molecular markers for rice quality traits...microsatellite simple sequence ct-repeats (ssr) in the...

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Molecular Markers for Molecular Markers for Rice Quality TraitsRice Quality Traits

M. Chen, R. M. Chen, R. FjellstromFjellstrom, C. Bergman,, C. Bergman,S. Pinson, A. McClungS. Pinson, A. McClung

USDAUSDA--ARSARS--Rice Research UnitRice Research UnitBeaumont, TexasBeaumont, Texas

Rice Quality Traits Rice Quality Traits

AromaAromaCooked kernel elongationCooked kernel elongationApparent amylose contentApparent amylose contentPasting propertiesPasting propertiesGelatinization temperatureGelatinization temperature

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Rice Quality TraitsRice Quality Traits

Phenotypic traitsPhenotypic traitsGood for explaining functional properties of riceGood for explaining functional properties of riceEffect of phenotypic traits =Effect of phenotypic traits =

Genotype x EnvironmentGenotype x EnvironmentThe effects of environment, minor or modifier genes can The effects of environment, minor or modifier genes can make progeny selection difficult.make progeny selection difficult.During cultivar development quality traits must be During cultivar development quality traits must be measured on grain from multiple years and locations to measured on grain from multiple years and locations to be sure the desired characteristics have been captured.be sure the desired characteristics have been captured.

Molecular Markers for Specialty Molecular Markers for Specialty Rice Rice -- TypesTypes

AromaAromaCooked kernel elongationCooked kernel elongationApparent Amylose ContentApparent Amylose ContentPasting propertiesPasting propertiesGelatinization TemperatureGelatinization Temperature

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AromaAromaChemistryChemistry–– primary compound: 2primary compound: 2--acetylacetyl--11--pyrrolinepyrroline–– Developed Gas chromatographic method for Developed Gas chromatographic method for

quantitatingquantitating this compound.this compound.

GC Method: Bergman et al. 2000. Cereal Chemistry

AromaAromaGeneticsGenetics–– fgrfgr gene identified on chromosome 8 gene identified on chromosome 8

associated with 2APassociated with 2AP–– RFLP marker (RG 28) linked with this locus RFLP marker (RG 28) linked with this locus

(Ahn et al 1992 TAG )(Ahn et al 1992 TAG )

–– PCR based marker have been mapped and PCR based marker have been mapped and linked to the RG28 and linked to the RG28 and fgrfgr gene gene (Garland et al. (Garland et al. 2000 TAG)2000 TAG)

–– This marker has now been mapped and used This marker has now been mapped and used in U.S. populationsin U.S. populations Marker (RG28)

Phenotype R2 = 0.80Bergman et al. 2002. RTWG.

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RM223 Alternative Marker for RM223 Alternative Marker for AromaAroma

In some crosses RG28 is In some crosses RG28 is not polymorphicnot polymorphicRM223 wasRM223 was identified from Rice Genes identified from Rice Genes databasedatabaseIn A301/Lablelle cross, 60% of In A301/Lablelle cross, 60% of phenotypic variance in aroma was phenotypic variance in aroma was explained by RM223explained by RM223In Rosemont/DE100 RM223 correctly In Rosemont/DE100 RM223 correctly identified 88% of the aromatic progenyidentified 88% of the aromatic progeny

Bergman et al. 2002. RTWG.

Cooked Kernel ElongationCooked Kernel ElongationChemistryChemistry–– physical or chemical cause physical or chemical cause

unknownunknown–– tedious phenotypic tedious phenotypic

measurementmeasurementGeneticsGenetics

–– RFLP on chromosome 8 RFLP on chromosome 8 explained 13.6 of phenotypic explained 13.6 of phenotypic and 45% of genetic variation and 45% of genetic variation (Ahn et al 1993, TAG)(Ahn et al 1993, TAG)

–– RFLP markers laborious, RFLP markers laborious, need to develop rapidly need to develop rapidly assayed PCR markerassayed PCR marker

Importedbasmati Sierra

Typical longgrain

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QTL Analysis of QTL Analysis of Cooked Kernel Cooked Kernel

ElongationElongation

• Peak at RM44

• LRS 34.3 = LOD 7.4

Bergman et al. 2002. RTWG.

