Arabidopsis thaliana cell lines

Plant cell lines

• rpc00008: Arabidopsis thaliana T87 cell suspension culture

• rpc00050: Arabidopsis thaliana YG1 cell suspension culture

• rpc00055: Arabidopsis thaliana gnom callus culture

• rpc00056: Arabidopsis thaliana At tom cell suspension culture

• rpc00103: Arabidopsis thaliana MM2d cell suspension culture

• rpc00104: Arabidopsis thaliana MM2d-LS cell suspension culture

Genotypes

Cell line	rbcL	GNOM	TOM	Transgene insertion	InDel/SSR
T87	Arabidopsis thaliana	—	—	—	T87
YG1	Arabidopsis thaliana	—	—	T-DNA (not detected)	At tom/YG1
gnom	Arabidopsis thaliana	gnom	—	—	—
At tom	Arabidopsis thaliana	—	tom1-2 tom3-1	T-DNA (homozygous)	—
MM2d	Arabidopsis thaliana	—	—	—	MM2d
MM2d-LS	Arabidopsis thaliana	—	—	—	MM2d-LS

Protocols

DNA barcoding — rbcL

DNA barcoding — rbcL

DNA sequencing — GNOM gene

1. PCR

   • Primer sets 《Primer sequences》

     Target	Forward primer	Reverse primer	Product size
     GNOM	gnom_At1g133980_F	gnom_At1g133980_R	500 bp

   • PCR reaction mixture

     Component	10 µL reaction
     TaKaRa Ex Taq™ Premix	5 µL
     Forward primer (100 µM)	0.125 µL
     Reverse primer (100 µM)	0.125 µL
     Template genome DNA	1 µL
     Sterile distilled water	3.75 µL

   • PCR reaction

     Temperature	Time	Cycles
     94°C	1 min
     94°C	30 sec	35 cycles
     55°C	30 sec
     72°C	1 min
     72°C	7 min

2. Check the DNA sequence of the PCR product.

   • gnom | GNOM At1g133980: p.Arg647Ter (c.1939C>T)

DNA sequencing — TOM genes

1. PCR

   • Primer sets 《Primer sequences》

     Target	Forward primer	Reverse primer	Product size
     TOM1	tom1-2_F	tom1-2_R	450 bp
     TOM3	Tom3-1_F	Tom3-1_R	550 bp

   • PCR reaction mixture

     Component	10 µL reaction
     TaKaRa Ex Taq™ Premix	5 µL
     Forward primer (100 µM)	0.125 µL
     Reverse primer (100 µM)	0.125 µL
     Template genome DNA	1 µL
     Sterile distilled water	3.75 µL

   • PCR reaction

     Temperature	Time	Cycles
     94°C	1 min
     94°C	30 sec	35 cycles
     55°C	30 sec
     72°C	1 min
     72°C	7 min

2. Check the DNA sequence of the PCR product.

   • tom1-2 | TOM1 At4g21790: p.Trp68Ter (c.204G>A)

   • tom3-1 | TOM3 At2g02180: p.Trp45Ter (c.135G>A)

Transgene insertion — T-DNA (GABI-Kat 046D04)

1. PCR

   • Primer sets 《Primer sequences》

     Target	Forward primer	Reverse primer	Product size
     Left border	2625	046D04_flankingseq_side	900 bp
     Zygosity	046D04_RBside	046D04_flankingseq_side	Not detected (850 bp)

   • PCR reaction mixture

     Component	10 µL reaction
     TaKaRa Ex Taq™ Premix	5 µL
     Forward primer (100 µM)	0.125 µL
     Reverse primer (100 µM)	0.125 µL
     Template genome DNA	1 µL
     Sterile distilled water	3.75 µL

   • PCR reaction

     Temperature	Time	Cycles
     94°C	1 min
     94°C	30 sec	35 cycles
     55°C	30 sec
     72°C	1 min
     72°C	7 min

2. Check the size of the PCR product.

Fragment analysis — InDel (1 loci)

1. PCR

   • Primer sets 《Primer sequences》

     • 3483/3484 primer mix

       Primer name	Concentration
       3483	1 µM
       3484	0.1 µM

     • uni-FAM primer: Fluorescent labeled primer at 5′ end with 6-FAM™

       Primer name	Concentration
       uni-FAM	10 µM

   • PCR reaction mixture

     Component	10 µL reaction
     AmpliTaq Gold™ 360 Master Mix	5 µL
     uni-FAM	0.2 µL
     Primer mix	2 µL
     Template DNA	1 µL
     Sterile distilled water	1.8 µL

   • PCR reaction

     Temperature	Time	Cycles
     95°C	10 min
     95°C	30 sec	40 cycles
     55°C	30 sec
     72°C	30 sec
     72°C	10 min

2. Capillary electrophoresis of the PCR product with the Applied Biosystems™ 3130xl Genetic Analyzer

3. Data analysis with the GeneMapper® Software

Fragment analysis — SSR (16 loci)

1. PCR with each of the primer mix

   • Primer sets: Fluorescent labeled primers at 5′ end

     Primer mix	Marker	Fluorescent label	Concentration
     PS1	nga280	6-FAM™	1 µM
     	nga1126	VIC®	2.5 µM
     	nga6	NED™	1 µM
     	nga1111	PET™	1 µM
     PS2	nga106	6-FAM™	1 µM
     	nga63	VIC®	2.5 µM
     	nga168	NED™	1 µM
     	nga172	PET™	1 µM
     PS3	nga1107	6-FAM™	1 µM
     	nga129	VIC®	0.5 µM
     	nga162	NED™	0.5 µM
     	nga1139	PET™	1 µM
     PS4	nga111	6-FAM™	2 µM
     	nga1145	VIC®	1 µM
     	nga158	NED™	1 µM
     	Det1.2	PET™	1 µM

   • PCR reaction mixture

     Component	10 µL reaction
     2×QIAGEN Multiplex	10 µL
     Primer mix	2 µL
     Template DNA	1 µL
     Sterile distilled water	7 µL

   • PCR reaction

     Temperature	Time	Cycles
     95°C	15 min
     94°C	30 sec	35 cycles
     54–62°C [1]	90 sec
     72°C	1 min
     60°C	30 min

     [1]

     • PS1: 54°C

     • PS2: 60°C/55°C

     • PS3: 58°C

     • PS4: 62°C

2. Capillary electrophoresis of each of the PCR products with the Applied Biosystems™ 3130xl Genetic Analyzer

3. Data analysis with the GeneMapper^® Software