characterization of novel rice germplasm from west africa and genetic marker associations with rice...
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Characterization of Novel Rice Characterization of Novel Rice Germplasm from West Africa Germplasm from West Africa
and Genetic Marker and Genetic Marker Associations with Rice Associations with Rice
Cooking QualityCooking Quality
Dr. Karim Traore IER, MaliDr. Karim Traore IER, MaliDr. Anna McClung USDA Beaumont, TXDr. Anna McClung USDA Beaumont, TX
Dr. Robert Fjellstrom USDA Beaumont, TXDr. Robert Fjellstrom USDA Beaumont, TX
Consumers Around the World Have Different Consumers Around the World Have Different Preferences in Rice Cooking QualityPreferences in Rice Cooking Quality
The Japanese people prefer soft and sticky rice with The Japanese people prefer soft and sticky rice with short grain (japonica types)short grain (japonica types)
In the USA, medium and long grain rice varieties are In the USA, medium and long grain rice varieties are preferredpreferred
In South America and the Middle East, people prefer firm In South America and the Middle East, people prefer firm and non-sticky riceand non-sticky rice
Thai people prefer long grain soft and nonstickyThai people prefer long grain soft and nonsticky
In India, Pakistan, fragrant or scented rice is preferredIn India, Pakistan, fragrant or scented rice is preferred
In Brazil, people prefer long, slender, and translucent In Brazil, people prefer long, slender, and translucent graingrain
Consumer Preferences in West Consumer Preferences in West AfricaAfrica
In West Africa consumers prefer:In West Africa consumers prefer: long, slender, intermediate amylose long, slender, intermediate amylose AromaAroma Sticky rice to make rice porridge Sticky rice to make rice porridge Parboiled riceParboiled rice BrokensBrokens Farmers like rice that is slow to Farmers like rice that is slow to
digest giving longer satisfactiondigest giving longer satisfaction
Experiments ConductedExperiments Conducted
11. Conduct genotypic and phenotypic evaluation of . Conduct genotypic and phenotypic evaluation of West African germplasm for agronomic and West African germplasm for agronomic and quality traits to identify characteristics that can quality traits to identify characteristics that can benefit WA and USA rice breeding programs.benefit WA and USA rice breeding programs.
22. Determine genetic marker associations with key . Determine genetic marker associations with key cooking quality traits that can be used in rice cooking quality traits that can be used in rice cultivar improvement programs.cultivar improvement programs.
Data collected for the Quality testsData collected for the Quality tests
Alkali Spreading Value (ASV)Alkali Spreading Value (ASV)- - qualitative indicator of starch gelatinization temperaturequalitative indicator of starch gelatinization temperature, dispersion , dispersion of milled grain in 1.5% KOH solutionof milled grain in 1.5% KOH solution
Apparent Amylose Content (AA), Soluble Amylose (SA)Apparent Amylose Content (AA), Soluble Amylose (SA)- - indicator of cooked rice textureindicator of cooked rice texture, using , using wet chemistry auto analyzerwet chemistry auto analyzer
Rapid Visco Analyzer (RVA)Rapid Visco Analyzer (RVA)- - determines the viscosity of rice flour pasting subjected to cycles of determines the viscosity of rice flour pasting subjected to cycles of heating and cooling heating and cooling
Differential Scanning Calorimetry DSCDifferential Scanning Calorimetry DSC- - quantitativequantitative indicator of starch gelatinization temperatureindicator of starch gelatinization temperature, , using DSC 6 analyzer determines the temperature and heat of gelatinizationusing DSC 6 analyzer determines the temperature and heat of gelatinization
Cooking TimeCooking Time- - time required for 10 milled rice grains to be completely gelatinizedtime required for 10 milled rice grains to be completely gelatinized
Total and Whole Milling YieldTotal and Whole Milling Yield – – indicator of crop value,indicator of crop value, using McGill#2 Mill for 1 min. using McGill#2 Mill for 1 min.