DLMT x B8462T3-710

MicrosatelliteMicrosatellite RM44:RM44:Phenotypic variance: Phenotypic variance: RR2 = 0.22= 0.22Genetic variance: Genetic variance: R2R2 = 0.74= 0.74

Markers for Aroma and Markers for Aroma and ElongationElongation

RG28 and RM223 are associated with aroma RG28 and RM223 are associated with aroma gene which is otherwise expensive to gene which is otherwise expensive to accurately analyze using GC methodaccurately analyze using GC methodRM44 is associated with kernel elongation RM44 is associated with kernel elongation which is otherwise very tedious to measurewhich is otherwise very tedious to measureThese markers are suitable for use in These markers are suitable for use in many crosses but not in allmany crosses but not in allNeed to know the marker genotype of Need to know the marker genotype of your parents before you use theseyour parents before you use these

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Rice Historical Quality TraitsRice Historical Quality Traits

AromaAromaCooked kernel elongationCooked kernel elongationApparent amylose contentApparent amylose contentPasting propertiesPasting propertiesGelatinization temperatureGelatinization temperature

Three Market Classes

6 - 7 (low) 3 - 5 (intermediate) 2 (high)

< 5 waxy-types

10 - 19 (low)

conventional medium grains conventional short grains

jasmine-types Japanese premium quality-types

cadet-types

20 - 24 (intermediate)

conventional long grains basmati-types

> 24 (high)

superior processing quality-types

App

aren

t Am

yose

(%)

Alkali Spreading Value

Other traits commonly used for rice quality categorization: grain dimensions, cooked kernel elongation, firmness and stickiness, pasting properties, protein and 2-acetyl-1-pyrroline content.

CP231, Hidalgo type

Conventional long grains

Basmati-type

Superior processing quality-type

Waxy-types

Conv. Med.&short grainsJasmine-typesJapanese premium type

App

aren

t Am

ylos

e (%

)

< 5

10-19(low)

20-24(inter-mediate)

>24(high)

6 – 7 (low) 3 – 5 (intermediate) 2 (high)Alkali Spreading Value

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Rice Quality and StarchRice Quality and StarchRice cooking and eating quality largely controlled by Rice cooking and eating quality largely controlled by physicalphysical--chemical properties of starchchemical properties of starch

Starch composed of mostly linear amylose and highly Starch composed of mostly linear amylose and highly branched amylopectinbranched amylopectin

Amylose synthesized by granuleAmylose synthesized by granule--bound starch bound starch synthase encoded by the synthase encoded by the WaxyWaxy genegene

Amylopectin synthesized by combination of: Amylopectin synthesized by combination of: soluble soluble starch synthase, ADPstarch synthase, ADP--glucose glucose pyrophosphorylasepyrophosphorylase, and , and starch branching and starch branching and debranchingdebranching enzymesenzymes

Apparent Amylose ContentApparent Amylose Content

ChemistryChemistry–– Major determinant of rice textureMajor determinant of rice texture–– IodineIodine--based colorimetric methodbased colorimetric method–– Lipid and longLipid and long--chain amylopectin affect the chain amylopectin affect the

determination of determination of ““apparentapparent”” amylose contentamylose content–– No pure rice amylose is available for use as a No pure rice amylose is available for use as a

standardstandard–– Effects of environment + minor or modifier Effects of environment + minor or modifier

genesgenes

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Amylose and Waxy GeneAmylose and Waxy Gene

• The Waxy gene on chromosome 6 encodes the granule-bound starch synthase enzyme and controls much of the variation in rice apparent amylose content.

• Waxy gene sequence variations that affect amylose content:• Exon 1 non-coding region: Waxy SSR

Marker• Intron 1 splice site: G/T• Exon 6• Exon 10

Microsatellite simple sequence CT-repeats (SSR) in the non-coding region of exon 1 of Waxy gene explained 82.9% of the variation in apparent amylose content of the 89 non-glutinous US rice cultivars (Ayres et al., 1997).

Waxy Gene – Waxy SSR Marker

----- (CT)n------- (CT)n--

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Microsatellite simple sequence CT-repeats (SSR) in the non-coding region of exon 1 of Waxy gene explained 82.9% of the variation in apparent amylose content of the 89 US rice cultivars (Ayres et al., 1997).

This Waxy SSR marker is being used for selection by U.S. and international breeding programs.

