Table 5.

Selected markers for 21 allele types at the 10 blast resistance loci

Locus	Marker name	Primer sequences (5′ to 3′)		Marker type	Temp. (°C)b	Gel (%)b	Genotype A (‘Nipponbare’ type)c		Genotype Bc		Genotype Cc		Allele types distinguishable by marker combinations
		Forward	Reverse				Size (bp)	Allele type	Size (bp)	Allele type	Size (bp)	Allele type
Pit	Pit-ID003	CCTGAAACACATTATCTATGTTG	AGAAAGAAGCATAAGTTTAAAATAGA	InDel	55	4	160		190	Pit			1) Pit
Pish	Pish-ID007	TACACCGCTCGGCTTTTCACC	ATGCCCCTCGTTGCAGCC	InDel	↓	↓	100	Pish or Pi35	87				1) Pish 2) Pi35
	Pish-ID010	TGCTACATATATATGATAATTGTCGAGG	TCAATCTACACCGTTAGATCAT	InDel	50	↓	94	Pish	81	Pi35
	Pish-ID011	AGCACTTGACACTCCACAGCAG	GGCAAAACCCGTGTTTCTGACG	InDel	55	↓	102	Pish	116	Pi35
Pib	Pib-ID11	AGAGTGGTTGGTTGGAGGTG	GCCCATATTGCTTTGCTCCAAA	InDel	↓	↓	103		92	Pib			1) Pib
Piz	Piz-ID18	CTGCTGCTACCGTTTGGAAGTCA	CTCCTGGCCCCACGCGTC	InDel	↓	↓	98	Piz-t, Piz-5, or Pi9	84	Piz			1) Piz 2) Piz-t or Piz-5 3) Pi9
	Piz-ID22	ATGTGGGGTTTCTGATTCCAAT	CTTGATTAGTGAGATCCATTGTTCC	InDel	↓	↓	127	Piz	100	Piz-t or Piz-5	118	Pi9
	Piz-ID31	CCAATTTCAGGCTTAGTTTGAT	AGCTATTTATTAAGCTGATTTCTCA	InDel	↓	↓	107	Piz or Pi9	84	Piz-t or Piz-5
Pi13	Pi13-ID008	GTGAGCTGGAATACTAGATCGA	GTCAAAGTTCCTGCAATTTTGTGA	InDel	↓	↓	114		88	Pi13			1) Pi13
Pii	Pii-ID07	TTCGGTCATTAGCCGGTGCT	GGCGGCAGGTATGGTACTTCA	InDel	↓	3 or 4	450		289	Pii, Pii*, Pi5, or Pi3	493		1) Pii 2) Pii* 3) Pi5 or Pi3
	Pii-ID21	AAGCGAACGACTCTAGCTAGAA	TCTCCATATGTATGTATAACTGGCTT	InDel	↓	4	84	Pii*	96	Pii, Pi5, or Pi3
	Pii-ID24	ATGAGGAGATGACAACGAGGAG	GAAGAGGGGAACGCCGAG	InDel	↓	↓	100	Pii*, Pi5, or Pi3	88	Pii
Pia	Pia-ID01_2	ACGGTAGAGCAATTTAGAAGCAGTGA	AGTGCGACTGACACTTTCAATAGCA	InDel	55	↓	195		152	Pia			1) Pia
Pik	Pik-ID001	CTTCTTAGCCTCCAGATTTGCA	TCATGTGCATCAAAATGGGCTA	InDel	↓	↓	100	Pik, Pik-h, Pi7, or Pi1	88	Pik-m, Pik-p, or Pik-s			1) Pik 2) Pik-m or Pik-s 3) Pik-p 4) Pi1 5) Pik-h or Pi7
	Pik-ID007	AACGAATATTTATGACTAAAGAAAGT	AGAAGCTTGACTCCGTTAG	InDel	↓	↓	120	Pik	406	Pik-m, Pik-p, Pik-s, Pik-h, Pi7, or Pi1
	Pik-ID011	GGTTAAATAGGACTCCCTCTA	GCATCCAATAGAATCAGAGA	InDel	↓	↓	169	Pik, Pik-m, Pik-s, or Pi1	150	Pik-p, Pik-h, or Pi7
	Pik-ID014	TTCTTCTTTATCCCGTCTTCTT	ATGAGGAAAACGAAGATGAGAG	InDel	↓	↓	344	Pik, Pik-p, Pik-h, or Pi7	149, 150	Pik-m, Pik-s, or Pi1
	Pik-ID018	ATCCTCTGTGTCTGAAGCCAT	AGGCTTCTCTGCTCCTATAACA	InDel	↓	↓	110	Pik, Pik-m, Pik-p, Pik-s, Pik-h, Pi7, or Pi1	100
Pita	Pita-ID13	AGGCAAGAGTACAATGGAAAC	TGCCCTCTGAAAATAAAGTTT	InDel	↓	↓	100	Pita	112	(Pi20)			1) Pita 2) (Pi20)
	Pita-ID04a	CGTGAAGAGGATTCCGGTAGCA	TGCCGTGGCTTCTATCTTTACgTT	PCR-CTPP	↓	↓	137	(Pi20)	216	Pita
		CAAGTCAGGTTGAAGATGCATtGC	CCGACGCCGAGCACTCTTAT
Pita-2	Pita-2-ID009	CACATAGCATTAGAGCACTAAACT	AACCATCATACCGGCCTATTTA	InDel	↓	↓	102	Pi19 or Pita-2	114	(Pi20)			1) Pita-2 2) Pi19 3) (Pi20)
	Pita-2-ID016_3	AAAGTTCATCGCATCGAATTTA	ACACGCGTTGGGATCTTCCTC	InDel	50	↓	135	Pi19 or (Pi20)	123	Pita-2
	Pita-2-ID011	TGCAATTAGTCTGGTGTTGTTGA	ACATGAATCAGTCTGACGTCAT	InDel	55	↓	115	Pi19	85	Pita-2	104	(Pi20)

^*Pii allele type with an asterisk (‘Nerica 1’) differs in marker genotypes from that in the reference varieties for Pii (‘Fujisaka 5’ and ‘Ishikarishiroke’) despite its typical reaction against differential pathogen races.

^aLowercase letters in primer sequences indicate mismatch bases against target complementary sequences. PCR-CTPP, polymerase chain reaction with confronting two-pair primers to distinguish co-dominant markers (Hamajima et al. 2000). PCR conditions for this marker: 95°C for 2 min, followed by 35 cycles of 94°C for 30 s, 55°C for 1 min 30 s, 72°C for 1 min, and finally 68°C for 5 min.

^bThe condition with a downward arrow is the same as the one above. Temp., annealing temperature; Gel, agarose gel.

^cGenotype A represents the ‘Nipponbare’-type, and B and C represent genotypes that differ from each other and from genotype A in terms of product size.

The genotype A obtained by the three markers (Pik-ID007, Pik-ID011, and Pik-ID014) represents the genotype of ‘Kusabue’ (HM048900) or ‘Kanto 51’ (AB616659), whose product sizes are indicated in gray boxes.

The genotype B obtained by the marker Pik-ID014 includes amplicons of two sizes that could not be clearly discriminated in an 4% agarose gel.