Barley1 Leaf gcRMAn SCRI (Dec06)

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Summary

Barley1 Leaf MAS 5.0 SCRI (Dec 06) - integrated probe set value for each gene has been calculated using MAS 5.0 algorithm which uses pixel values from both, PM and MM probes. Descriptions of probe set signal calculation can be found on this page below, section 'About Data Processing'.

The SCRI barley data set provides estimates of mRNA abundance in doubled haploid recombinant lines of cultivated barley. Embryo-derived tissues at four days after imbibition (150 lines) and seedling leaves at 12 days after imbibition (subset of 34 lines) and three biological replicates of each parental cultivar (Steptoe and Morex) for each tissue were used for the isolation of total RNA and hybridization to the Barley1 22K GeneChip (GEO GPL1340).

Experiment design

RNA Sample Processing:

Trizol RNA isolation and RNeasy clean up protocol for whole plants (embryo-derived tissue dissected from 4 days old germinating grains) and the seedling leaves (12 days after planting).


☐ Grind tissue (9 embryos) with a mortar and pestle in liquid nitrogen
☐ Add 5 ml TRIzol (pre-heated to 60oC) to all samples, vortex until all the tissue is thawed, place in the 60oC waterbath..
☐ Incubate samples at 60oC for 10 minutes, vortexing three times.
☐ Centrifuge @ 4000 x rpm @ 4C for 30 minutes (in Eppendorf 5810R).
☐ While centrifuging, label new set of 15 ml tubes
☐ Transfer supernatant to 15 ml centrifuge tube
☐ Add 1 ml of chloroform. Vortex the sample until color shade is uniform at least 5
seconds, and incubate at room temperature for 5 minutes.
☐ Centrifuge @ 4000 x rpm for 30 minutes @ 4oC.
☐ While centrifuging, label new 15 ml tubes
☐ Collect the upper aqueous layer (there will be about 3 mls) and transfer to a new 15 ml tube.
☐ Add 0.6 volumes (2 ml) of isopropanol, mix gently, incubate at room temperature for 20 minutes.
☐ Centrifuge @ 4000 rpm for 30 minutes @ 4oC.
☐ Wash the pellet with 10 ml of cold 75% ethanol. Swirl & centrifuge at
4000 rpm for 15 minutes @ 4oC.
☐ Discard supernatant, centrifuge for 5 min, remove the rest of the ethanol
☐ Air-dry the pellet for 10 minutes, inverted on a kimwipe.
☐ Dissolve pellet in 400 ul of DEPC-treated H2O. Resuspend by pipeting up & down a
few times.
☐ Add 2 ul SuperaseIn. Incubate at 60oC for 10 minutes to resuspend.
☐ Set water bath to 37oC.
☐ Add 50 ul 10X DnaseI Buffer, 45 ul H2O and 5 ul of DnaseI, incubate at 37oC for 1 hr.
☐ Prepare Buffer RLT (Rneasy Clean-up Midi Kit) by adding b-mercaptoethanol (10ul/1ml RLT).
☐ Add 2.0 ml Buffer RLT to the RNA prep and mix thoroughly.
☐ Add 1.4 ml ethanol (96-100%) to the diluted RNA. Mix thoroughly.
☐ Label 15 ml tubes from the kit and place midi columns in them
☐ Apply sample to a Midi column, close tube gently and centrifuge for 20 min at 3000 rpm.
☐ Discard the flow-through.
☐ Add 2.5 ml Buffer RPE to the RNA easy column, close the centrifuge tube gently,
incubate for 3 min
☐ Centrifuge for 10 min at 3000 rpm. Discard the flow-through.
☐ Add another 2.5 ml Buffer RPE to the RNeasy column. Close the centrifuge tube
gently, incubate for 3 min
☐ Centrifuge for 10 min at 3000 rpm, remove flow-through
☐ Centrifuge again for another 5 min.
☐ Label new 15 ml tubes from the kit.
☐ Transfer the RNA easy column to a new tube and pipet 250 ul volume of
RNase-free water directly onto the RNeasy silica-membrane incubate for 1 min
☐ Centrifuge for 5 min at 3000 rpm.
☐ To the same tube add again 250 ul H2O, incubate for 1 min.
☐ Centrifuge for 5 min at 3000 rpm.
☐ Label two sets of 1.5 ml Eppendorf tubes.
☐ Transfer 490 ul to the one tube and 10 ul to another one. Use 10 ul tube for the RNA

