Supplementary materials
1 Materials and methods
1.1 Plant materials
The mutant gra(k) was obtained from the tissue-cultured rice Kitaake. The gra phenotype was
genetically stable for more than ten generations in both greenhouse and field. The F1 hybrids
and F2 populations derived from the crosses, gra(k) × Jodan and gra(k) × 02428, were used
for genetic analysis and molecular mapping of Gra(k). Temperature treatment was performed
in an illuminated plant growth chamber (12 light/12 dark) with 75% humility at different
temperatures, 24°C, 28°C and 30 °C, respectively. Examinations of the phenotypic traits,
including tillering number, plant height, seed setting rate and thousand-grain weight, were
performed on ten Kitaake plants and ten gra(k) plants randomly selected, respectively.
1.2 Measurement of chlorophyll content
The samples for chlorophyll content measurement were collected from the leaves at the twoleaf (albino), the four-leaf (green-reversible) and the six-leaf (complete green) stages,
respectively. Leaf samples with 200 mg each from the mutant gra(k) and the wild type
Kitaake were respectively soaked in 30 ml acetone (80%) for 48 h in the dark until samples
became colorless. Chlorophyll concentration was measured following the method as
described previously [1].
1.3 Transmission Electron Microscopy
The samples were collected from the leaves before and after green-revertible albino presented
and prefixed with a mixed solution of 3% glutaraldehyde. The samples were then fixed in 1%
osmium tetroxide, dehydrated in series acetone, infiltrated in Epox812 for four hours, and
embedded followed by cut section [2]. The sections were then stained using methylene blue
and cut using a diamond knife to get ultrathin sections. The ultrathin sections were stained
using uranyl acetate and lead citrate. The stained ultrathin sections were then examined under
a transmission electron microscope (TEM; HITACHI, H-600IV, Japan).
1.4 Genetic analysis and Gene mapping
F1 hybrids from the reciprocal crosses between gra(k) and Jodan and F2 populations derived
from the crosses of gra(k) × Jodan and gra(k) × 02428 were planted in Wenjiang, Sichuan
province. Segregation ratio of the leaf color in F2 populations was examined at the greenrevertible stage.
The F2 individuals with gra phenotypes from the cross gra(k) × Jodan were used for gene
mapping. The SSR primers were synthesized according to the information available from
Gramene database (http://www.gramene.org/microsat) (Table S4). Indel markers were
obtained by aligning the reference sequences between the genomes, japonica (Nipponbare)
(http://www.rgp.dna.affrc.go.jp) and indica (93-11) (http://www.rise.genomics.org.cn).
Primers were synthesized by Shanghai Invitrogen Company. PCR reaction was performed
following the method described previously [3] and amplified products were visualized in 3%
agarose gels.
Bulk segregant analysis (BSA) was used to rapidly determine the chromosomal localization
of the target locus. For this purpose, equal amounts of leaf blades from each of eight wild type
F2 individuals and eight mutant F2 individuals were respectively collected for DNA extraction
to form the wild-type DNA pool and the mutant DNA pool. Fine mapping was then
performed by analyzing the 5168 F2 individuals with albino phenotypes using additional
markers surrounding the locus of Gra(k).
1.5 Gene cloning and sequencing
Primers for cloning were shown in Table S5. PCR was performed following the method
described previously [4]. Images were photographed and captured by Gel Doc 2000TM (BioRad, USA). Sequencing was carried out by a commercial company (BGI Life Tech Co., Ltd,
China).
1.6 RNA isolation and quantitative RT-PCR
The mRNA samples were extracted using TRIzol_reagent (Invitrogen Life Technologies,
Shanghai, China) following the manufacturer’s protocols. cDNA was synthesized using
cDNA reverse transcription Kit (Invitrogen Life Technologies, Shanghai, China). The
quantitative reverse transcription PCR (qRT-PCR) was performed on C1000TM Thermal
Cycler machine. The primer pairs used were listed in Table S6. All qRT-PCR results were
normalized with the expression data of the reference gene Ubq5 [5]. The 2-∆∆CT method was
used to calculate relative expression level with three experimental duplicates [6].
1.7 Complementation test of Gra(k)
For complementary test, the CDSs of the candidate genes were amplified using the primer
pair listed in Table S7 and cloned into Getway-Ubic1300 carrying the promoter of Ubiquitin,
respectively. The resulting construct Ubic1300-Gra(k) was introduced into the mutant gra(k)
through agrobacterium-mediated transformation following the method as described previously
[7]. Hygromycin was used for screening the regenerated transformants. The positive
transgenic plants were verified by using PCR-based genotyping.
References
1.
Yangimi CM, Chang KW, Yin MH, et al (1998) Taiwania 43: I16-122
2.