Grain dimensions- Grain dimensions- indicator of crop value, indicator of crop value, using WinSEEDLEusing WinSEEDLE
Crude Protein Content-Crude Protein Content- indicator of nutritional value, indicator of nutritional value, using nitrogen gas analyzer LECO 528using nitrogen gas analyzer LECO 528
Aroma (2-Acetyl-1-pyrroline)-Aroma (2-Acetyl-1-pyrroline)-indicator of market value, indicator of market value, using gas chromatographyusing gas chromatography
Heading from the Agronomic Heading from the Agronomic Evaluation Field of WA GermplasmEvaluation Field of WA Germplasm
0
20
40
60
80
100
120
IMP INT LOC USA
Heading (days)
102
74
103
76
Same color =No differences
IMP = Improved Africa(6)Land = Landrace(13)INT = Interspecific (7)USA = checks (8)
020406080
100120140160180
IMP INT Land USA
Height (cm)
122 119
162
105
Same color =No differences
IMP = Improved Africa(6)Land = Landrace(13)INT = Interspecific (7)USA = checks (8)
Plant Height from the Agronomic Plant Height from the Agronomic Evaluation Field of WA GermplasmEvaluation Field of WA Germplasm
Plots: 6 rows of 4.57 m length; spacing Plots: 6 rows of 4.57 m length; spacing 17.78 cm between rows17.78 cm between rowsRCBD, 4 rep.RCBD, 4 rep.
0
1000
2000
3000
4000
5000
6000
7000
IMP INT Land USA
Yield (kg/ha)
43424579
2586
6200
Same color =No differences
IMP = Improved Africa(6)Land = Landrace(13)INT = Interspecific (7)USA = checks (8)
Grain Yield from the Agronomic Evaluation Grain Yield from the Agronomic Evaluation of WA Germplasmof WA Germplasm
0
.5
1
1.5
2
2.5
3
IMP INT Land USA
100 seeds (g)
2.59 2.78
2.372.51
Same color =No difference
IMP = Improved Africa(6)Land = Landrace(13)INT = Interspecific (7)USA = checks (8)
Grain weight from the Agronomic Evaluation Grain weight from the Agronomic Evaluation Field of WA GermplasmField of WA Germplasm
US variety CCDR and African US variety CCDR and African improved WAB 56-104improved WAB 56-104
CCDR7004 kg/ha74 days99.25 cm
WAB 56-1044540 kg/ha69 days127 cm
African Landrace Gninni Zeba and African Landrace Gninni Zeba and interspecific NERICA 5interspecific NERICA 5
Gninni Zeba3063 kg/ha105 days162 cm
NERICA 56229 kg/ha69 days109 cm
Total and Whole Milling Yield of Total and Whole Milling Yield of Varieties Grown in the USAVarieties Grown in the USA
% Total Mill-Top 10% Total Mill-Top 10Gnanle Gnan-Man (78)Gnanle Gnan-Man (78)Baldo (77)Baldo (77)Bengal (76.6)Bengal (76.6)Nerica 2 (76.5)Nerica 2 (76.5)Mahafin (76.4)Mahafin (76.4)Nerica 5 (76.2)Nerica 5 (76.2)ZHE733(BMT) (76)ZHE733(BMT) (76)Nerica 1 (76)Nerica 1 (76)Mokossi (76)Mokossi (76)Nerica 3 (76)Nerica 3 (76)
% Whole Mill-Top 10% Whole Mill-Top 10Bengal (70)Bengal (70)CPRS (68)CPRS (68)Saber (68)Saber (68)Nerica 3 (67.7)Nerica 3 (67.7)Saber (BMT) (67.5)Saber (BMT) (67.5)Cheniere (66.7)Cheniere (66.7)Nerica 4 (66.5)Nerica 4 (66.5)WAB 638-1 (66.1)WAB 638-1 (66.1)Bakue Danane (66.05)Bakue Danane (66.05)CCDR (65)CCDR (65)
Plots: 3 rows of 4.57 m length; spacing 17.78 cm Plots: 3 rows of 4.57 m length; spacing 17.78 cm between rowsbetween rowsRCBD, 4 RepRCBD, 4 Rep
Grain Characteristics like Grain Width and Grain Characteristics like Grain Width and Total Mill can affect Cooking timeTotal Mill can affect Cooking time
CTCT GLGL GWGW GLWRGLWR TOTALTOTAL