Detailed descriptions of these procedures can be found under the ArrayExpress (http://www.ebi.ac.uk/aerep/?) protocol P-MEXP-4631 (Caldo et al. 2004).

Replication and Sample Balance:

3 independent replicates of both parental cultivars Steptoe and Morex were generated for both tissues, embryo and seedling leaf.

Experimental Design and Batch Structure:

The following are ArrayExpress (http://www.ebi.ac.uk/aerep/?) experiment IDs: E-TABM-111 (leaf, 41 chips) and E-TABM-112 (embryo derived, 156 chips).

About cases

The SM cross was originally made to map barley grain quality traits; Steptoe is high-yielding barley cultivar used for animal feeding, but Morex has good malting barley characteristics (Hayes et al 1993). Many agronomic quality traits have been mapped using this population (for the lists see BeerGenes web-site http://gnome.agrenv.mcgill.ca/bg/).

The sample used in this study consists of 150 Steptoe x Morex doubled haploid recombinant lines (Kleinhofs et al. 1993) was used to obtain embryo-derived tissue. For the seedling leaf tissue a subset of 35 lines was used. This subset was selected based on evenly spaced crossovers along each of seven barley chromosomes. The expression data of 11 DH lines has been removed from both, embryo and leaf, leaving for the analysis 129 lines with embryo expression data and a subset of 30 lines with seedling leaf expression data. The lines were removed from the analysis after error checking; discrepancies with genotyping data were found. We left all 150 lines in the embryo Apr06 data set and the full data set is available from the ArrayExpress. The following table lists line IDs and corresponding CEL file IDs, also indicating:
1) pedigree; shows the direction of the cross that was used to produce the original F1. The parental plants were given letter codes of A - Z. For example, SM1 was derived from an F1 that was generated by crossing Steptoe plant "B" as a female with Morex plant "F" as a male.
2) 'minimapper' subset - MINI;
3) lines that have expression data removed - ERROR:

