This document describes a study that used marker-assisted backcrossing to transfer brown midrib (bmr) traits from donor parents into high biomass sorghum cultivars. KASPar assays were developed for SNPs in genes associated with bmr6 and bmr12. These markers were used to select plants in backcross generations. Three improved sorghum lines with introgressed bmr6 or bmr12 traits and high biomass were developed. The bmr traits are expected to increase sugar release and digestibility for biofuel production or livestock feed.

1. DEVELOPMENT OF BROWN MIDRIB (bmr) 6 AND 12 HYBRID
PARENTS THROUGH MARKER-ASSISTED BACKCROSSING (MABC)
IN SORGHUM
ABSTRACT
Production of renewable fuels, especially from plant biomass are gaining importance due to high energy balance
and reduced greenhouse gas emissions. However, lignocellulose based ethanol production is still in pilot scale, as
the pre-treatment cost is high due to the presence of lignin that confers mechanical strength to the cell wall. Low
lignin feed stocks are not only useful in biofuel sector, but also preferable as fodder as they are more palatable
and possess high in vitro organic matter digestibility. In this context, natural mutants with altered expression of
genes involved in lignin biosynthesis pathway are highly desirable. Allelic studies in sorghum have revealed four
different loci bmr2, bmr6, bmr12 and bmr19 that contribute to the bmr phenotype. Brown midrib (bmr) is a
visible marker associated with the reduction of lignin. In this study we have successfully transferred bmr
phenotype (bmr6 and bmr12) into high biomass-low lignin sorghum cultivars through marker-assisted
backcrossing (MABC) using KASPar assays. These introgressed bmr cultivars could be highly amenable to biofuel
production due to their altered lignin content.
Subhasini V Reddy 1, 2, Santosh P Deshpande1, B V S Reddy 1, D Manohar
Rao 2, A Ashok, Kumar 1*
1 International Crops Research Institute for the Semi-Arid Tropics, Telangana, India
2 Osmania University, Hyderabad, Telangana, India, * Corresponding author email: a.ashokkumar@cgiar.org
MATERIALS AND METHODS
Parent Materials
Donor parents (♀) bmr 6: IS 23789; bmr 12 : N 593 and N 595
Recurrent parents (♂) High biomass lines: IS 8813, IS 13762 and IS 21893
Selection of molecular markers for foreground selection
Two KASPar SNP markers present on target bmr region on chromosomes SBI-04 (bmr 6) (Gorthy et al 2013) and
SBI-07 (bmr 12) (Saballos et al 2008) (Table 1) were used for foreground selection across donor-recurrent parent
combination (Fig-2). KASPar assays for the targeted SNPs were carried out at KBioscience, UK.
Marker-assisted Background selection
Background screening of the bmr6 and bmr 12 introgressed lines will be carried out using SSR markers to select
the plants with higher recurrent parent genome recovery.
RESULT AND DISCUSSION
ACKNOWLEDGEMENTS
We are thankful for the study was funded by the Department of scientist and Technology (DST), Reference No: DST No: SR/WOS-A/LS-408/2012 (G), New Delhi, India and Department of Biotechnology funded Joint Clean Energy
Research and Development Center (JCERDC) project, ICRISAT.
Chromosome
number
Marker names
(Primer ID)
SNP
position
Allele in
IS 23789,
N 593 and
N 595
Allele in
IS 8813, IS
13762 and
IS 21893
The sequence used for primer
designing
SBI-04 bmr6_39_3699 3699 CAD
gene
G C
AAAGGCCTTTGGGCTGACGGCCCCAGG
CTCCGCGGTGGCATCGTGGGCCTGGGC
[G/C]
GCGTGGGCCACATGGGCGTGAAGGTGG
CGAAGGCCATGGGCCACC
SBI-07 bmr12_691 691 COMT
gene
T C
CACCCCTAACGAGGACGGCGTCTCCATG
GCCGCCCTCGCGCTCATGAAC [C/T]
AGGACAAGGTCCTCATGGAGAGCTGGT
GAGTAGTCGTCGTCAGAGCACA
Table 2 Details of genotyping, selection for foreground (FGS) selection and crossing of lines in different
generations during marker-assisted backcrossing for bmr 6 and 12 introgression.
Crosses for bmr6
and bmr12
No of
Kaspar
SNPs used
for FGS
No of plants and True hybrids observed in each generation
F1s BC1F1s BC2F1s BC2F2s
No of
plant
s
True
hybrids
No of
plants
True
hybrids
No of
plants
True
hybrids
No of
plants
True
hybrids
IS 23789 x IS
8813
1 6 3 66 29 24 11 568 284
N 593 x IS 13762 1 6 4 8 6 11 7 430 215
N 595 x IS 21893 1 6 2 21 7 7 4 1310 655
Fig. 1 Graphical representation (chromatogram) on Hybridity screening of BC2F2s for bmr6 & bmr12 with Kaspar
SNPs, analyzed through infiniteF200PRO. The dots show polymorphism at a locus on chromosomes SBI 04 and SBI
07. The Red spots denotes homozygous allele from donor parents IS 23789 (bmr6) and N 593 (bmr12), N 595
(bmr12) and green spots is heterozygous allele from both parents whereas blue spots denotes the homozygous
allele from High biomass Recurrent parent i.e., IS 8813, IS 13526, and IS 21893
CONCLUSION
In this study, we successfully developed three high biomass lines with brown midrib trait using marker assisted
backcrossing.
• The two KASPar SNPs present in the candidate genes CAD and COMT have been validated and can be used for
marker-assisted selection of bmr introgressed lines.
• The CAD and COMT alleles in introgression lines might reduce the activity of the CAD2 enzyme due to G-to-C
(G191R) substitution in the CAD2 protein sequence and C-to-T at 691 of COMT gene thus leading to the bmr
phenotype.
• Three improved low lignin, high biomass sorghum introgression lines were IS 8813, IS 13762 and IS 21893 have a
practical breeding value and can be used to increase the conversion efficiency of biomass into ethanol.
Table 1 List and details of KASPar markers used for undertaking the foreground selection in marker-assisted
backcrossing programs to transfer to the bmr region for high biomass sorghum.
INTRODUCTION
High biomass (energy) sorghum produces higher biomass with greater dry matter yields than non-bmr and bmr
forage sorghums. The brown midrib mutation with altered lignin content is associated with an increase in the
accumulation of fermentable sugars and improved digestibility. Mutations in cinnamyl alcohol dehydrogenase
(CAD2) and caffeic O-methyltransferase (COMT) genes contribute to the phenotype of bmr6 and bmr12 groups,
respectively. In the present study, the marker-assisted backcross technique was applied to introgress bmr6 and
bmr12 associated genes from bmr sources to high biomass elite lines. Foreground screening was done using
KASPar markers for the target bmr regions of bmr 6 (Chr. SBI-04) and bmr 12 (Chr. SBI-07) lines in high biomass
lines. These traits are useful to improve the conversion efficiency of the biomass during fermentation and forage
digestibility for livestock.
KASPar assays designed and SNP validation
Kompetitive Allele Specific PCR (KASP) assays were developed and tested for all the identified SNPs using bmr
introgression phenotypes. Two SNPs, IS 23789 for bmr 6 in (bmr 6_39_3699, Chr-4, G-to-C at 3699 of CAD gene) N
593 and N 595 for bmr 12 in (bmr 12_691, Chr-7, C-to-T at 691 of COMT gene), were then selected for marker
validation (Table-1).
Introgression lines (BC2F2s)Parent 1 (Recurrent) Parent 2 (Donor)
× =