Journal: Molecular Breeding Molecular characterization of allelic variation in spontaneous brown midrib mutants of sorghum (Sorghum bicolor L Moench) Sunita Gorthy 1 , Karthikeyan Mayandi 1 , Dhara Faldu 2 and Monika Dalal 1, 2  1 Directorate of Sorghum Research, Rajendra Nagar, Hyderabad , Andhra Pradesh, India 2 National Research Center on Plant Biotechnology, Indian Agricultural Research Institute, New Delhi , India  Joint First Authors  Corresponding Author: Telephone No: Fax:

Wild type CGGAGCAAGCGGCGCCGCTGCTGTGCGCGGGCGTAACGGTGTACAGCCCGCTAAAG Template IS21549 CGGAGCAAGCGGCGCCGCTGCTGTGCGTTGGCGTAACGGTGTACAGCCCGCTAAAG Template dCAPS primer AGCGGCGCCGCTGCTGAACG Wild type CGGAGCAAGCGGCGCCGCTGCTGAACGCGGGCGTAACGGTGTACAGCCCGCTAAAG Product IS21549 CGGAGCAAGCGGCGCCGCTGCTGAACGTTGGCGTAACGGTGTACAGCCCGCTAAAG Product Wild type CGGAGCAAGCGGCGCCGCTGCTGAACGCGGGCGTAACGGTGTACAGCCCGCTAAAG Product IS21549 CGGAGCAAGCGGCGCCGCTGCTGAA & CGTTGGCGTAACGGTGTACAGCCCGCTAAAG Product Digestion with AclI (AA ↓ CGTT) Supplementary Fig. S1

Supplementary Fig. S2 IS21549 PEQAAPLLCVGVTVYSPLKAFGLTA 179 Sorghum bicolor PEQAAPLLCAGVTVYSPLKAFGLTA 179 Saccharum officinarum PEQAAPLLCAGVTVYSPLKAFGLTT 179 Zea mays PEQAAPLLCAGVTVYSPLKHFGLTT 179 Panicum virgatum PEQAAPLLCAGVTVYSPLKHFGLTA 179 Triticum aestivum PEQAAPLLCAGVTVYSPLKHFGLMT 180 Hordeum vulgare PEQAAPLLCAGVTVYSPLKHFGLMT 180 Festuca arundinacea PEQAAPLLCAGVTVYSPLKHFGLMT 180 Lolium perenne PEQAAPLLCAGVTVYSPLKHFGLMT 180 Brachypodium distachyon PEQAAPLLCAGVTVYSPLKHFGLMS 180 Oryza sativa PEQAAPLLCAGLTVYSPLKHFGLMS 175 Oryza glaberrima PEQAAPLLCAGLTVYSPLKHFGLMS 179 Bambusa glaucescens PEQAAPLLCAGVTVYSPLKHFGLMA 179 Gossypium hirsutum PEQVAPLLCAGVTVYSPLNHFGLMG 179 Hibiscus cannabinus PEQVAPLLCAGITVYSPLNHFGLTA 188 Linum album PEQAAPLLCAGITVYSPLNHFGLKQ 179 Ocimum basilicum PEQAAPLLCAGVTVYSPLNHFGLKQ 179 Striga asiatica PEQAAPLLCAGVTVYSPLNHFGLKQ 174 Hevea brasiliensis AEQAAPLLCAGLTVYSPLSHFGLNK 179 Citrus sinensis LEQAAPLLCAGVTVFSPLSHFGLKQ 179 Ricinus communis PEQAAPLLCAGVTVYSPLSHFGLKK 179 Vitis vinifera PEQAAPLLCAGVTVYSPLSHFGLKK 179 Populus nigra PEQAAPLLCAGLTVYSPLKHFGLKQ 179 Arabidopsis lyrata VEQAAPLLCAGVTVYSPLSHFGLMA 179 Arabidopsis thaliana VEQAAPLLCAGVTVYSPLSHFGLMA 180 Artemisia annua PEQVAPLLCAGVTVYSPLNYFGLKK 179 Aralia cordata PEQAAPLLCAGVTVYSPLTHFGLKE 179 Nicotiana tabacum PEQAAPLLCAGITVYSPLNHFGFKQ 179 Capsicum annuum PEQAAPLLCAGVTVYSPLNHFGFNQ 179 Medicago sativa PEQVAPLLCAGVTVYSPLSHFGLKT 180 Medicago truncatula PEQVAPLLCAGVTVYSPLSHFGLKT 180 Glycine max PEQVAPLLCAGVTVYSPLVRFGLKE 179 Leucaena leucocephala PEQVAPLLCAGVTVYSPLSHFGLRE 179 Eucalyptus globulus SEQAAPLMCAGVTVYSPLVRFGLKQ 179 Pinus radiata LEQAAPLLCAGVTVFSPMKHFAMTE 179 Picea abies LEQAAPLLCAGVTVYSPMKHFGMTE 179 **.***:*.*:**:**: *.:

