Southern Cross University found that an eight base pair deletion and three SNPs in a gene encoding a betaine aldehyde dehydrogenase 2 (BAD2) homolog was the likely cause of fragrance in Jasmine and Basmati style rice. Non-fragrant rice varieties possess what appears to be a fully functional copy of the gene encoding BAD2 while fragrant varieties possess a copy of the gene encoding BAD2 which contains the deletion that presumably disables the BAD2 enzyme. This polymorphism provided an opportunity for the construction of a perfect marker for fragrance in rice. A competitive allele specific PCR assay for the polymorphism was developed and accurately predicted the fragrance status of each of the individuals within a population of 168 plants derived from a cross of fragrant (Kyeema) and nonfragrant (Gulfmont) parents which were segregating for fragrance. The assay also accurately predicted the fragrant status of 88 fragrant and non-fragrant rice varieties. The assay can be used to detect heterozygous individuals and mixed populations. Rice starch has a semi-crystalline structure which is disrupted by cooking, transforming it into a softer edible gel like material. The temperature at which rice starch gelatinises is an important component of rice eating quality because it is associated with the cooking time and texture of cooked rice. Although rice starch gelatinisation temperature (GT) is genetically determined, it also displays a high level of variability in response to the influence of the environment in which the plant is grown. It was known the major gene that controls rice amylopectin structure and starch GT codes for soluble starch synthase IIa (SSIIa), it was not known how many versions of the gene were found in commercial rice varieties and which were responsible for high or low GT starch. DNA sequence analysis of 70 rice varieties that differed by GT allowed identification of DNA differences which led to amino acid changes that were associated with two statistically significant GT classes. The high GT class (average GT of 78 oC) was genotype G/GC only, while the low GT class (average GT of 70 oC) was either genotype A/GC or G/TT. A competitive allele specific PCR was chosen as the assay for this trait. The nature of the mutation and the kinetics of each PCR meant each possible DNA difference was assayed independently. The markers detect all possible genotypes which impact upon GT. The parents within a breeding program can be screened in order to identify which versions of SSIIa each carry. Using the information derived from this process, rice breeders will be able to identify undesirable genotypes to be removed from the breeding program. Based on the experience of the Californian rice industry which suffered its first blast outbreak in 1996 after being blast free for 85 years and experience with other crops, breeding rice varieties not carrying any resistance to internationally important diseases is undesirable. If a disease was introduced to the Australian rice industry, the consequences could be serious. For those diseases which do not currently exist in the Australian rice industry, use of molecular markers tightly linked to resistance genes avoids the need for selection using a pathogen challenge, a technique which would expose the industry to obvious risk because the pathogen would be introduced into the country. Molecular markers also offer

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other benefits including allowing the rice breeder to identify plants in the breeding program which have broken the linkage between disease resistance genes and undesirable genes, determining if a resistant plant derives its resistance from one or more resistance genes, and enabling more than one disease resistance gene to be efficiently incorporated into a single line of rice. This is particularly useful as incorporation of several resistance genes has been shown to reduce the likelihood of pathogens overcoming resistance. It has been determined that blast poses the greatest threat to the Australian rice industry. Under blast favourable conditions, outbreaks have caused crop losses of up to 60%. Because of this risk, varieties which carry a range of genes for blast resistance were procured by the rice breeding program as the first step in initiating a program of pre-emptive rice blast disease resistance breeding. Rice varieties BL14 and BL24 were chosen as donors of resistance as they carry blast resistance genes which are well characterised at the molecular level and hence allow accurate application of molecular marker technology. Hybrid rice lines are used widely in China because they yield up to 20% higher than traditional inbred rice lines. The type of hybrid rice system most suited to the Australian rice growing regions is the cytoplasmic male sterile or three line, system. For the system to work efficiently it is important no B line rice contaminates the A line as the yield will be reduced due to the non-hybrid self fertilised low yielding B line diluting the high yielding hybrid seed. A simple laboratory test has been developed which distinguishes between the A and B lines so A line purity can be checked without having to grow the rice. We have checked and found the laboratory test that distinguishes between the A and B lines works with 12 hybrid rice lines that were imported from China.