Order # Line ID Permanent Oregon ID Cross direction
CEL file names
 Mini-mapper set Error check
embryo data-set leaf data-set embryo data-set leaf data-set
1 SM001 2907001 Steptoe/Morex(BxF) AD_SCRI_82.CEL     OK  
2 SM002 2907002 Steptoe/Morex(BxF) AD_SCRI_1.CEL     OK  
3 SM003 2907003 Morex/Steptoe(CxF) AD_SCRI_19.CEL     OK  
4 SM004 2907004 Morex/Steptoe(CxF) AD_SCRI_3.CEL 0521-1_SetA1.CEL SMmini OK OK
5 SM005 2907005 Steptoe/Morex(BxH) AD_SCRI_88.CEL     OK  
6 SM006 2907006 Morex/Steptoe(CxF) AD_SCRI_48.CEL     OK  
7 SM007 2907007 Steptoe/Morex(BxH) AD_SCRI_35.CEL 0521-2_SetA2.CEL SMmini OK OK
8 SM009 2907009 Steptoe/Morex(BxF) AD_SCRI_2.CEL     OK  
9 SM010 2907010 Morex/Steptoe(IxE) AD_SCRI_42.CEL     OK  
10 SM011 2907011 Steptoe/Morex(QxG) AD_SCRI_10.CEL     OK  
11 SM012 2907012 Morex/Steptoe(CxF) AD_SCRI_45.CEL 0521-3_SetA3.CEL SMmini ERROR ERROR
12 SM013 2907013 Morex/Steptoe(IxE) AD_SCRI_78.CEL 0521-4_SetA4.CEL SMmini ERROR ERROR
13 SM014 2907014 Steptoe/Morex(BxH) AD_SCRI_18.CEL     OK  
14 SM015 2907015 Steptoe/Morex(BxH) AD_SCRI_5.CEL     OK  
15 SM016 2907016 Steptoe/Morex(BxH) AD_SCRI_21.CEL     OK  
16 SM020 2907020 Steptoe/Morex(OxJ) AD_SCRI_77.CEL     OK  
17 SM021 2907021 Morex/Steptoe(IxE) AD_SCRI_30.CEL     OK  
18 SM022 2907022 Morex/Steptoe(IxE) AD_SCRI_31.CEL 0521-5_SetA5.CEL SMmini OK OK
19 SM023 2907023 Steptoe/Morex(BxH) AD_SCRI_32.CEL     OK  
20 SM024 2907024 Morex/Steptoe(IxE) AD_SCRI_33.CEL 0521-6_SetA6.CEL SMmini OK OK
21 SM025 2907025 Morex/Steptoe(CxF) AD_SCRI_34.CEL     OK  
22 SM027 2907027 Steptoe/Morex(OxJ) AD_SCRI_12.CEL 0521-7_SetA7.CEL SMmini OK OK
23 SM030 2907030 Morex/Steptoe(IxE) AD_SCRI_79.CEL     OK  
24 SM031 2907031 Steptoe/Morex(OxJ) AD_SCRI_16.CEL     OK  
25 SM032 2907032 Morex/Steptoe(IxE) AD_SCRI_13.CEL     OK  
26 SM035 2907035 Morex/Steptoe(CxF) AD_SCRI_15.CEL     ERROR  
27 SM039 2907039 Morex/Steptoe(CxF) AD_SCRI_41.CEL     OK  
28 SM040 2907040 Steptoe/Morex(BxH) AD_SCRI_83.CEL     OK  
29 SM041 2907041 Steptoe/Morex(OxJ) AD_SCRI_11_redo.CEL 0521-8_SetA8.CEL SMmini OK OK
30 SM042 2907042 Morex/Steptoe(CxF) AD_SCRI_57.CEL     OK  
31 SM043 2907043 Morex/Steptoe(JxE) AD_SCRI_49.CEL 0521-9_SetA9.CEL SMmini OK OK
32 SM044 2907044 Steptoe/Morex(OxJ) AD_SCRI_50.CEL 0521-10_SetA10.CEL SMmini OK OK
33 SM045 2907045 Steptoe/Morex(BxH) AD_SCRI_51.CEL     OK  
34 SM046 2907046 Steptoe/Morex(OxJ) AD_SCRI_52.CEL 0521-11_SetA11.CEL SMmini OK OK
35 SM048 2907048 Steptoe/Morex(BxF) AD_SCRI_53.CEL     ERROR  
36 SM050 2907050 Morex/Steptoe(IxE) AD_SCRI_46.CEL     OK  
37 SM054 2907054 Morex/Steptoe(CxF) AD_SCRI_60.CEL     OK  
