A multiplex PCR for rapid identification of Brassica species in the triangle of U

Table 2 Primers used in this study

Primer^a,b	Sequence (5′–3′)	Length	Tm (°C)^c	Species	Chromosome	Gene	Genomic location	Product (bp)
A6-1	F: CCAGCGAAGGATTTGACGAC	20	59.3	B. rapa	A06	Bra019579	A06:12049397-120493649	253
	R: GACGAATCGAGTGCCCTG	18	57.9
A6-2	F: GTTTTGGCCGTAAATCCCAC	20	57.6	B. rapa	A06	Bra019582	A06:12019296-12019481	186
	R: GTTACGGGTAGCGTGTGTC	19	58.3
C1	F: TGCTGCGCCGAACAATAG	18	58.5	B. oleracea	C01	Bo1g016520	C1:5373673-5373829	157
	R: CCGATCGTGGTTCATATTGC	20	57.1
C9	F: GTTAACGCACTTAAGGACCATG	22	57.7	B. oleracea	C09	Bo9g098720	C9:32340576-32341187	612
	R: ATTGACAACACCACCTCCCG	20	60.3
B	F: GGCATCTGAAGAGAGAGTC	19	54.4	B. nigra	All	–	–	331
	R: CCATCTTCTTCTTGCCATG	19	53.7

^aA and C genome specific primers were designed based on selected B. rapa and B. oleracea genes from a previous study [17]. Genomic and sequence information for Bra019579, Bra019582, Bo1g016520 and B09g098720 were obtained from EnsemblPlants online database (http://plants.ensembl.org/index.html)
^bB genome specific primers were designed based on a cloned B. nigra fragment, pBNBH35 that is present on all eight of the B genome chromosomes [20]
^cAnnealing temperatures (Tm) for primers are based on Primer3 prediction but all primers were multiplexed successfully at Tm = 58 °C

ISSN: 1746-4811