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1. A perfect molecular marker for fragrance 1.1 Background Consumers are willing to pay a premium for fragrant rices. Rice breeders need a simple, accurate and inexpensive method to distinguish fragrant from non-fragrant rice within the breeding program if they are to develop fragrant rice varieties suited to Australian rice growing conditions. The flavour and fragrance of Basmati and Jasmine style rice have been associated with increased levels of the compound, 2-acetyl-1-pyrroline (2AP) [1,2,3,4]. A number of sensory methods have been developed to assist breeders in selecting fragrant rice but they have limitations when processing large numbers of samples. For example, tasting individual grains is one of the original methods for the selection of fragrant rice varieties within the Australian breeding program [5] and is still the principal means of identifying fragrance in many breeding programs worldwide. However, the objective evaluation of fragrance using this method is labour intensive, difficult and unreliable. A panel of analysts is required as the ability to detect fragrance varies between individuals. For any individual analyst, the ability to distinguish between fragrant and non-fragrant samples diminishes with each successive analysis because the senses become saturated. In addition, physical damage occurs from abrasions to the tongue which can result from chewing the hard grain. Other methods involve smelling leaf tissue or grains after heating in water or reacting with solutions of KOH or I2-KI [6] but these can cause damage to the nasal passages. An objective method of 2AP identification using gas chromatography is available but the assay requires large tissue samples and is time consuming [2,3]. More recently molecular markers, such as single nucleotide polymorphisms (SNPs) and simple sequence repeats (SSRs), which are genetically linked to fragrance have been developed for the selection of fragrant rice [7]. Although these markers have the advantage of being inexpensive, simple, rapid and only requiring small amounts of tissue, they are only linked with the fragrance gene (fgr) and therefore do not allow prediction of fragrance with 100% accuracy. A perfect molecular marker is a marker that is within the gene that codes for a trait. Workers at Southern Cross University found that an eight base pair deletion and three SNPs in a gene encoding a putative betaine aldehyde dehydrogenase 2 (BAD2) was the likely cause of fragrance in Jasmine and Basmati style rice [8]. Non-fragrant rice varieties possess what appears to be a fully functional copy of the gene encoding BAD2 while fragrant varieties possess a copy of the gene encoding BAD2 which contains the deletion and SNPs, resulting in a frame shift that generates a premature stop codon that presumably disables the BAD2 enzyme (Figure 1.1). This polymorphism provided an opportunity for the construction of a perfect marker for fragrance in rice.

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Figure 1.1. Schematic diagram of the BAD2 encoding gene in rice. Part of the exon seven sequence which harbours the deletion which results in elevated levels of 2AP is shown. The premature stop codon, “TAA”, is shown in red.

1.2 Approach and outcomes The nature of the lesion which causes fragrance and the need for an inexpensive assay meant competitive allele specific PCR would deliver the most efficient assay. Competitive allele specific PCR is a modified PCR where two PCR primers amplify the target region of DNA while one or two primers internal to these primers anneals to internal DNA sequence. This distinguishes between each of two alternative gene sequences. In the case of the gene for fragrance, four primers, two that annealed to sequences common to both fragrant and non-fragrant varieties and external to the mutated region and two that are specific to one of the two known alleles were designed and synthesised (Figure 1.2). The two external primers act as a positive control, amplifying a region of approximately 580 bp in both fragrant (577 bp) and non-fragrant (585 bp) genotypes. Individually, these external primers also pair with internal primers to give products of varying size, depending upon the genotype of the DNA sample. The internal primers, IFAP and INSP (Figure 1.2), anneal only to their specified genotype producing DNA fragments with their corresponding external primer pair, ESP and EAP. Using these four primers in a PCR results in three possible outcomes. In all cases a positive control band of approximately 580 bp is produced. A band of 355 bp indicates a variety or individual is homozygous non-fragrant. A band of 257 bp indicates a variety or individual is homozygous fragrant. When both bands of 355 bp and 257 bp are produced, this indicates an individual is heterozygous or a sample is a mixture of non-fragrant and fragrant plants. The assay was tested on a population of 168 plants derived from a cross of fragrant (Kyeema) and non-fragrant (Gulfmont) parents which were segregating for fragrance. A segregating population has a mixture of individuals that differ by a trait, in this case fragrance. The assay accurately predicted the