GLGL NSNS
GWGW 0.54**0.54** -0.41**-0.41**
GLWRGLWR NSNS 0.76**0.76** -0.89**-0.89**
TOTALTOTAL 0.66**0.66** NSNS 0.41**0.41** NSNS
WHOLEWHOLE NSNS NSNS -0.34*-0.34* 0.34*0.34* NSNS
1% change in breakage can cause a $100,000 difference in profit for an average-sized rice mill (Hosney 1998)
Aroma Content 2-AP (ng/g) ofAroma Content 2-AP (ng/g) ofCultivars grown in the USACultivars grown in the USA
SierraSierra 1258.83 a1258.83 a
Bakue DananeBakue Danane 1140 ab1140 ab
CocoteCocote 1102 ab1102 ab
WAB 638-1WAB 638-1 1075.33 b1075.33 b
Jasmine 85Jasmine 85 494 c494 c
Nerica 1Nerica 1 444 c444 c
Protein Content (%) ofProtein Content (%) ofCultivars Grown in the USACultivars Grown in the USA
Observations5.5
6
6.5
7
7.5
8
8.5
9
9.5
prot
ein
Mean
+1 SD
-1 SD
Cheniere (9.1)Cheniere (9.1)Jaya (9)Jaya (9)Nerica 2 (9)Nerica 2 (9)ZHE733 (BMT)(9)ZHE733 (BMT)(9)ZHE733 (8.8)ZHE733 (8.8)IITA 123(a) (8.8)IITA 123(a) (8.8)Bengal (8.7)Bengal (8.7)BG 90-2 (b) (8.6)BG 90-2 (b) (8.6)IITA 123 (b) (8.5)IITA 123 (b) (8.5)
Amylose classes and Amylose classes and WaxyWaxy gene gene
Starch = amylose + amylopectin (60-80%) of edible weight of Starch = amylose + amylopectin (60-80%) of edible weight of cereal.cereal.
Starch comprises 90% of the total dry matter of milled rice (Bao et Starch comprises 90% of the total dry matter of milled rice (Bao et al. 2002). al. 2002). The cooking and eating quality of rice is mainly The cooking and eating quality of rice is mainly influenced by the properties of starchinfluenced by the properties of starch. .
Smith et al. (1997): GBSS= Smith et al. (1997): GBSS= wxwx protein is the product of waxy protein is the product of waxy gene, plays roles in the synthesis of amylose.gene, plays roles in the synthesis of amylose.
Starch branching enzyme, soluble starch synthase, and starch Starch branching enzyme, soluble starch synthase, and starch debranching enzyme plays major role in the synthesis of debranching enzyme plays major role in the synthesis of amylopectin.amylopectin.
No amylose (waxyNo amylose (waxy): very soft and extremely sticky (0%)): very soft and extremely sticky (0%) Low amyloseLow amylose: firm, separate, non sticky (10-19%): firm, separate, non sticky (10-19%) Intermediate:Intermediate: (20-24%) (20-24%) High amyloseHigh amylose: extra firm, low solid loss during processing, : extra firm, low solid loss during processing,
superior kernel stability (>24%)superior kernel stability (>24%)
AmyloseAmylopectin
Glucose molecule
Distribution of Distribution of WaxyWaxy Alleles in Alleles in WARDA Materials Grown in AfricaWARDA Materials Grown in Africa
02468
101214
103 105 114 116 118 122 124 HEWaxy allele
Co
un
t
DXBL
Soft Cooking
Conv. LG
PB/Canning
Distribution of the Distribution of the Waxy Waxy allele among allele among the interspecifics Grown in Africathe interspecifics Grown in Africa
21.1 21.7 22.2 22.6 23.5 23.6 24.7 25.3 26.1
0
5
10
15
20
25
30
WAB 5
6-10
4
NERICA 3
NERICA 7
NERICA 4
NERICA 6
NERICA 2
NERICA 5
NERICA 1
CG14
AA
%
Waxy 103-105Waxy 124
Marker Associations with Cooking Marker Associations with Cooking Quality TraitsQuality Traits
Cocodrie CCDRCocodrie CCDR: Cypress//L-202/Tebonnet at Louisiana in : Cypress//L-202/Tebonnet at Louisiana in
1990. 1990.
