Supplementary Table 1

(a) Primers used for cloning of BjNRAMP and BjYSL family members

Degenerate primers for RT-PCR and Nested PCR
Gene / Forward Primer Sequence / Reverse Primer Sequence
NRAMP / NF1
5’ATGAGHATYGCKTTTMTYGATCCAGGRAA3’ / NR1
5’GGTCCAATYTTGAADSHRCCCATGAT3’
NF2
5’TRGCKGAGCTKTGTCGWGMWGAGTA3’ / NR2
5’ATTATAAAYTGTCCRGCRTAWGTWCCGGT3’
YSL / YF1
5’CTWCAARCTDACATAYCCIAGTGG3’ / YR1
5’TCWCCACAKATCARHCCWGAWGC3’
YF2
5’TCTCTTGGGGDHTHMTGTGGCC3’ / YR2
5’AAYGGWAYAGCCATHGCCAT3’
A (Anchor primer)
5’CGGAATTCGGGACTAGTACTTT3’

H= A, C or T; Y= C or T; N= A or C; K= G or T; R= A or G; D= A, G or T; S=A, G or C; W=A or T; I= Inosine

(b) PCR conditions used for cloning of BjNRAMP and BjYSL family members

Primer Combinations / PCR Condition
NF1-A / 940C for 4mins; 940C for 30sec, 570C for 45sec, 680C for 3mins for 35 cycles; 720C for 10mins
NF2-A / 940C for 4mins; 940C for 30sec, 570C for 45sec, 680C for 3mins for 35 cycles; 720C for 10mins
NF1-NR1 / 940C for 4mins; 940C for 30sec, 570C for 45sec, 680C for 2mins and 30secs for 35 cycles; 720C for 10mins
NF1-NR2 / 940C for 4mins; 940C for 30sec, 570C for 45sec, 680C for 2mins and 30secs for 35 cycles; 720C for 10mins
NF2-NR1 / 940C for 4mins; 940C for 30sec, 570C for 45sec, 680C for 2mins and 30secs for 35 cycles; 720C for 10mins
NF2-NR2 / 940C for 4mins; 940C for 30sec, 570C for 45sec, 680C for 2mins and 30secs for 35 cycles; 720C for 10mins
YF1-A / 940C for 2mins; 940C for 45sec, 550C for 30sec, 680C for 4mins for 40 cycles; 720C for 5mins
YF2-A / 940C for 2mins; 940C for 45sec, 580C for 30sec, 680C for 4mins for 40 cycles; 720C for 5mins
YF1-YR1 / 940C for 2mins; 940C for 45sec, 550C for 30sec, 680C for 3mins for 40 cycles; 720C for 5mins
YF1-YR2 / 940C for 2mins; 940C for 45sec, 550C for 30sec, 680C for 3mins for 40 cycles; 720C for 5mins
YF2-YR1 / 940C for 2mins; 940C for 45sec, 580C for 30sec, 680C for 3mins for 40 cycles; 720C for 5mins
YF2-YR2 / 940C for 2mins; 940C for 45sec, 580C for 30sec, 680C for 3mins for 40 cycles; 720C for 5mins

Supplementary Table 2

(a) Primers and PCR conditions for 5’ end cloning of BjNRAMP and BjYSL isoforms

Gene Name / PCR / Primer Sequence / PCR Condition
Forward Primer / Reverse Primer
BjNRAMP4.1 / Primary PCR / 5’ATGTCGGAGMCDGAAGAGMG 3’ / 5’ATCAGAAGAATCAAAGACAAGAGCC 3’ / 940C for 5mins; 940C for 45sec, 58.50C for 45sec, 720C for 1min and 30sec for 30 cycles; 720C for 7mins
Nested PCR / 5’ATGTCGGAGMCDGAAGAGMG 3’’ / 5’AAGATCTTGTCTAATGGGTTGG 3’ / 940C for 5mins; 940C for 45sec, 58.50C for 45sec, 720C for 1min and 20sec for 30 cycles; 720C for 7mins
BjYSL6.1 / Primary PCR / 5’ATGGGACGGAGATCCCAC3’ / 5’CAGAGGCTTCGTCACTACAA3’ / 940C for 2mins; 940C for 45sec, 590C for 45sec, 720C for 1min and 20 sec for 40 cycles; 720C for 5mins
Nested PCR / 5’ATGGGACGGAGATCCCA 3’ / 5’ATCCACATGCATCTCCAAC3’ / 940C for 2mins; 940C for 45sec, 590C for 45sec, 720C for 1min, for 30 cycles; 720C for 5mins
BjYSL6.4 / Primary PCR / 5’ATGGGACGGAGATCCCAC3’ / 5’CAGAGGCTTCGTCACTACAA3’’ / 940C for 2mins; 940C for 45sec, 590C for 45sec, 720C for 1min and 20 sec for 40 cycles; 720C for 5mins
Nested PCR / 5’ATGGGACGGAGATCCCAC3’’ / 5’ATCCACATGCATCTCCAAC3’ / 940C for 2mins; 940C for 45sec, 590C for 45sec, 720C for 1min, for 30 cycles; 720C for 5mins