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fragrance status of each of the individuals within this population (Figure 1.3). Fragrance was evaluated according to Berner and Hoff [9]. The phenotype of F2 individuals were classified as fragrant, segregating or non-fragrant by tasting dehulled F3 seed. At least 12 F3 seeds from individual F2 plants were chewed individually. F2 plants were rated homozygous fragrant or non-fragrant if all 12 F3 seeds were fragrant or non-fragrant, respectively. F3 seeds from heterozygous F2 plants were expected to contain both fragrant and non-fragrant seeds and so if the sample from a single F2 plant was a mixture of fragrant and non-fragrant, the F2 plant was considered heterozygous.The assay also accurately predicted the fragrant status of 88 fragrant and non-fragrant rice varieties.

577 bp or 585 bp 257 bp ESP

IFAP

INSP

EAP 355 bp

Figure 1.2. Schematic diagram of competitive allele specific assay design for elevated 2AP levels, fragrance, in rice. Primers ESP and EAP amplify a fragment of 577 bp in fragrant rice and 585 bp in non-fragrant rice. In combination with primer ESP, primer IFAP amplifies a fragment of 257 bp in fragrant rice only. In combination with primer EAP, primer INSP amplifies a fragment of 355 bp in non-fragrant rice only.

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positive control band ~580 bp Non-fragrant 355 bp Fragrant 257 bp

Heterozygous individual

Fragrant individual

Non-fragrant individual

Figure 1.3. Agarose gel showing a population segregating for fgr using competitive allele specific PCR assay. The PCR product of approximately 580 bp serves as a positive control and is present in every sample. Fragrant individuals have a second a product of 257 bp in size while non-fragrant individuals give a product of 355 bp in size. Heterozygotes are identified by the presence of all three PCR products.

1.3 Significance and utility of outcomes Heterozygous individuals, plants that carry both the fragrant and non-fragrant version of the gene, can be identified because this assay uses two rather than the usual one internal primer. Heterozygous individuals need to be avoided because they are non-fragrant and give rise to a mixture of fragrant and non-fragrant plants. The assay can also identify mixtures of fragrant and non-fragrant plants which is useful for pure seed maintenance. The assay is robust and utilises DNA from whole seeds which has been isolated using a simple procedure as described by Bergman et al [10] and leaves using a 10 min boiling protocol. The assay uses the most simple DNA size separation apparatus so can operate in any molecular biology laboratory.

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2. Molecular markers for rice starch gelatinisation temperature 2.1 Background Starch is composed of a mixture of two forms of glucose polymer, amylose and amylopectin. Amylose is principally a linear polymer of α(1-4) linked glucose with some α(1-6) linkages. Amylopectin is a more complex mixture due to the extensive branching introduced by many more α(1-6) linkages of the α(1-4) linked chains of glucose. In its native state, rice starch has a semi-crystalline structure which is disrupted by cooking, transforming the starch into a softer edible gel like material. Because it is associated with the cooking time and texture of cooked rice, the temperature at which rice starch gelatinises is an important component of rice eating quality [11]. Although rice starch gelatinisation temperature (GT) is genetically determined, it also displays a high level of variability in response to the influence of the environment in which the rice plant is growing. Starch is synthesised by the activity of several enzymes, each of which occur as a number of different isoforms that display tissue specific expression [12,13]. A link between the GT of rice starch and enzymes of starch bio-synthesis was made when it was found that the major gene that controls rice starch gelatinisation temperature via amylopectin structure, codes for soluble starch synthase IIa (SSIIa) [14]. The evidence this gene affected starch GT in rice was strong, however, it was not known how many versions of the gene were found in commercial rice varieties and which were responsible for high or low GT starch. Umemoto and co-workers [15] identified four versions of the gene that codes for SSIIa. However, gelatinization temperature and chain length distribution were not unique to any version of the gene in that study. Rices of version two and three were composed of both low and high GT types with corresponding structural differences of amylopectin, suggesting the natural variations in the rice SSIIa gene which affect GT had not been fully elucidated. In order to construct a molecular marker for rice GT, each version of the gene had to be identified and its relationship with GT determined. To that end SSIIa was characterised in relationship to starch GT.

