This presentation discusses marker assisted selection (MAS) for herbicide resistance. MAS uses DNA markers linked to target genes to predict phenotypes, allowing selection earlier than phenotypic screening. The presentation provides advantages of MAS like simpler screening and selection at seedling stage. A case study of MAS for herbicide resistance in sunflower is described, where different markers like SSRs, CAPS and SNPs were developed for alleles conferring resistance. Transgenic approaches for herbicide resistance using selectable marker genes like pat/bar for phosphinothricin and epsps/aroA for glyphosate resistance are also summarized.

1. MARKER ASSISTED
SELECTION
FOR
HERBICIDE RESISTANCE
PRESENTED BY:
SHREYA LODH
UNDER THE GUIDANCE OF:
DR.T.R.SHARMA

2. HIGHLIGHTS OF THE
PRESENTATION
 Overview of MAS , its advantages and
basic procedure along with important
aspects of a marker.
 MAS for herbicide resistance in sunflower
(case study).
 Introduction to Transgenics for herbicide
resistance.

3. MARKER ASSISTED
SELECTION
(MAS)
The use of DNA markers that are tightly-linked
to target loci as a substitute for or to assist
phenotypic screening
Assumption
DNA markers can reliably predict phenotype

4. PROCEDURE

5. Advantages of MAS
 Simpler method compared to phenotypic screening
 Especially for traits with laborious screening
 May save time and resources
 Selection at seedling stage
 Important for traits such as grain quality
 Can select before transplanting in rice
 Increased reliability
 No environmental effects
 Can discriminate between homozygotes and heterozygotes
and select single plants
 Accurate and efficient selection of specific
genotypes
 May lead to accelerated variety development

6. Important Aspects Of A Marker
Markers must be tightly-linked to target loci!
• Using a pair of flanking markers can greatly improve reliability
but increases time and cost
Marker A
QTL
5 cM
RELIABILITY FOR
SELECTION
Using marker A only:
1 – rA = ~95%
Marker A
QTL
Marker B
5 cM 5 cM
Using markers A and B:
1 - 2 rArB = ~99.5%

7. Markers must be polymorphic
1 2 3 4 5 6
7 8
1 2 3 4 5 6 7
8
RM84 RM296
Not polymorphic Polymorphic!

8. CASE STUDY : SUNFLOWER
 IMISUN, SURES, and CLPlus are three herbicide
tolerance traits in sunflower which are determined
by the expression of different alleles at the same
locus, Ahasl1(multiallelic locus).
 The Ahasl1 gene sequences from lines carrying
different alleles for susceptibility or resistance
showed single nucleotide polymorphisms and length
variations for a simple sequence repeat.
 These differences were utilized to develop three
types of PCR markers (SSRs, CAPS and SNPs)
which allow the precise identification of each allele
at the Ahasl1 locus.

9. Differences among Ahasl1 alleles sequences
Figure 1. Partial nucleotide sequences alignment of HaAhASL1 for four
different alleles:
ahasl1 (susceptible), Ahasl1-1 (IMISUN), Ahasl1-2 (SURES), and Ahasl1-3
(CLPLus). The position of the A122T single nucleotide polymorphism
is also shown (marked out in a box). Numbers at the end of the
sequences indicate
the expected size of each PCR fragment

10. SSR Marker Analysis
 The presence of an ACC repetition pattern in the
nucleotide sequence of the putative transit peptide of the
AHASL protein sequence permitted to develop an SSR
marker.
Figure 2:
Lane1: ahasl1
Lane3: Ahasl1-3
 No difference in fragment size were observed for lines
carrying ahasl1 and Ahasl1-3 allele since both of them
harbor the same number of (ACC) repeats in the
nucleotide sequence. In this case use another type of
marker or phenotypic assays in order to identify these
alleles or their combination.

11. CAPS Marker Analysis
 The sequence obtained from the lines carrying
Ahasl1-3 (CLPlus) presents a single nucleotide
polymorphism when compared with the sequence
obtained from the line carrying the ahasl1 allele
(wild type).
Figure 1:
Two bands(183 + 138bp) were obtained for the wild type allele ahasl1;
three different bands(183 + 77 + 62 bp) were observed for the Ahasl1-3
allele present in the CLPlus lines.

12. SNP Marker Analysis
 SNPs markers developed also can be used for line
conversions, rapid sterilization of maintainer lines
and characterization of the resistant gene present in
lines extracted from populations segregating for two
or more resistant traits.
 Because of the dominant nature of the SNP markers
they can also be applied for checking seed purity of
any particular resistant line in order to assure it
carries the correct trait (IMISUN, SURES CLPlus) in
any specified level.

13. Herbicide Resistance By Transgenics
• Plants that have been genetically
engineered by incorporating genes from
another species to express agriculturally-
desirable traits, including resistance to
herbicides are known as TRANSGENIC
PLANTS.
• In this selectable marker genes are usually an
integral part of plant transformation system.
They are present in the vector along with the
target gene.

14. Herbicide Resistance Markers
Genes that confer resistance to herbicides are in use as markers for the
selection of transgenic plants.
 Phosphinothricin acetyltransferase (pat/bar gene):
Bialophos, phosphinothricin and glufosinate are commonly used
herbicides. The pat/bar genes code for phosphinothricin
acetyltransferase which converts these herbicides into acetylated forms
that are non-herbicidal. Thus, pat/bar genes confer resistance to the
transformed plants.
 Enolpyruvylshikimate phosphate synthase (epsps/aroA genes):
The herbicide glyphosate inhibits photosynthesis. It blocks the activity of
enolpyruvylshikimate phosphate (EPSP) synthase, a key enzyme involved
in the biosynthesis of phenylalanine, tyrosine and tryptophan. Mutant
strains of Agrobacterium and Petunia hybrida that are resistant to
glyphosate have been identified. The genes epsps/aroA confer resistance
to transgenic plants which can be selected.
 Bromoxynil nitrilase (bxn gene):
The herbicide bromoxynil inhibits photosynthesis (photosystem II).
Bromoxynil nitrilase enzyme coded by the gene bxn inactivates this
herbicide. The gene bxn can be successfully used as a selectable marker
for the selection of transformed plants.

15. Selectable marker genes

17. Thank You