38 SM055 2907055 Steptoe/Morex(OxJ) AD_SCRI_55.CEL     OK  
39 SM056 2907056 Steptoe/Morex(BxH) AD_SCRI_23.CEL     OK  
40 SM057 2907057 Morex/Steptoe(CxF) AD_SCRI_24.CEL     OK  
41 SM058 2907058 Steptoe/Morex(BxF) AD_SCRI_22.CEL     OK  
42 SM059 2907059 Steptoe/Morex(BxH) AD_SCRI_27.CEL     OK  
43 SM061 2907061 Morex/Steptoe(LxF) AD_SCRI_81.CEL 0521-12_SetA12.CEL SMmini OK OK
44 SM062 2907062 Morex/Steptoe(CxF) AD_SCRI_44.CEL     OK  
45 SM063 2907063 Steptoe/Morex(OxJ) AD_SCRI_40.CEL 0521-13_SetA13.CEL SMmini OK OK
46 SM064 2907064 Morex/Steptoe(CxF) AD_SCRI_87_redo.CEL     OK  
47 SM065 2907065 Morex/Steptoe(CxF) AD_SCRI_54.CEL     OK  
48 SM067 2907067 Steptoe/Morex(OxJ) AD_SCRI_73.CEL     OK  
49 SM068 2907068 Steptoe/Morex(OxG) AD_SCRI_56.CEL     ERROR  
50 SM069 2907069 Steptoe/Morex(BxH) AD_SCRI_71.CEL     OK  
51 SM070 2907070 Steptoe/Morex(BxF) AD_SCRI_64.CEL     OK  
52 SM071 2907071 Steptoe/Morex(BxH) AD_SCRI_58.CEL     OK  
53 SM072 2907072 Morex/Steptoe(CxF) AD_SCRI_59.CEL     OK  
54 SM073 2907073 Steptoe/Morex(BxF) AD_SCRI_74.CEL 0521-14_SetA14.CEL SMmini OK ERROR
55 SM074 2907074 Morex/Steptoe(CxF) AD_SCRI_25.CEL 0521-15_SetA15.CEL SMmini OK OK
56 SM075 2907075 Steptoe/Morex(QxG) AD_SCRI_120.CEL     OK  
57 SM076 2907076 Steptoe/Morex(BxF) AD_SCRI_112.CEL     OK  
58 SM077 2907077 Morex/Steptoe(CxF) AD_SCRI_142.CEL     OK  
59 SM078 2907078 Steptoe/Morex(BxF) AD_SCRI_86.CEL     OK  
60 SM079 2907079 Morex/Steptoe(CxF) AD_SCRI_153.CEL 0521-16_SetA16.CEL SMmini OK ERROR
61 SM080 2907080 Steptoe/Morex(BxF) AD_SCRI_107.CEL     OK  
62 SM081 2907081 Morex/Steptoe(CxF) AD_SCRI_105.CEL     OK  
63 SM082 2907082 Steptoe/Morex(BxF) AD_SCRI_97.CEL     OK  
64 SM083 2907083 Steptoe/Morex(BxF) AD_SCRI_89.CEL     OK  
65 SM084 2907084 Morex/Steptoe(CxF) AD_SCRI_155.CEL     OK  
66 SM085 2907085 Morex/Steptoe(IxE) AD_SCRI_149.CEL 0521-17_SetA17.CEL SMmini OK OK
67 SM087 2907087 Steptoe/Morex(OxJ) AD_SCRI_113.CEL     OK  
68 SM088 2907088 Morex/Steptoe(CxF) AD_SCRI_93.CEL 0521-18_SetA18.CEL SMmini OK OK
69 SM089 2907089 Steptoe/Morex(OxJ) AD_SCRI_148.CEL 0521-19_SetA19.CEL SMmini OK OK
70 SM091 2907091 Morex/Steptoe(CxF) AD_SCRI_110.CEL     OK  
71 SM092 2907092 Steptoe/Morex(OxJ) AD_SCRI_7.CEL     OK  
72 SM093 2907093 Steptoe/Morex(BxF) AD_SCRI_122.CEL     OK  
73 SM094 2907094 Morex/Steptoe(CxF) AD_SCRI_150.CEL     OK  
74 SM097 2907097 Morex/Steptoe(CxF) AD_SCRI_158.CEL     OK  
75 SM098 2907098 Morex/Steptoe(CxF) AD_SCRI_121.CEL     OK  
76 SM099 2907099 Steptoe/Morex(QxG) AD_SCRI_137.CEL     OK  
77 SM103 2907103 Morex/Steptoe(IxE) AD_SCRI_156.CEL     OK  