2.2 Approach and outcomes The DNA sequence of the SSIIa encoding gene was obtained in the Australian rice varieties Opus, Doongara, and Langi and the sequences aligned and compared to each other and the variety Nipponbare coding sequence found in public data bases. The GT of Nipponbare, Opus, Doongara, and Langi are 68 oC, 69 oC, 74 oC and 78 oC respectively. A single DNA difference (SNP) was identified, a ‘G’ to ‘A’ transition in exon 8. Langi and Doongara carried the ‘G’ allele while Opus and Nipponbare carried the ‘A’ allele. This difference was also identified by Umemoto and co-workers [15]. This SNP results in an amino acid change of methionine (ATG) to valine (GTG). Sequence analysis of a further 66 varieties that differed by GT, allowed identification of another DNA difference which led to an amino acid change. This DNA difference consists of two adjacent bases which were found to be either ‘GC’ or ‘TT’. Only the second base of the two affected the amino acid sequence of the protein with the alternative amino acids being leucine (CTC) or phenylalanine (TTC). Umemoto and co-workers [15] identified an additional SNP which resulted in an amino acid change. This DNA difference (A/G) was assayed in these same 70 genotypes. Analysis of these three DNA differences in all 70 different genotypes found that only four of the eight possible combinations were represented G/G/GC, A/G/GC, A/A/GC, A/G/TT.

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95% Confidence interval for pop. mean GT (0C) 95% CI GT

When grouped by genotype class, two statistically significant GT classes were evident (Figure 2.1). The high GT class had an average GT of 78 oC and was composed of eight varieties of genotype G/G/GC and 23 varieties of genotype A/G/GC. The low GT class had an average GT of 70 oC and was composed of 26 varieties of genotype A/G/TT and 13 varieties of genotype A/A/GC. From this data it could be seen that only the second two of these three DNA differences were required for differentiation into each of the classes. The high GT class was G/GC only while the low GT class was either A/GC or G/TT. This knowledge allowed the construction of a molecular marker for GT class.

82 80 78 76 74 72 70 68 66 N=

Haplotype

8

23

26

1

2

3

G/G/GC

A/G/GC

A/G/TT

HAPLOTYP Gly/Val/Leu Ser/Val/Leu Ser/Val/Phe Amino acid pattern

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4

A/A/GC Ser/Met/Leu

Figure 2.1. Relationship between haplotype and gelatinisation temperature (GT) for 70 rice genotypes. Mean (M) GT (°C) and standard deviation (SD) for each haplotype were as follows; haplotype 1 (G/G/GC), M = 78.50, SD = 2.976; haplotype 2 (A/G/GC), M = 77.96, SD = 2.246; haplotype 3 (A/G/TT) M = 69.00, SD = 2.966, haplotype 4 A/A/GC, M = 70.00, SD = 2.000. Bars represent the 95% confidence interval around the mean for each haplotype.

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As for fragrance, competitive allele specific PCR was chosen as the assay for this trait. An assay was constructed for each of the possible DNA differences. The nature of the mutation and the kinetics of each PCR meant each possible DNA difference was assayed independently (Figure 2.2- Figure 2.5)

385 bp

FF1

SNP3F3

FR1 241 bp

385 bp band 241 bp band “A” present No 241 bp band “G” present

Figure 2.2. Schematic diagram and agarose gel showing competitive allele specific PCR assay for SSIIa SNP3 which impacts on GT. Primers FF1 and FR1 generate a 385 bp band in all PCRs independent of the SNP. Primer SNP3F3 in combination with FR1 generates a 241 bp band only when “A” is present at the SNP site. A 385 bp band and no 241 bp band indicates a “G” is present at the SNP site.