D= A, G or T; M=A or C

(b) Primers and PCR conditions for 3’ end cloning of BjYSL isoforms

Gene Name / PCR / Primer Sequence / PCR Condition
Forward Primer / Reverse Primer
BjYSL6.1 / Primary PCR / 5’GCTATCTCGACCGCAACG3’ / 5’CGGAATTCGGGACTAAGTACTTT3’ / 940C for 2mins; 940C for 45sec, 600C for 45sec, 720C for 1min and 20 sec for 40 cycles; 720C for 5mins
Nested PCR / 5’TGGTACTTTGTCCTGTGTT 3’ / 5’CGGAATTCGGGACTAAGTACTTT3’ / 940C for 2mins; 940C for 45sec, 590C for 45sec, 720C for 1min, for 35 cycles; 720C for 5mins
BjYSL6.4 / Primary PCR / 5’GTTTGGAGTTGTTTCAAGTGGT3’ / 5’CGGAATTCGGGACTAAGTACTTT3 / 940C for 2mins; 940C for 45sec, 580C for 45sec, 720C for 1min and 55 sec for 35 cycles; 720C for 5mins
Nested PCR / 5’TGGTACTTTGTCCTGTGTT 3’ / 5’CGGAATTCGGGACTAAGTACTTT3 / 940C for 2mins; 940C for 45sec, 590C for 45sec, 720C for 1min, for 35 cycles; 720C for 5mins

Supplementary Table 3

Details of Brassica juncea (Bj) specific amino acid change among both NRAMP and YSL family with respect to closest ortholog