Waxy Gene

----- (CT)n------- (CT)n--

----- (CT)n------- (CT)n--

SSR CT Class

(CT)n = 8, 11(CT)n = 14, 20(CT)n = 17, 18, 19

US Rice Market Class Apparent Amylose (%)

> 2420 - 2410 -19

high amyloseintermediate amylose

low amylose

Waxy SSR CT-repeat marker

Waxy Gene – Waxy SSR Marker

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G/T single nucleotide polymorphism (G/T SNP) in the leader intron 5′ splice site.

G/Ttatac---------- (CT)n-- G/Ttatac---------- (CT)n--

Waxy Gene – G/T SNP

G/T single nucleotide polymorphism (G/T SNP) in the leader intron 5′ splice site.

Low amylose cultivars have the nucleotide sequence T at this G/T SNP; intermediate and high amylose cultivars have the G. This G/T SNP explained 79.7% of the total variation in apparent amylose content of 89 non-glutinous US cultivars (Ayres et al., 1997).

G/Ttatac---------- (CT)n-- G/Ttatac---------- (CT)n--

Waxy Gene – G/T SNP

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Single Nucleotide Polymorphism in Exon 6

Nucleotide A C

Amino Acid Tyr Ser224*

Cultivars All others Conv. Long Grains

Exon 6 SNP: A → CExon 6 SNP: A → C

Waxy Gene – Exon 6 SNP

(Larkin, TAMU dissertation, 1999.)

Apparent Amylose Content in Diverse Rice Germplasm

Examined the nucleotide polymorphism in Waxy gene of international rice germplasm: – Waxy SSR marker: microsatellite CT-repeat allele

in exon 1 – G/T SNP at the leader intron splice site – Exon 6 SNP

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Association of the Apparent Amylose Content to the Waxy SSR Marker in Non-Glutinous Diverse Rice Germplasm

App

aren

t Am

ylos

e (%

)

810121416182022242628

8 10 11 14 16 17 18 19 20

Waxy SSR Marker

R2=69.2

High

Intermediate

Low

Chen et al. 2004. RTWG.

Association of the Apparent Amylose Content to the Waxy G/T SNP in Non-Glutinous Diverse Rice Germplasm

810121416182022242628

G T

App

aren

t Am

ylos

e (%

)

G/T SNP

Low

R2=75.7

High

Intermediate+


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Molecular Markers for Amylose -Conclusion

All of the SSR markers (CTs) are associated with intermediate or high amylose content except when G/T = T

All low amylose content cultivars have been found to possess G/T = T and these have only been identified in CT 17, CT18, and CT 19

There are two Exon 6 alleles“C” version results in the “Conventional” long

grain RVA.“A” version is associated with “All” other RVA

types. Chen et al. 2004. RTWG.

Molecular Markers for Amylose –Conclusion

Higher amylose content is generally associated with decreased peak paste viscosity

CT 20 allele (=intermediate amylose) + Exon 6 = A, results in higher amylose content and a flattened RVA profile (L202 type)

The G/T SNP + Exon 6 SNP haplotypesdifferentiate between genotypes and predict there end-use quality (functionality).


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Pasting Properties and Pasting Properties and Waxy Waxy GeneGene

Phenotypic trait:Phenotypic trait:–– Determined using a Rapid Determined using a Rapid ViscoVisco AnalyAnalyzzer er

(RVA)(RVA)–– Differentiate high amylose superiorDifferentiate high amylose superior--

processing type from other high amylose processing type from other high amylose cultivars.cultivars.

–– Affected by environment, storage, different Affected by environment, storage, different mills, RVA calibration.mills, RVA calibration.

‘Rexmont’

‘Jodon’

Vi s

cosi

ty (

RV

U)

Tem

pera

ture

(°

C)

Time (min.)

A

B

C

A: Peak viscosity

B: Hotpaste viscosity B-A: Breakdown

C: Coolpaste viscosity C-A: Setback

Rapid Visco Analyzer

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Pasting Properties and Pasting Properties and Waxy Waxy GeneGene

Genetics:Genetics:–– Single nucleotide Single nucleotide differencesdifferences in in ExonExon

10 of 10 of WaxyWaxy gene resultgene resultss in changes in changes in the in the amino acid amino acid codoncodon..

–– This has a dramatic effect on RVA This has a dramatic effect on RVA pasting propertiespasting properties

(Larkin et al., 2003).(Larkin et al., 2003).