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385 bp

FF1

SNP4R8

FR1

181 bp

385 bp band 181 bp band “G” present No 181 bp band “A” present

Figure 2.3. Schematic diagram and agarose gel showing competitive allele specific PCR assay for SSIIa SNP3 which impacts on GT. PCR Primers FF1 and FR1 generate a 385 bp band in all PCRs independent of the SNP. Primer SNP3R8 in combination with FF1 generates a 181 bp band only when “G” is present at the SNP site. A 385 bp band and no 181 bp band indicates an “A” is present at the SNP site.

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385 bp

FF1

SNP4R3

FR1

308 bp

385 bp band

308 bp band “TT” present No 308 bp band “GC” present

Figure 2.4. Schematic diagram and agarose gel showing competitive allele specific PCR assay for SSIIa SNP4 which impacts on GT. PCR primers FF1 and FR1 generate a 385 bp band in all PCRs independent of the polymorphism. Primer SNP4R3 in combination with FF1 generates a 308 bp band only when “TT” is present at the polymorphic site. A 385 bp band and no 308 bp band indicates “GC” is present at the polymorphic site.

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385 bp

FF1

SNP4R14

FR1

307 bp

385 bp band 307 bp band “GC” present No 307 bp band “TT” present

Figure 2.5. Schematic diagram and photograph of agarose gel showing competitive allele specific PCR assay for SSIIa SNP4 which impacts upon GT. PCR Primers FF1 and FR1 generate a 385 bp band in all PCRs independent of the SNP. Primer SNP4R14 in combination with FF1 generates a 307 bp band only when “GC” is present at the polymorphic site. A 385 bp band and no 307 bp band indicates “TT” is present at the polymorphic site.

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2.3 Significance and utility outcomes The key finding is the identification of the link between different versions of the SSIIa encoding gene and GT class. The markers that were constructed based on this knowledge will allow the GT class of offspring of parents that differ by GT class to be identified reliably within a breeding program. These markers are dominant, they do not distinguish between homozygotes and heterozygotes if a particular allele is present, however this is not critical because all SNPs which impact upon GT can be detected. Rice breeders can therefore identify an undesirable SNP and remove it from the breeding program.

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3. Markers for disease resistance 3.1 Background The Australian rice industry enjoys freedom from many of the major rice diseases. However, based on experience with other crops and the experience of the Californian rice industry which suffered its first blast outbreak in 1996 after being blast free for 85 years [16], breeding varieties not carrying any resistance to internationally important diseases is undesirable. If a disease was introduced to the Australian rice industry, the consequences could be serious. Three major disease threats to the Australian rice industry have been identified. They are, Bacterial blight (Xanthomonas oryzae pv Oryzae), Blast (Magnaporthae grisea (Herbert) Borr (anamorphe Pyricularia oryza Cav.) and Sheath blight (Rizoctonia solani). Twenty one lines carrying disease resistance genes were imported from India, Egypt, the Philippines and the USA. They included sources of major genes for blast resistance (Pi-1, Pi-z-5, Pi-ta, Pi-6(t), Pi-7(t), Pi-5(t), Pi-44(t)), major genes for bacterial blight resistance (Xa4, xa5, Xa21, xa13, Xa22(t)) and quantitative trait loci for resistance to blast, bacterial blight and sheath blight. Incorporation of resistance genes by conventional breeding is challenging. Firstly, without molecular markers it is very difficult to break the linkage of resistance genes with genes which code for undesirable traits, even with many generations of backcrossing [17]. Secondly, molecular markers allow one or more different disease resistance genes to be efficiently incorporated into a single line of rice because they can be used to determine if a resistant plant derives resistance from one or more genes. This is particularly useful because incorporation of several resistance genes has been shown to reduce the likelihood of pathogens overcoming resistance [18,19]. Thirdly, for diseases which do not currently exist in the Australian rice industry, marker assisted selection is the most effective way to incorporate disease resistance into Australian rice lines and cultivars. The only way to determine if a plant is resistant to a disease is to either challenge that plant with disease or use molecular markers tightly linked to resistance genes. Because molecular markers negate the need for pathogen selection, it is unnecessary to introduce and use the pathogen within the country which would risk a disease outbreak, or utilise expensive off shore testing. Rice blast is caused by the fungus Magnaporthe grisea. Based in large part on the experience of the Californian industry[16], it has been determined that blast poses the greatest threat to the Australian rice industry. Under favourable conditions blast outbreaks have caused crop losses of up to 60% [16]. Because of this risk, varieties which carry a range of genes for blast resistance were procured by the rice breeding program as the first step in initiating a program of pre-emptive rice blast disease resistance breeding. Varieties carrying blast resistance genes which were well characterised at the molecular level, hence allowing accurate application of molecular marker technology, were chosen as donors of resistance in this pre-emptive breeding program.