Dixiebelle DXBLDixiebelle DXBL: RU8303181/CB801 at Beaumont in 1983: RU8303181/CB801 at Beaumont in 1983
Brown rice was used for DNA extraction using QiagenBrown rice was used for DNA extraction using Qiagen
Kit. Kit. PCR was used for amplification followed by evaluation for PCR was used for amplification followed by evaluation for
polymorphisms using ABI sequencerpolymorphisms using ABI sequencer
Diagrammatic Representation of the Waxy Gene
Exon 6: A C transversion and substitution changes a Tyrosine to SerineDifferences in DNA sequence of Rexmont, JODON, and Toro-2 from lemont-intermediate-High/lowLarkin and Park. 2003
CT repeats associated with apparent amylose content(CT)10 & (CT)11= high(CT)14 & (CT) 20= int(CT)17 & (ct)18= low
Ayres et al. (1997)Bergman et al. (2001)
Exon 1G T substitution is associated with low amylose varieties.-interm. /high-low amyloseAyres et al. (1997)
Exon 10: C T transition and substitution changes a Proline to Serine. Differences in DNA sequence of Rexmont from Jodon, Toro-2 and Lemont.-high amyl.strong RVA-othersLarkin and Park (2003)
G/TTATAC------ (CT)n--
Rexmont= high amylose strong RVALemont= intermediate amyloseJodon, L202= high amylose, weak RVAToro-2= low amylose Adapted from Chen (2004)
CCDR et DXBL ont la même teneur en CCDR et DXBL ont la même teneur en Amylose (~26%) mais DiffAmylose (~26%) mais Diffèèrent en RVArent en RVA
0
100
200
300
400
45
60
75
90
105
00 3 6 9 12 1515Time minutes
Temp oC
CCDR
DXBLTemperature profile
RVU
Peak
Hot
Cool
Bkdn=Peak- hotStbk= Cool-PeakCS= Cool- Hot
PCR Primers Used for Molecular PCR Primers Used for Molecular Marker AnalysisMarker Analysis
PrimersPrimers AnnealinAnnealing temp.g temp.
SequenceSequence Starch Starch metabolismetabolism genem gene
Map Map location location /Chromo. /Chromo. Loc.Loc.
WaxyWaxy 5555 5’-CTTTGTCTATCTCAAGACAC-5’-CTTTGTCTATCTCAAGACAC-3’3’
5’-TTGCAGATGTTCTTCCTGATG-5’-TTGCAGATGTTCTTCCTGATG-3’3’
GBSSGBSS 6-8.26-8.2
Exon 10Exon 10 6666 5’-GCGGCCATGACGTCTGG-3’5’-GCGGCCATGACGTCTGG-3’
5’-GGCGGCCATGACGTCTGA-3’5’-GGCGGCCATGACGTCTGA-3’GBSSGBSS 6-8.26-8.2
AB2628AB262855
5555 5’-CTAGCCATGCTCTCGTACC-3’5’-CTAGCCATGCTCTCGTACC-3’
5’-5’-CAACTTACTGTGACTGACTTGG-CAACTTACTGTGACTGACTTGG-3’3’
SSSISSSI 6-15.36-15.3
21 PCR markers were selected and screened for marker 21 PCR markers were selected and screened for marker association study. The markers were either:association study. The markers were either:-near starch metabolism (like SSS, SBE )-near starch metabolism (like SSS, SBE )-at a map position with significant effects on starch properties -at a map position with significant effects on starch properties (like amylose content, or RVA pasting properties(like amylose content, or RVA pasting properties
Waxy, exon10, and AB26285 showed association with the amylose and RVA properties.