Matin MN, Saief SA, Rahman MM, et al (2010). Mol Cells 30: 533-543
3.
Li WT, Peng YY, Wei YM, et al (2009). Genet Resour Crop Evol 56: 465-480
4.
Li WT, Wei YM, Wang JR, et al (2010). Theor Appl Genet 121: 907-917
5.
Jain M, Nijhawan A, Tyagi AK, et al (2006). Biochem Bioph Res Co 345: 646-651
6.
Livak KJ, Schmittgen TD (2001). Methods 25: 402-408
7.
Chern M, Fitzgerald HA, Canlas PE, et al (2005). Mol Plant Microbe In 18: 511-520
Supplementary Tables
Supplementary Table 1 Genetic analysis of the gra(k) locus
Cross
F1
F2
Total Number Number of Number
of plants
of χ2(3:1)
normal
green-revertible
plant
albino
gra(k)×Jodan
Normal
1523
1109
439
1.592
Jodan×gra(k)
Normal
920
703
217
0.979
gra(k)×02428
Normal
1041
805
236
2.989
Note: P value of χ2 is 0.001.
Supplementary Table 2 Genes predicted in the mapped Gra(k) region (200 kb)
Gene No.
Gene ID
Annotation
1
LOC_Os05g23620
cytochrome c1-1, heme protein, mitochondrial precursor, putative
2
LOC_Os05g23640
retrotransposon protein, putative, unclassified
3
LOC_Os05g23650
expressed protein
4
LOC_Os05g23670
transcription factor jumonji, putative, expressed
5
LOC_Os05g23680
retrotransposon protein, putative, unclassified, expressed
6
LOC_Os05g23690
retrotransposon protein, putative, unclassified, expressed
7
LOC_Os05g23700 DNA-binding storekeeper protein-related, putative, expressed
8
LOC_Os05g23720 mitochondrial carrier protein, putative, expressed
9
LOC_Os05g23740 DnaK family protein, putative, expressed
10
LOC_Os05g23750
transposon protein, putative, CACTA, En/Spm sub-class,
expressed
11
LOC_Os05g23760
latency associated nuclear antigen, putative, expressed
12
LOC_Os05g23770
transposon protein, putative, CACTA, En/Spm sub-class,
expressed
13
LOC_Os05g23780
OsMADS70 - MADS-box family gene with M-alpha type-box,
expresse
14
LOC_Os05g23790
retrotransposon, putative, centromere-specific, expressed
15
LOC_Os05g23800
RNA recognition motif containing protein, putative, expressed
Note: Genes with functional annotation in the deleted region are in dark bold.
1
Supplementary Table 3 Co-segregation analyses between the transgene and the phenotypes
2
using T1 plants derived from some independent transgenic plants carrying the transgene
3
Os05g23740CDS in gra(k) genetic background
Plants No. Line1(hygromycin) Line2(hygromycin) Line3(hygromycin)
4
1
Albino(-)
Albino(-)
Green(+)
2
Albino(-)
Green(+)
Green(+)
3
Green(+)
Green(+)
Green(+)
4
Green(+)
Albino(-)
Green(+)
5
Green(+)
Green(+)
Green(+)
6
Green(+)
Albino(-)
Albino(-)
7
Green(+)
Albino(-)
Green(+)
8
Albino(-)
Green(+)
Green(+)
9
Green(+)
Green(+)
Green(+)
10
Green(+)
Green(+)
Albino(-)
11
Green(+)
Albino(-)
Green(+)
12
Albino(-)
Green(+)
Green(+)
13
Albino(-)
Green(+)
Green(+)
14
Green(+)
Albino(-)
Albino(-)
15
Green(+)
Green(+)
Albino(-)
16
Green(+)
Green(+)
Green(+)
17
Green(+)
Green(+)
Green(+)
18
Green(+)
Green(+)
Albino(-)
19
Albino(-)
Green(+)
Green(+)
20
Green(+)
Green(+)
Green(+)
21
Green(+)
Green(+)
Green(+)
22
Green(+)
Green(+)
Green(+)
23
Albino(-)
Green(+)
Green(+)
24
Green(+)
Green(+)
Green(+)
Note: +: transgene-positive, -: transgene-negative
5
6
6
Supplementary Table 4 Molecular markers used for mapping of the gene Gra(k)
Marker
Forward Primer
Reverse Primer
Distance(Mb
RM163
CGCCTTTATGAGGAGGAGATGG
AAACTCTTCGACACGCCTTGC
19.11
RM440
GGTAGGCACCAAAGAGTTTGACG
GGCATCACCTTATCCAATCACC
19.83
BI-12
GCTTCAAAACAATCATCA
CAACCACCCACTTATGTA
9.60
BI-19
CCCAGTGGAAAGGAAGCA
TGAATTTTGGTGGACTAG
15.40
BI-31
TGGCACTCGTATTTCTCAA
GACGAGGTTGTTTATTAGTGC
13.48
P9-1
TTTTATGCCCCTACCGCTAC
GAGGAGCATTCCTACAAACATAG 13.64
P11
ACATAGGAAGCACTCGCACA
CAACTATCTGCTTGCCTCCA
13.68
P12
ACCAACAAAGAAGGGAGAGG
CTCCATGATGACGCCACT
13.70
P14
TATTCCTCATTTGCGTGACG
AGATCACCATCGTGTAGTTGC
13.71
ACACACAACAGCTGCTCACTGG
13.78
RM18349 CGTAAACACGAGCACACAAAGG
RM18353 AGATCTCACTATTGAGTAGCCCATGC CACCTTGCCCTTAAATACCAACC
13.84
RM18360 TCGAGACTGATCGGAGTTTAGGC
14.10
7
8
7
CGCTCCTCCCTAACACCTCTACG
9
Supplementary Table 5 Primers used for qRT-PCR.