78 SM104 2907104 Steptoe/Morex(BxH) AD_SCRI_70.CEL     ERROR  
79 SM105 2907105 Morex/Steptoe(IxE) AD_SCRI_69.CEL     OK  
80 SM110 2907110 Morex/Steptoe(CxF) AD_SCRI_75.CEL     ERROR  
81 SM112 2907112 Steptoe/Morex(BxF) AD_SCRI_84.CEL     OK  
82 SM116 2907116 Morex/Steptoe(CxF) AD_SCRI_117.CEL 0521-20_SetA20.CEL SMmini OK OK
83 SM120 2907120 Steptoe/Morex(OxJ) AD_SCRI_138.CEL     OK  
84 SM124 2907124 Steptoe/Morex(BxF) AD_SCRI_146.CEL     OK  
85 SM125 2907125 Morex/Steptoe(IxE) AD_SCRI_43.CEL     OK  
86 SM126 2907126 Steptoe/Morex(OxJ) AD_SCRI_144_redo.CEL     OK  
87 SM127 2907127 Steptoe/Morex(BxH) AD_SCRI_129.CEL     OK  
88 SM129 2907129 Steptoe/Morex(OxJ) AD_SCRI_132.CEL     OK  
89 SM130 2907130 Morex/Steptoe(CxF) AD_SCRI_101.CEL 0521-21_SetA21.CEL SMmini OK OK
90 SM131 2907131 Steptoe/Morex(OxJ) AD_SCRI_102.CEL     OK  
91 SM132 2907132 Steptoe/Morex(QxG) AD_SCRI_4_redo.CEL     OK  
92 SM133 2907133 Morex/Steptoe(CxF) AD_SCRI_157.CEL     OK  
93 SM134 2907134 Morex/Steptoe(IxE) AD_SCRI_159.CEL     OK  
94 SM135 2907135 Steptoe/Morex(BxF) AD_SCRI_72.CEL 0521-22_SetA22.CEL SMmini OK OK
95 SM136 2907136 Steptoe/Morex(QxG) AD_SCRI_123.CEL 0521-23_SetA23.CEL SMmini OK OK
96 SM137 2907137 Steptoe/Morex(BxH) AD_SCRI_39.CEL     OK  
97 SM139 2907139 Morex/Steptoe(CxF) AD_SCRI_133.CEL     OK  
98 SM140 2907140 Morex/Steptoe(CxF) AD_SCRI_134.CEL 0521-24_SetA24.CEL SMmini OK OK
99 SM141 2907141 Steptoe/Morex(BxH) AD_SCRI_136.CEL 0521-25_SetA25.CEL SMmini OK OK
100 SM142 2907142 Morex/Steptoe(IxE) AD_SCRI_6.CEL     OK  
101 SM143 2907143 Steptoe/Morex(BxH) AD_SCRI_145.CEL     OK  
102 SM144 2907144 Steptoe/Morex(BxF) AD_SCRI_103.CEL     OK  
103 SM145 2907145 Steptoe/Morex(QxG) AD_SCRI_108.CEL     OK  
104 SM146 2907146 Morex/Steptoe(BxF) AD_SCRI_91.CEL 0521-26_SetA26.CEL SMmini OK OK
105 SM147 2907147 Steptoe/Morex(OxJ) AD_SCRI_139.CEL     OK  
106 SM149 2907149 Steptoe/Morex(BxF) AD_SCRI_131.CEL     ERROR  
107 SM150 2907150 Morex/Steptoe(CxF) AD_SCRI_37.CEL     OK  
108 SM151 2907151 Morex/Steptoe(IxE) AD_SCRI_28.CEL     OK  
109 SM152 2907152 Steptoe/Morex(BxH) AD_SCRI_9_redo.CEL 0521-27_SetA27.CEL SMmini OK OK
110 SM153 2907153 Steptoe/Morex(BxH) AD_SCRI_135.CEL     OK  
111 SM154 2907154 Steptoe/Morex(BxH) AD_SCRI_114.CEL     OK  
112 SM155 2907155 Steptoe/Morex(BxH) AD_SCRI_119.CEL 0521-28_SetA28.CEL SMmini OK OK
113 SM156 2907156 Steptoe/Morex(BxH) AD_SCRI_140.CEL     OK  
114 SM157 2907157 Morex/Steptoe(CxF) AD_SCRI_106_redo.CEL     OK  
115 SM158 2907158 Morex/Steptoe(CxF) AD_SCRI_65.CEL     OK  
116 SM159 2907159 Morex/Steptoe(IxE) AD_SCRI_168.CEL     OK  