Isoforms / Closest ortholog / Extracellular loop / Transmembrane domain / Cytoplasmic loop
Position of the changed residue in Arabidopsis ortholog) / Amino acid in Brassica sub members / Position of the changed residue in Arabidopsis ortholog / Amino acid in Brassica sub members / Position of the changed residue in Arabidopsis ortholog / Amino acid in Brassica sub members
BjNRAMP2 / AtNRAMP2 / 177F / V / 137D / D/A
185V / V/I / 153L / L/I
208V / V/M / 233E / E/Q
336Q / Q/E
BjNRAMP3 / NcNRAMP3 / 132R / G / 165I / I/T / 265D / N/D
162S / T / 170A / A/V / 276L / I/K
224I / V / 173V / V/I / 434S / T/A
312N / D / 202A / G
325F / D / 205G / A/G
207S / A
288F / I
289I / V
292L / M
331V / L
339A / G
BjNRAMP4 / AtNRAMP4 / 259E / K / 162V / I/V / 300S / A
265R / R/S / 195I / I/V / 305E / D
266F / F/V / 380I / V / 466N / S
426Q / Q/H / 467L / V
430S / T / 478I / V
BjYSL2 / NcYSL2 / 260E / D / 238V / M / 320K / K/R
361L / L/F / 241K / R / 327A / A/S
370V / I/K / 245I / F / 330P / E
372L / L/I / 248F / L / 437H / H/Q
375T / T/E / 348I / I/M / 440H / H/D
376A / D / 354L / L/V / 452I / T/V
377S / D / 417T / T/S / 453C / C/F
378V / D /T / 421V / V/G / 530K / R/K
379S / T/W / 425I / I/V / 610I / V/I
380R / S/R / 430T / T/V / 616W / P
381T / D /T / 534V / I/V / 618R / R/K/Q
382T / N/T / 552W / W/L / 619W / F/W
384L / L/F / 623I / V
386A / R/T
391P / P/T
395L / L/F
396S / T
398M / K
399Q / Q/I
400S / P/S
414E / Q
556K / N/K
559D / D/E
566T / S
588S / A/S
652I / V
BjYSL5 / AtYSL5 / 258E / E/D / 236V / V/M / 325D / A
373P / D /T / 243L / F/L / 326N / D
374T / D /T / 246F / L / 328P / E
375T / D /L/P / 419V / V/M / 329S / P/S
376S / T/S / 424T / I/T / 528K / R/K
377I / F/S/T / 426A / A/S / 531F / F/L
380T / R/A / 428T / T/A / 614H / P
381S / F/S / 532V / I/V / 616N / R/K/Q
382F / F/T / 550W / W/L / 617W / F/W
383T / V/T / 601G / G/S / 621I / V
384L / V / 647I / V
385E / E/Q
385-386 / EEE/E/D
389H / H/T
393L / Q/Y
394S / T/S
397Q / Q/E
554K / N/K
557D / D/E
564T / S
571A / A/T
586A / A/S
650I / V
660E / E/G
BjYSL6 / AtYSL6 / 205T / S/T / 291I / F / 410I / L
211G / A / 391E / G / 505F / S
226I / V / 394I / V
237I / V / 395A / S
267I / V / 463S / S/T
350S / N / 517V / I
351R / S / 524W / Y
354R / H / 574I / V
359I / V
361T / A
362D / N
363G / D
365D / V
367S / D
528T / S
633E / D
635F / Y
BjYSL8 / AtYSL8 / 241K / E / 245V / V/M / 265A / G/A
355T / M / 252F / L / 267E / E/D
369I / S / 255F / L / 272N / S
372F / I / 257G / G/S / 273S / S/N
376I / S / 429V / G / 445H / Q
377S / K / 436S / A / 448S / D
382K / R / 456I / L / 622C / G
383A / T / 460I / V / 631V / I
384G / S / 464I / V
385-391 SRSSLAH / D / 470A / T/A
392K / E / 604M / V
394D / P / 608V / G
395-398 PPAS / D / 618L / V
573N / K / 619I / V

Supplementary Table 4

Primers and conditions of Semiquantitative RT-PCR of BjNRAMP and BjYSL isoforms

Gene Name / Primer Sequence / PCR Condition
Forward Primer / Reverse Primer
BjNRAMP3.5 / 5’GACTTGGGCGGGTATGGTCT 3’ / 5’TTGATCATGAAGGAGACGGTG 3’ / 940C for 2mins; 940C for 45sec, 540C for 45sec, 720C for 30sec for 35 cycles; 720C for 5mins
BjNRAMP4.1 / 5’AAGATCTTGTCTAATGGGTTGG’3’ / 5’GCTATGTCTGTCCCGTAAAAG 3’ / 940C for 2mins; 940C for 45sec, 570C for 45sec, 720C for 30sec for 30 cycles; 720C for 5mins
BjNRAMP4.3 / 5’ACAATAAAACAAGCTGTTGGG 3’ / 5’ACCCATTTCTTCATCTTGAGA 3’ / 940C for 2mins; 940C for 45sec, 570C for 45sec, 720C for 30sec for 30 cycles; 720C for 5mins
BjYSL2.1 / 5’'TTCGACTTACGGAAAGCTA 3’ / 5’ATAAGACTAGACATTCTCTTGGTAAT 3’ / 940C for 2mins; 940C for 45sec, 550C for 45sec, 720C for 40sec, for 35 cycles; 720C for 5mins
BjYSL5.8 / 5’CGTCGAAGAAGATCCAACC 3’ / 5’TTCTGTACACTGTGGCGAAT 3’ / 940C for 2mins; 940C for 45sec, 510C for 45sec, 720C for 1min, for 38 cycles; 720C for 5mins
BjYSL6.1 / 5’GCTATCTCGACCGCAACG 3’ / 5’TCCCTCAATACCGAGAATC 3’ / 940C for 2mins; 940C for 45sec, 560C for 45sec, 720C for 45sec, for 40 cycles; 720C for 5mins
Actin2 / 5’AGTGGTCGTACAACCGGTATTGT 3’ / 5’GAGGAAGAGCATTCCCCTCGTA 3’ / 940C for 2mins; 940C for 45sec, 600C for 45sec, 720C for 15sec, for 40 cycles; 720C for 5mins