Waxy Gene

Exon 10:C → T substitution changes a Pro to

Ser. Rexmont (strong RVA) has a

nucleotide T, while other cultivars have a nucleotide C (Larkin, 1999, TAMU dissertation).

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Gelatinization TemperatureGelatinization Temperature

Phenotype:Phenotype:Gel Temp: critical temperature during cooking Gel Temp: critical temperature during cooking when starch granules undergo an irreversible when starch granules undergo an irreversible process process –– gelatinizationgelatinizationAlkali Spreading Value (ASV) is a rating of grain Alkali Spreading Value (ASV) is a rating of grain dispersion in 1.5 or 1.7% KOH solution after 16dispersion in 1.5 or 1.7% KOH solution after 16--24 24 hourshoursASV provides a simple means of classifying rice ASV provides a simple means of classifying rice into high, intermediate and low gel temp typesinto high, intermediate and low gel temp types

ASV MeasurementASV Measurement

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ASV InheritanceASV Inheritance

ASV reported to be primarily under control of ASV reported to be primarily under control of one or two genes depending on the crossone or two genes depending on the cross

Both loci map to rice chromosome 6Both loci map to rice chromosome 6–– WaxyWaxy gene gene -- granule bound starch synthasegranule bound starch synthase

–– AlkAlk gene gene -- soluble starch synthase IIa (soluble starch synthase IIa (SSSIIaSSSIIa) ) UmemotoUmemoto et al. (2002)et al. (2002)

ASV in Diverse Rice GermplasmASV in Diverse Rice Germplasm

Rice Foundation sponsored study of grain Rice Foundation sponsored study of grain quality traits associated with starch quality traits associated with starch metabolism gene variation for 190 diverse rice metabolism gene variation for 190 diverse rice accessionsaccessionsSSSIIaSSSIIa gene sequences were compared gene sequences were compared between between NipponbareNipponbare and 93and 93--11 (sequenced 11 (sequenced Chinese Chinese indicaindica line) using two public line) using two public databasesdatabasesTwo sequence polymorphisms found Two sequence polymorphisms found conferring amino acid substitutionsconferring amino acid substitutions

Chen et al. 2003. PAG.

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Identified three alleles (Identified three alleles (haplotypeshaplotypes) at ) at the the SSSIIa/SSSIIa/AlkAlk locus (single nucleotide locus (single nucleotide polymorphic)polymorphic)–– Normal (wildNormal (wild--type) allele which results in type) allele which results in

intermediate ASVintermediate ASV–– Mutation which results in high ASV (low Mutation which results in high ASV (low

gel tempgel temp))–– Another mutation primarily found in Another mutation primarily found in

Japanese material also having high ASV Japanese material also having high ASV (low gel temp)(low gel temp)



Two alleles were found in US Two alleles were found in US germplasmgermplasm–– Lemont type allele Lemont type allele –– intermediate ASV (int. gel intermediate ASV (int. gel

temp)temp)–– M202 type allele M202 type allele –– high ASV (low gel temp)high ASV (low gel temp)

There is an interaction between There is an interaction between amyloseamylosecontent and ASVcontent and ASVCultivars having low Cultivars having low amyloseamylose content and content and the Lemont allele of the Lemont allele of SSSIIaSSSIIa results in a low results in a low ASV value (high gel temp)ASV value (high gel temp)


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Cultivars Possessing Different Waxy and Cultivars Possessing Different Waxy and SSSIIaSSSIIa Alleles Alleles Result in Different Types of FunctionalityResult in Different Types of Functionality

Waxy Exon1 SNPG T

SSSIIa Amylose ContentAllele High-Int Low

Cypress CP231LMNT type Wells Jacinto

Lemont HidalgoIRGA 409 Bengal

M202 type Teqing M ars IR 8 M 202

Conclusions Conclusions –– Gel TempGel Temp

Genetic variation at the Genetic variation at the AlkAlk gene explains gene explains most of the variation in ASVmost of the variation in ASVASV is secondarily controlled by variation at ASV is secondarily controlled by variation at WaxyWaxy genegeneAlthough ASV is a simple test to run, theAlthough ASV is a simple test to run, the AlkAlkmarker can make selection for desired ASV marker can make selection for desired ASV more efficient because it can be determined more efficient because it can be determined at the same time other markers are runat the same time other markers are run

Fjellstrom et al. 2004. RTWG.