3.2 Approach and practical significance of outcomes Rice lines resistant to blast (Giza 177, Giza 178 and Sakha) were initially sourced from Egypt for inclusion in the Australian pre-emptive breeding program. These lines were chosen because the environment of the Egyptian rice growing area is similar to the Australian growing area in terms of rainfall, humidity and so on. The environmental similarity means the Egyptian cultivars probably contain combinations of genes that will allow cultivars derived from these varieties and developed at Yanco to perform well under Australian conditions and it is likely the races of blast that effect the Egyptian industry are the same races most likely to affect the Australian industry.

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The pedigree information that accompanied the Egyptian lines suggested the genetic background of Giza 178 was too complex to be used in the application of molecular markers in pre-emptive breeding for blast disease resistance. The pedigrees of Giza 177 and Sakha 102 were less complex and suggested both received blast resistance from the rice variety Pi 4. The gene that confers blast resistance to Pi 4 is reported to be Pi-ta2 [20]. It is believed the blast resistance gene Pi-ta2 is the blast resistance gene Pi-ta plus another gene close by that slightly extends its spectrum of resistance [20,21]. Pi-ta and Pi-ta2 provide resistance to a broad spectrum of races of the blast pathogen. Both Giza 177 and Sakha 102 were crossed with the Australian rice variety Paragon. Data in the public domain suggests a single nucleotide within the resistance gene Pi-ta is responsible for resistance, “T” has been found in susceptible varieties while resistant varieties have a “G” [21]. However, analysis of DNA from the rice variety Pi 4 at Southern Cross University identified a "T" at the SNP position in Pi-ta, not a "G" as expected. The Egyptian lines Giza 177 and Sakha 102 also have the non-resistant “T”. Because the data derived from Pi 4, Giza 177 and Sakha 102 did not conform to the expected pattern, we investigated DNA variation both around and within the coding regions of the Pi-ta gene in Giza 177, Sakha 102 and Paragon. We sequenced the gene in these varieties and found no differences between the local parent (Paragon) and the imported rice. Likewise, we investigated the DNA variability around the gene using 23 microsatellites and could not identify any differences between Giza 177, Sakha 102 and Paragon while Pi 4 was different to all other varieties. The analysis was conducted on two independent samples derived from the Yanco breeding program, suggesting the results were not due to sampling error. In summary, the molecular data showed there was no difference within the Pi-ta gene when comparing Giza 177, Sakha 102 and Paragon, and there is no genetic difference between Giza 177, Sakha 102 and Paragon in the region around Pi-ta. Because of this it was not possible to develop markers for these varieties of rice that would allow the introgression of blast disease resistance genes relying solely on published data. The blast resistant varieties BL12, BL14 and BL24 were sourced directly from the International Rice Research Institute (IRRI) and found to conform to the published data [22]. BL12 has the susceptible version of the gene (pi-ta) having a “T” at the key position while BL14 and BL24 have the resistant gene Pi-ta with a “G” at the key position. Because BL14 and BL24 generate molecular data that conforms with the published data, they were chosen as donors of blast disease resistance in crosses with locally adapted varieties. It is possible to test for the nucleotide difference responsible for resistance directly in the offspring of any cross involving these lines using a simple published molecular test for Pi-ta [23] that is operational in the this germplasm. Pi-ta provides relatively broad spectrum resistance and, assuming the gene Pi-ta gives protection from the race of blast that ultimately arrives in Australia, this approach will allow the development of rice varieties that are blast resistant.