117 SM160 2907160 Steptoe/Morex(OxJ) AD_SCRI_47.CEL 0521-29_SetA29.CEL SMmini OK ERROR
118 SM161 2907161 Steptoe/Morex(BxH) AD_SCRI_76.CEL     ERROR  
119 SM162 2907162 Morex/Steptoe(CxF) AD_SCRI_147.CEL     OK  
120 SM164 2907164 Steptoe/Morex(OxJ) AD_SCRI_128.CEL     OK  
121 SM165 2907165 Steptoe/Morex(BxH) AD_SCRI_143.CEL     OK OK
122 SM166 2907166 Morex/Steptoe(CxF) AD_SCRI_115.CEL     OK  
123 SM167 2907167 Steptoe/Morex(BxH) AD_SCRI_127.CEL 0521-30_SetA30.CEL SMmini OK OK
124 SM168 2907168 Steptoe/Morex(BxH) AD_SCRI_130.CEL     OK  
125 SM169 2907169 Morex/Steptoe(CxF) AD_SCRI_118.CEL 0521-31_SetA31.CEL SMmini OK OK
126 SM170 2907170 Steptoe/Morex(BxF) AD_SCRI_151.CEL     OK  
127 SM171 2907171 Steptoe/Morex(BxF) AD_SCRI_165.CEL     ERROR  
128 SM172 2907172 Steptoe/Morex(OxJ) AD_SCRI_152.CEL     ERROR  
129 SM173 2907173 Steptoe/Morex(OxJ) AD_SCRI_104.CEL 0521-32_SetA32.CEL SMmini OK OK
130 SM174 2907174 Steptoe/Morex(BxH) AD_SCRI_154.CEL     OK  
131 SM176 2907176 Morex/Steptoe(CxF) AD_SCRI_141.CEL     OK  
132 SM177 2907177 Morex/Steptoe(CxF) AD_SCRI_111.CEL 0521-33_SetA33.CEL SMmini OK OK
133 SM179 2907179 Morex/Steptoe(CxF) AD_SCRI_166.CEL     OK  
134 SM180 2907180 Morex/Steptoe(IxE) AD_SCRI_161.CEL     OK  
135 SM181 2907181 Morex/Steptoe(IxE) AD_SCRI_162.CEL     OK  
136 SM182 2907182 Morex/Steptoe(CxF) AD_SCRI_163.CEL     OK  
137 SM183 2907183 Morex/Steptoe(CxF) AD_SCRI_164.CEL     OK  
138 SM184 2907184 Morex/Steptoe(IxE) AD_SCRI_160.CEL 0521-34_SetA34.CEL SMmini OK OK
139 SM185 2907185 Morex/Steptoe(IxE) AD_SCRI_167.CEL     OK  
140 SM186 2907186 Morex/Steptoe(IxE) AD_SCRI_62.CEL     OK  
141 SM187 2907187 Morex/Steptoe(IxE) AD_SCRI_61.CEL     OK  
142 SM188 2907188 Morex/Steptoe(CxF) AD_SCRI_63.CEL     OK  
143 SM189 2907189 Steptoe/Morex(QxG) AD_SCRI_80.CEL     OK  
144 SM193 2907193 Morex/Steptoe(IxE) AD_SCRI_36.CEL     OK  
145 SM194 2907194 Steptoe/Morex(OxJ) AD_SCRI_29.CEL     OK  
146 SM196 2907196 Steptoe/Morex(BxF) AD_SCRI_26.CEL     OK  
147 SM197 2907197 Steptoe/Morex(BxF) AD_SCRI_85.CEL     OK  
148 SM198 2907198 Morex/Steptoe(IxE) AD_SCRI_8.CEL     OK  
149 SM199 2907199 Steptoe/Morex(BxF) AD_SCRI_20.CEL     OK  
150 SM200 2907200 Morex/Steptoe(IxE) AD_SCRI_38.CEL 0521-35_SetA35.CEL SMmini OK OK
parent Steptoe     AD_SCRI_17.CEL 0521-36_SetA36.CEL      
parent Steptoe     AD_SCRI_66.CEL 0521-37_SetA37.CEL      
parent Steptoe     AD_SCRI_68.CEL 0521-38_SetA38.CEL      
parent Morex     AD_SCRI_116.CEL 0521-39_SetA39.CEL      
parent Morex     AD_SCRI_14.CEL 0521-40_SetA40.CEL      
parent Morex     AD_SCRI_67.CEL 0521-41_SetA41.CEL      