Single Factor Analysis for the 3 Markers used for Single Factor Analysis for the 3 Markers used for Associations StudyAssociations Study
Source Source variationvariation
dfdf AAAA SASA IAIA PEAKPEAK HOTHOT COOLCOOL
WaxyWaxy 22 1.04*1.04* 78.00**78.00** 60.3360.33 116548.61**116548.61** 98433.24**98433.24** 26737.78**26737.78**
AdditiveAdditive 11 2.08*2.08* 155.75**155.75** 120.39120.39 232946.49**232946.49** 196828.50*196828.50***
52458.07**52458.07**
DominantDominant 11 NSNS NSNS NSNS 1124.02*1124.02* NSNS NSNS
R^2R^2 0.040.04 0.630.63 0.540.54 0.800.80 0.810.81 0.480.48
Exon 10Exon 10 22 1.18*1.18* 76.56**76.56** 58.02**58.02** 112729.13**112729.13** 95577.79**95577.79** 246835.08*246835.08***
AdditiveAdditive 11 2.35**2.35** 153.07**153.07** 115.99**115.99** 225123.11**225123.11** 190999.55*190999.55***
493627.87*493627.87***
DominantDominant 11 NSNS NSNS NSNS NSNS NSNS NSNS
R^2R^2 0.040.04 0.610.61 0.520.52 0.770.77 0.800.80 0.790.79
AB26285AB26285 22 NSNS 30.41**30.41** 24.52**24.52** 56690.88**56690.88** 46866.25**46866.25** 112115.44*112115.44***
AdditiveAdditive 11 NSNS 60.77**60.77** 48.92**48.92** 112538.50**112538.50** 93473.63**93473.63** 223082.71*223082.71***
DominantDominant 11 NSNS NSNS NSNS NSNS NSNS NSNS
R^2R^2 0.010.01 0.240.24 0.220.22 0.380.38 0.390.39 0.360.36
R^2= Total Phenotypic explanation (%)
Summary and ConclusionsSummary and Conclusions
Interspecifics were found interesting for reduced Interspecifics were found interesting for reduced water rice growing, more studies can elucidate water rice growing, more studies can elucidate these findingsthese findings
Nerica 2 had good agronomic and milling Nerica 2 had good agronomic and milling characteristicscharacteristics
Bakue Danane, Cocote, WAB 638-1 had strong Bakue Danane, Cocote, WAB 638-1 had strong aromaaroma
Jaya, Nerica 2, BG 90-2, IITA 123 had high level of Jaya, Nerica 2, BG 90-2, IITA 123 had high level of ProteinProtein
Summary and Conclusions (cont.)Summary and Conclusions (cont.)
Soluble Amylose (SA) explained more the Soluble Amylose (SA) explained more the difference in RVA profile than the Apparent difference in RVA profile than the Apparent Amylose (AA).Amylose (AA).
Different GBSS alleles may produce the same Different GBSS alleles may produce the same amount of total amylose but different proportions amount of total amylose but different proportions of soluble and insoluble amylose. of soluble and insoluble amylose.
The The Waxy Waxy microsatellite and waxy microsatellite and waxy exon 10 SNPexon 10 SNP markers are now useful molecular markers for markers are now useful molecular markers for rapid and efficient identification of cooking quality rapid and efficient identification of cooking quality traits that can be difficult to separate with only traits that can be difficult to separate with only physico-chemical data.physico-chemical data.
AcknowledgementsAcknowledgements
I wish to express my sincere I wish to express my sincere gratitude to:gratitude to:
WARDAWARDA Rockefeller Rockefeller
foundationfoundation Texas A&M (Soil and Texas A&M (Soil and
Crop Sciences)Crop Sciences) USDA-ARS BeaumontUSDA-ARS Beaumont