Potato Germplasm Enhancement Laboratory

Obihiro Univerisity of Agriculture and Veterinary Medicine\
West 2-11, Inada, Obihiro, Hokkaido 080-8555, Japan

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Multiplex PCR detection of five resistance genes (H1, Rx1, R1, R2, and Rychc) (Mori et al. 2011)

1. DNA extraction

Any DNA extraction method can be used. Recommended DNA concentration is 5 ng/μl. DNA extracted using One-minute DNA extraction method is sufficiently used without adjustment of the concentration.

2. Multiplex PCR

1. Place 2 μl of DNA into the bottom of PCR tube, and then, dispense 8 μl each of Reaction mix.

   Component	Volume	Reaction mix (xn)
   DNA (5 ng/μl)	2 μl	-
   2x Ampdirect Plus	5.0 μl (1x)	5.0 x n μl
   BIOTAQ HS DNA Polymerase (5 units/μl)	0.1 μl (0.5 units)	0.1 x n μl
   10x Multiplex primer	1.0 μl	1.0 x n μl
   Sterile water	1.9 μl	1.9 x n μl
   Total	10.0 μl	8 x n μl

   
   

   10x Multiplex primer

   Marker	Target gene	Size (bp)	Primer	Primer (5' - 3' sequence)	Vol. (μl)*	Conc. (μM)
   N146	H1	506	N146-17	AAGCTCTTGCCTAGTGCTC	5	0.5
   			N146-22	AGGCGGAACATGCCATG	5	0.5
   N195	H1	337	N195-09	TGGAAATGGCACCCACTA	5	0.5
   			N195-06	CATCATGGTTTCACTTGTCAC	5	0.5
   PVX	Rx1	1230	RxSP-S3	ATCTTGGTTTGAATACATGG	150	15
   			RxSP-A2	CACAATATTGGAAGGATTCA	150	15
   R1	R1	1400	76-2sf2	CACTCGTGACATATCCTCACTA	25	2.5
   			76-2SR	CAACCCTGGCATGCCACG	25	2.5
   R2-800	R2	800	R2SP-S7	TACTAACCTTTTCCTAGATG	25	2.5
   			R2SP-A9	AGAACTTTCTCACAGCTTTT	25	2.5
   Ry186	Rychc	587	RY186-11	TGGTAGGGATATTTTCCTTAGA	10	1.0
   			RY186-12	GCAAATCCTAGGTTATCAACTCA	10	1.0
   GBSS	GBSS	981	gbss-01	ATGGCAAGCATCACAG	15	1.5
   			gbss-02	CAAAACTTTAGGTGCCTC	15	1.5

   *Volume (μl) of the 100 μM original primer in 1 ml of 10x Multiplex primer


2. PCR [95°C 10 min, 5 cycles (94°C 30 sec, 68°C 30 sec, 72°C 1.5 min), 35 cycles (94°C 30 sec, 58°C 30 sec, 72°C 1.5 min), 72°C 5 min, soak at 4°C]

3. Agarose gel electrophoresis

1. Mix with 3 μl of PCR products loading dye (sterile water: 10x Dye = 17:13) (10x dye = 0.25% bromophenol blue, 0.25% xylene cyanol, and 25% Ficoll Type 400).


2. Electrophoresis in a 1.4% agarose gel in 1x TAE buffer.


The left-most lane in each gel contained HindIII-digested lambda DNA.


Lane	Sample	R1	PVX	R2-800	Ry186	N146	N195
1	Hanashibetsu	-	+	+	-	+	+
2	Aiyutaka	+	+	-	-	+	+
3	Saikai 35	-	-	-	+	+	+
4	Pike	+	+	-	-	+	+
5	Saya-akane	+	+	+	-	+	+
6	10H15	+	+	-	+	+	+
7	Harimaru	-	+	-	+	+	+
8	10H17	+	+	-	+	+	+