bmr6-ref MGSLASERKVVGWAARDATGHLSPYTYTLRNTGPEDVVVKVLYCGICHTDIHQAKNHLGA 60 bmr6-3 MGSLASERKVVGWAARDATGHLSPYTYTLRNTGPEDVVVKVLYCGICHTDIHQAKNHLGA 60 BTx623 MGSLASERKVVGWAARDATGHLSPYTYTLRNTGPEDVVVKVLYCGICHTDIHQAKNHLGA 60 IS23253 MGSLASERKVVGWAARDATGHLSPYTYTLRNTGPEDVVVKVLYCGICHTDIHQAKNHLGA 60 IS23789 MGSLASERKVVGWAARDATGHLSPYTYTLRNTGPEDVVVKVLYCGICHTDIHQAKNHLGA 60 IS21549 MGSLASERKVVGWAARDATGHLSPYTYTLRNTGPEDVVVKVLYCGICHTDIHQAKNHLGA 60 ************************************************************ bmr6-ref SKYPMVPGHEVVGEVVEVGPEVSKYGVGDVVGVGVIVGCCRECSPCKANVEQYCNKKIWS 120 bmr6-3 SKYPMVPGHEVVGEVVEVGPEVSKYGVGDVVGVGVIVGCCRECSPCKANVEQYCNKKIWS 120 BTx623 SKYPMVPGHEVVGEVVEVGPEVSKYGVGDVVGVGVIVGCCRECSPCKANVEQYCNKKIWS 120 IS23253 SKYPMVPGHEVVGEVVEVGPEVSKYGVGDVVGVGVIVGCCRECSPCKANVEQYCNKKIWS 120 IS23789 SKYPMVPGHEVVGEVVEVGPEVSKYGVGDVVGVGVIVGCCRECSPCKANVEQYCNKKIWS 120 IS21549 SKYPMVPGHEVVGEVVEVGPEVSKYGVGDVVGVGVIVGCCRECSPCKANVEQYCNKKIWS 120 ************************************************************ bmr6-ref YNDVYTDGRPT-GGFASTMVVDQKFVVKIPAGLAPEQAAPLLCAGVTVYSPLKAFGLTAP 179 bmr6-3 YNDVYTDGRPTQGGFASTMVVDQKFVVKIPAGLAPEQAAPLLCAGVTVYSPLKAFGLTAP 180 BTx623 YNDVYTDGRPTQGGFASTMVVDQKFVVKIPAGLAPEQAAPLLCAGVTVYSPLKAFGLTAP 180 IS23253 YNDVYTDGRPTQGGFASTMVVDQKFVVKIPAGLAPEQAAPLLCAGVTVYSPLKAFGLTAP 180 IS23789 YNDVYTDGRPTQGGFASTMVVDQKFVVKIPAGLAPEQAAPLLCAGVTVYSPLKAFGLTAP 180 IS21549 YNDVYTDGRPTQGGFASTMVVDQKFVVKIPAGLAPEQAAPLLCVGVTVYSPLKAFGLTAP 180 *********** *******************************.**************** bmr6-ref GLRGGIVGLGGVGHMGVKVAKAMGHHVTVISSSSKKRAEAMDHLGADAYLVSTDAAAMAA 239 bmr6-3 GLRGGIVGLGSVGHMGVKVAKAMGHHVTVISSSSKKRAEAMDHLGADAYLVSTDAAAMAA 240 BTx623 GLRGGIVGLGGVGHMGVKVAKAMGHHVTVISSSSKKRAEAMDHLGADAYLVSTDAAAMAA 240 IS23253 GLRGGIVGLGRVGHMGVKVAKAMGHHVTVISSSSKKRAEAMDHLGADAYLVSTDAAAMAA 240 IS23789 GLRGGIVGLGRVGHMGVKVAKAMGHHVTVISSSSKKRAEAMDHLGADAYLVSTDAAAMAA 240 IS21549 GLRGGIVGLGGVGHMGVKVAKAMGHHVTVISSSSKKRAEAMDHLGADAYLVSTDAAAMAA 240 ********** ************************************************* bmr6-ref AADSLDYIIDTVPVHHPLEPYLSLLRLDGKHVLLGVIGEPLSFVSPMVMLGRKAITGSFI 299 bmr6-3 AADSLDYIIDTVPVHHPLEPYLSLLRLDGKHVLLGVIGEPLSFVSPMVMLGRKAITGSFI 300 BTx623 AADSLDYIIDTVPVHHPLEPYLSLLRLDGKHVLLGVIGEPLSFVSPMVMLGRKAITGSFI 300 IS23253 AADSLDYIIDTVPVHHPLEPYLSLLRLDGKHVLLGVIGEPLSFVSPMVMLGRKAITGSFI 300 IS23789 AADSLDYIIDTVPVHHPLEPYLSLLRLDGKHVLLGVIGEPLSFVSPMVMLGRKAITGSFI 300 IS21549 AADSLDYIIDTVPVHHPLEPYLSLLRLDGKHVLLGVIGEPLSFVSPMVMLGRKAITGSFI 300 ************************************************************ bmr6-ref GSIDETAEVLQFCVDKGLTSQIEVVKMGYVNEALERLERNDVRYRFVVDVAGSNVEEDAA 359 bmr6-3 GSIDETAEVLQFCVDKGLTSQIEVVKMGYVNEALERLERNDVRYRFVVDVAGSNVEEDAA 360 BTx623 GSIDETAEVLQFCVDKGLTSQIEVVKMGYVNEALERLERNDVRYRFVVDVAGSNVEEDAA 360 IS23253 GSIDETAEVLQFCVDKGLTSQIEVVKMGYVNEALERLERNDVRYRFVVDVAGSNVEEDAA 360 IS23789 GSIDETAEVLQFCVDKGLTSQIEVVKMGYVNEALERLERNDVRYRFVVDVAGSNVEEDAA 360 IS21549 GSIDETAEVLQFCVDKGLTSQIEVVKMGYVNEALERLERNDVRYRFVVDVAGSNVEEDAA 360 ************************************************************ bmr6-ref DAPSN- 364 bmr6-3 DAPSN- 365 BTx623 DAPSN- 365 IS23253 DAPSN- 365 IS23789 DAPSN- 365 IS21549 DAPSN- 365 ***** Supplementary Fig. S3