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4. Molecular markers for hybrid rice Hybrid rice is used widely in China because they yield up to 20% higher than traditional inbred rice varieties. The type of hybrid rice system that is most suited to the Australian rice growing regions is the cytoplasmic male sterile, or three line, system (Figure 4.1). As the name suggests, three lines of rice are used to produce this hybrid, the A, B and C lines. The B and C lines reproduce in a conventional fashion by self-pollination. The A line does not produce its own pollen so it must receive pollen from another line of rice. To create more of the A line, the B line is used to donate pollen to the A line. The offspring of this cross is more of the sterile A line. When there is sufficient A line seed available, the A line is crossed to the C line. The seed from this cross is the hybrid seed that the farmer receives and plants for the hybrid crop itself. For the system to work efficiently it is important no B line rice contaminates the A line. If it does, then the yield will be reduced because the non-hybrid low yielding B line dilutes the high yielding hybrid seed. In the past it has been necessary to grow a sub-sample of A line rice to check there is no contaminating B line. This can take many months as the rice needs to grow to maturity. To circumvent this problem a simple laboratory test has been developed which distinguishes between the A and B lines. This means the purity of the A line can be checked without having to grow the rice.

Figure 4.1. An outline of the three line hybrid breeding system.

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We have checked and found the laboratory test [24] that distinguishes between the A and B lines works with 12 hybrid rice lines that were imported from China. The tools are in place to trial whether or not the yield advantage offered by hybrids in other parts of the world also apply to Australia.

No band indicating absence of male sterile line 386 bp band indicating presence of male sterile line

Figure 4.2. Agarose gel showing male sterile and maintainer line discriminating PCR assay. The 386 base pair band indicates the presence of the male sterile line while the larger product in the absence of the 386 bp band indicates the absence of a male sterile line.

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References 1. Buttery, R.G, Ling, L.C., Juliano, B.O., and Turnbaugh, J.G. (1983) Cooked rice aroma and 2acetyl-1-pyrroline. Journal of Agriculture and Food Chemistry. 31: 823-826. 2. Lorieux, M., Petrov, M., Huang, N., Guiderdoni, E. and Ghesquière, A. (1996) Aroma in rice: genetic analysis of a quantitative trait. Theoretical and Applied Genetics. 93: 1145-1151. 3. Widjaja, R., Craske, J.D. and Wootton, D. (1996) Comparative studies on volatile components of non-fragrant and fragrant rices. Journal of the Science of Food and Agriculture. 70: 151-161. 4. Yoshihashi, T. (2002) Quantitative analysis of 2-acetyl-1-pyrroline of an aromatic rice by stable isotope dilution method and model studies on its formation during cooking. Journal of Food Science. 67: 619–622. 5. Reinke, R.F., Welsh, L.A., Reece, J.E., Lewin, L.G. and Blakeney, A.B. (1991) Procedures for quality selection of aromatic rice varieties. International Rice Research Newsletter. 16: 10–11. 6. Sood, B.C. and Sidiq, E.A. (1978) A rapid technique for scent determination in rice. Indian Journal of Genetical Plant Breeding. 38: 268-271. 7. Cordeiro, G.M., Christopher, M.J., Henry, R.J. and Reinke, R.F. (2002) Identification of microsatellite markers for fragrance in rice by analysis of the rice genome sequence. Molecular Breeding. 9: 245–250. 8. Bradbury, L.M.T., Fitzgerald, T.L., Henry, R.J., Jin, Q. and Waters, D.L.E. (2005) The gene for fragrance in rice. Plant Biotechnology Journal. 3:363–370. 9. Berner, D.K. and Hoff, B.J. (1986) Inheritance of scent in American long grain rice. Crop Science. 26: 876–878. 10. Bergmann, C.J., Delgado, J.T., McClung, A.M. and Fjellstrom, R.G. (2001) An improved method for using a microsatellite in the rice waxy gene to determine amylose class. Cereal Chemistry. 78: 257–260. 11. Maningat, C.C. and Juliano, B.O. (1978) Properties of lintnerized starch granules from rices of different amylose content and gelatinization temperature. International Rice Research Newsletter. 3: 7–8. 12. Ball, S.G. and Morell, M.K. (2003) From bacterial glycogen to starch: understanding the biogenesis of the plant starch granule. Annual Review of Plant Biology. 54: 207–233. 13. Fitzgerald, M.A. (2004) Starch. In Rice Chemistry and Technology (Champagne, E.T., ed.), pp. 109–141. St Paul, MN: American Association of Cereal Chemists. 14. Umemoto, T., Yano, M., Satoh, H., Shomura, A. and Nakamura, Y. (2002) Mapping of a gene responsible for the difference in amylopectin structure between japonica-type and indica-type rice varieties. Theoretical and Applied Genetics. 104: 1–8. 15. Umemoto, T., Aoki, N., Lin, H.X., Nakamura, Y., Inouchi, N., Sato, Y., Yano, M., Hirabayashi, H. and Maruyama, S. (2004) Natural variation in rice starch synthase IIa affects enzyme and starch properties. Functional Plant Biology. 31: 671–684.