 

About tissue

Plant material according to the current plant ontologies: Embryo-derived tissues: whole plant (PO:0000003) at the development stage 1.05-coleoptile emerged from seed (GRO:0007056); Seedling leaves: primary shoot (PO:0006341) at the developmental stage 2.02-first leaf unfolded (GRO:0007060) (Druka et al. 2006).

To obtain embryo-derived tissue, growth room#2, AN building, SCRI, with the standard laboratory bench positioned in the middle of the room was used to germinate sterilized seeds. Seeds were placed between three layers of wet 3MM filter paper in the 156 10 mm Petri plates. Thirty to fifty seeds per line (per Petri plate) were used. Germination was in the dark, 16 hours at 17 deg C and 8 hours at 12 deg C. After 96 hours, embryo-derived tissue (mesocotyl, coleoptile, and seminal roots) from three grains was dissected and flash frozen in the liquid nitrogen. Germination and collection was repeated two more times. Complete randomization of the Petri plates was done for each germination event. Tissues from all three germinations (collections) were bulked before RNA isolation. Three replicates of the parental cultivars were germinated for each collection.

To obtain seedling leaves, three Microclima 1000 growth chambers (Snijders Scientific B.V., Tilburg, Holland) were used for the experiment. Each cabinet accomodated 40 (13x13 cm) pots. Humidity was set to 70%, with light conditions for 16 hours light at 17C and 8 hours dark at 12C. The cycle started at 10 am with lights on. Light intensity was 337-377 mmol m-2 s-1, measured at the beginning of the experiment, 11 cm from the light source. Measurement was done using Sky Quantium light sensor at 15oC. Plants were placed 55 cm from the light source (from the bulb to the surface of the vermiculite). Ten sterilized seeds per pot were planted and 3 pots per genotype / per cabinet were used. After 12 days, leaf blade and sheath from 5-7 the same size plants was cut off, bulked and flash frozen in the liquid nitrogen.

 

About platform

Affymetrix 22K Barley1 GeneChip probe array (http://www.affymetrix.com/products/arrays/specific/barley.affx ; Affymetrix product #900515 GeneChip Barley Genome Array) representing 21,439 non-redundant Barley1 exemplar sequences was derived from worldwide contribution of 350,000 high-quality ESTs from 84 cDNA libraries, in addition to 1,145 barley gene sequences from the National Center for Biotechnology Information non-redundant database (Close et al 2004). Abbreviated annotations were created based on the exemplar sequence homology by Arnis Druka using data from the Harvest (http://harvest.ucr.edu/) data depository.

About data processing

Types of the expression data-sets
Data processing description
Barley1 Embryo gcRMA SCRI (Dec 06)
Barley1 Leaf gcRMA SCRI (Dec 06)

 

The Affymetrix' CEL files that were generated using MAS 5.0 Suite were imported into the GeneSpring GX 7.3 (Agilent Technologies, Palo Alto, CA) and processed using the RMA algorithm.