Genes
Forward primer (5’-3’)
LOC_05g23700
GGTGCGCATTTCTTGGATCG TGCTCTGCTTCCGTTCTCTG
LOC_05g23720
CTGGCCTTGCTTCATACCCA
LOC_05g23740
GTCTCCTACCGCGTCATCAG AGCTTTCTAAGCACCTGGGC
LOC_Os06g46770(Ubq5) AACCAGCTGAGGCCCAAGA
10
11
8
Reverse primer (5’-3’)
TGAAGAGTGACTTCGCACCC
ACGATTGATTTAACCAGTCCATGA
12
Supplementary Table 6 Primers used for cloning of the CDSs of LOC_Os05g23700, LOC_Os05g23720 and LOC_Os05g23740
Gene
Forward Primer
Reverse Primer
Amplified fragment length
LOC_Os05g23700 CACCATGGCGGTGGTGGGGGTGCT
CTACGTGCTTTTCTTCGTCATGCTCT
771bp
LOC_Os05g23720 CACCATGGCTGACGATTTGGGCCCTC
TTAGCCACCACCAGAACCGTACTTCTTAC 1143bp
LOC_Os05g23740 CACCGCACCAGCGGTCTCTCTCGGTGG ACTACTCTCCTGCTCATTGGCTGT
13
14
9
2117bp
15
Supplementary Table 7 Primers used for genomic amplifying of LOC_Os05g23700, LOC_Os05g23720 and LOC_Os05g23740
Gene
Forward Primer
Reverse Primer
Amplified fragment length
LOC_Os05g23700-1 CAAACCCTCTATAATCCTCTACCT AAGTAGGCGACGCTGTAGT
677
LOC_Os05g23700-2 CACGTCCTCGACTACAGCGT
AGAACTCGCCATCAAGGAGTG
772
LOC_Os05g23720-1 GCATGTCATTATCGTATCCAGTT
TCACTAACCTGAAGGTTGCC
1007
LOC_Os05g23720-2 GTTGGTATCATTGTCTATCGTGG
TCCCGAAGATGCTACAATGG
890
LOC_Os05g23740-1 TCTCGCCATTTCTCCCCCA
GAAGGGAACCCACAGGATTTG
1163
LOC_Os05g23740-2 TGAACAGCCCTTGCTCTTTG
CCTCAAAAACACCATCACCAACT
1284
LOC_Os05g23740-3 CTTGAAGTTGGTGATGGTGTTT
CAATATCTCCAGAACCTGACAAA 1170
LOC_Os05g23740-4 TGGTTTGAAATGGATGCGAT
ACAATGCTACCAATTTGCCAG
16
17
18
19
10
1537
20
21
Supplementary Figure 1 Determination whether the green-revertible albino phenotype co-segregates with the hygromycin gene in F2 segregating population.
22
WT: wild type. +: Positive control of hygromycin, -: Negative control of hygromycin; Number “1” through “22” represent 22 F2 green-revertible albino
23
individuals from the cross of Jodan × gra(k).
24
11
25
26
27
Supplementary Figure 2 Comparison of the main agronomic traits between gra(k) and Kitaake plants. The main agronomic traits, including tillering
28
number, plant height, setting rate and thousand-grain weight, were respectively determined. WT: wild type.
29
30
12
31
32
Supplementary Figure 3 PCR amplification of genomic sequences of LOC_Os05g23700, LOC_Os05g23720 and LOC_Os05g23740 from the wild type
33
Kitaake and the mutant gra(k), respectively. Genomic sequences of LOC_Os05g23700 and LOC_Os05g23720 were amplified using two primer pairs
34
whereas genomic sequence of LOC_Os05g23740 was amplified using four primer pairs.
35
13
36
14