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16. Greer, C.A. and Webster, R.K. (2001) Occurrence, distribution, epidemiology, cultivar reaction and management of rice blast in California. Plant Disease. 85(10): 1096-1102. 17. Young, N.D. and Tanksley, S.D. (1989) RFLP analysis of the size of chromosomal segments retained around the Tm-2 locus of tomato during backcross breeding. Theoretical and Applied Genetics. 77: 353-359. 18. Yoshimura, S., Yoshimura, A., Iwata, N., McCouch, S.R., Abenes, M.L., Baraoidan, M.R., Mew, T.W., Nelson, R.J. (1995) Tagging and combining bacterial blight resistance genes in rice using RAPD and RFLP markers. Molecular Breeding. 1(4): 375-387. 19. Huang, N., Angeles, E.R., Domingo, J., Magpantay, G., Singh, S., Zhang, G., Kumaravadivel, N., Bennett, J., Khush, G.S. (1997) Pyramiding of bacterial blight resistance genes in rice: markerassisted selection using RFLP and PCR. Theoretical and Applied Genetics. 95: 313-320. 20. Rybka, K., Miyamoto, M., Ando, I., Saito, A., and Kawasaki, S. (1997) High resolution mapping of the indica derived rice blast resistance genes II. Pi-ta and Pi-ta2 and a consideration of their origin. Molecular Plant-Microbe Interactions. 10(4): 517-524. 21. Bryan, G.T., Wu, K.S., Farrall, L, Jia, Y.L., Hershey, H.P, McAdams, S.A, Faulk, K.N., Donaldson, G.K., Tarchini, R. and Valent, B. (2000) A single amino acid difference distinguishes resistant and susceptible alleles of the rice blast resistance gene Pi-ta. Plant Cell. 12(11): 2033-2045. 22. Hittalmani, S., Parco, A., Mew, T.V., Zeigler, R.S., and Huang, N. (2000) Fine mapping and DNA marker-assisted pyramiding of the three major genes for blast resistance in rice. Theoretical and Applied Genetics. 100(4): 1121-1128. 23. Jia, Y.L., Redus, M., Wang, Z.H. and Rutger, J.N. (2004) Development of a SNLP marker from the Pi-ta blast resistance gene by tri-primer PCR. Euphytica. 138(1): 97–105. 24. Yashitola, J., Sundaram, R. M., Biradar, S. K., Thirumurugan, T., Vishnupriya, M. R., Rajeshwari, R., Viraktamath, B. C., Sarma, N. P. and Sonti, R. V. (2004) A sequence specific PCR marker for distinguishing rice lines on the basis of wild abortive cytoplasm from their cognate maintainer lines. Crop Science. 44: 920–924.

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