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Summary of DNA Markers for Summary of DNA Markers for Grain Quality TraitsGrain Quality Traits

Apparent amylose content is determined by:Apparent amylose content is determined by:–– Waxy SSR MarkerWaxy SSR Marker–– Waxy Waxy ExonExon 1 G/T SNP1 G/T SNP–– Waxy Waxy ExonExon 6 SNP6 SNP

Superior processing properties as determined Superior processing properties as determined by RVA pasting propertiesby RVA pasting properties–– Waxy Waxy ExonExon 10 SNP 10 SNP

Gel Temperature:Gel Temperature:–– AlkAlk alleles combined with G/T SNPalleles combined with G/T SNP

What do these DNA sequence What do these DNA sequence substitutions have in common?substitutions have in common?

They are all within the They are all within the exonsexons of the of the genes involved in the syntheses of genes involved in the syntheses of amylose and amylopectin.amylose and amylopectin.

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What do these DNA sequence What do these DNA sequence differences have in common?differences have in common?

All but one (i.e. Waxy SSR marker) are All but one (i.e. Waxy SSR marker) are single nucleotide substitutions (i.e. single nucleotide substitutions (i.e. SNP).SNP).


The nucleotide substitutions alter the The nucleotide substitutions alter the coded amino acids.coded amino acids.

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These associations have been verified These associations have been verified in US cultivars, diverse rice accessions, in US cultivars, diverse rice accessions, and genetically segregating and genetically segregating populationspopulations..

Rice Grain Quality MarkersRice Grain Quality Markers

These associations were determined across U.S. and international germplasm indicating that breeding programs using U.S. material, as well as internationally sourced germplasmwill benefit from the using of these markers.

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Allele Specific MarkersAllele Specific Markers

Allele specific markers to each SNP have been Allele specific markers to each SNP have been developed.developed.–– Two fluorescently labeled primers in one PCR Two fluorescently labeled primers in one PCR

reaction (plus one unlabeled primer)reaction (plus one unlabeled primer)It can discriminate the homozygous plants It can discriminate the homozygous plants with different SNP alleles (color and size with different SNP alleles (color and size differences in the PCR products).differences in the PCR products).The heterozygous plants will have two The heterozygous plants will have two different colored, and sized PCR Products.different colored, and sized PCR Products.

Allele specific markersAllele specific markers

Allele specific markers to each SNP have been Allele specific markers to each SNP have been developed.developed.–– Two fluorescently labeled primers in one PCR Two fluorescently labeled primers in one PCR

reaction (plus one unlabeled primer)reaction (plus one unlabeled primer)It can discriminate the homozygous plants It can discriminate the homozygous plants with different SNP alleles (color and size with different SNP alleles (color and size differences in the PCR products).differences in the PCR products).The heterozygous plants will have two The heterozygous plants will have two different colored, and sized PCR Products.different colored, and sized PCR Products.

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The Benefits of Using Molecular The Benefits of Using Molecular Markers in Breeding for Quality TraitsMarkers in Breeding for Quality Traits

Selection is based upon the genotype and is not Selection is based upon the genotype and is not distorted by environmental conditions (distorted by environmental conditions (i,.ei,.e. . amyloseamylose, ASV), ASV)Progeny with undesirable quality traits can be Progeny with undesirable quality traits can be eliminated early in the breeding process so that eliminated early in the breeding process so that more effort can be focused on materials having more effort can be focused on materials having the right quality parameters for their market classthe right quality parameters for their market classOne can select for multiple traits using one One can select for multiple traits using one assay, requiring a trivial amount of sampleassay, requiring a trivial amount of sampleGrain quality traits can be selected before the Grain quality traits can be selected before the grain is producedgrain is produced

AcknowledgementsAcknowledgementsTechnical SupportRick Boyd Mickey Frank

Jodie Cammack Naomi Gipson

Pat Carre Ann Jund

Eric Christensen Fran Pontasch

Janis Delgado Faye Seaberg

Support from:Support from:The Rice Foundation,The Rice Foundation,USDAUSDA--ARS, TAMUARS, TAMU--TAESTAES

molecular markers for rice quality traits...microsatellite simple sequence ct-repeats (ssr) in the...

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