 
Barley1 Embryo MAS 5.0 SCRI (Dec 06)
Barley1 Leaf MAS 5.0 SCRI (Dec 06)

 

The MAS 5.0 values were calculated from the DAT files using Affymetrix' MAS 5.0 Suite.

 
Barley1 Embryo0 gcRMA SCRI (Apr 06)
Barley1 Leaf gcRMAn SCRI (Dec 06)

The Affymetrix' CEL files were imported into the GeneSpring GX 7.3 (Agilent Technologies, Palo Alto, CA) software and processed using the RMA algorithm. Per-chip and per-gene normalization was done following the standard GeneSpring procedure (citation of the GeneSpring normalization description):

  1. Values below 0.01 were set to 0.01.
  2. Each measurement was divided by the 50.0th percentile of all measurements in that sample.
  3. Each gene was divided by the median of its measurements in all samples. If the median of the raw values was below 10 then each measurement for that gene was divided by 10 if the numerator was above 10, otherwise the measurement was thrown out.

Citation

Druka A, Muehlbauer G, Druka I, Caldo R, Baumann U, Rostoks N, Schreiber A, Wise R, Close T, Kleinhofs A, Graner A, Schulman A, Langridge P, Sato K, Hayes P, McNicol J, Marshall D, Waugh R. (2006) An atlas of gene expression from seed to seed through barley development. Funct Integr Genomics, Jul;6(3):202-11.

Kleinhofs A, Kilian A, Saghai Maroof M, Biyashev R, Hayes P, Chen F, Lapitan N, Fenwick A, Blake T, Kanazin V, Ananiev E, Dahleen L, Kudrna D, Bollinger J, Knapp SJ, Liu BH, Sorrells M, Heun M, Franckowiak J, Hoffman D, Skadsen R, Steffenson B (1993) A molecular, isozyme, and morphological map of the barley (Hordeum vulgare) genome. Theor Appl Genet 86:705-712.

Caldo RA, Nettleton D, Wise RP (2004) Interaction-dependent gene expression in Mla-specified response to barley powdery mildew. Plant Cell 16:2514-2528.

Close TJ, Wanamaker SI, Caldo RA, Turner SM, Ashlock DA, Dickerson JA, Wing RA, Muehlbauer GJ, Kleinhofs A, Wise RP. (2004) A new resource for cereal genomics: 22K barley GeneChip comes of age. Plant Physiol 134:960-968.

Hayes PM, Liu BH, Knapp SJ, Chen F, Jones B, Blake T, Franckowiak J, Rasmusson D, Sorrells M, Ullrich SE, Wesenberg D, Kleinhofs A (1993) Quantitative trait locus effects and environmental interaction in a sample of North American barley germplasm. Theor Appl Genet 87:392-401

Acknowledgment

Plant maintenance, tissue collection, RNA isolation, and data submission to ArrayExpress was done at SCRI by Arnis Druka with support from BBSRC/SEERAD grant SCR/910/04 The genetics of gene expression in barley' to Michael Kearsey (University of Birmingham, UK) and Robbie Waugh (SCRI, UK). Probe synthesis, labeling and hybridization were performed according to manufacturer’s protocols (Affymetrix, Santa Clara, CA) at the Iowa State University GeneChip Core facility (Rico Caldo and Roger Wise). ArrayExpress (EBI, UK) team members Tim Rayner, Helen Parkinson, and Alvis Brazma are acknowledged for excellent help with data submission to ArrayExpress.

Notes

Arnis Druka
Genetics Programme
Scottish Crop Research Institute
Invergowrie, Dundee DD2 5DA
Angus, Scotland, United Kingdom
Tel +44 01382 562731
Fax +44 01382 568587
adruka@scri.sari.ac.uk

This text file originally generated by Arnis Druka on May 8, 2006. Modified Aug1 by AD. Entered by RWW Aug 4, 2006. Modified by AD Jan 29, 2007, Feb 01, 2007.