Marker assisted selection (2)

MARKER ASSISTED SELECTION FOR HERBICIDE RESISTANCE

PRESENTED BY: SHREYA LODHUNDER THE GUIDANCE OF: DR.T.R.SHARMA DR.R.K.CHAHOTA

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.

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

PROCEDURE

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

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

QTL5 cM

RELIABILITY FOR SELECTION

Using marker A only:

1 – rA = ~95%Marker AQTL

Marker B

5 cM 5 cMUsing markers A and B:

1 - 2 rArB = ~99.5%

Markers must be polymorphic

1 2 3 4 5 6 7 8

1 2 3 4 5 6 7 8RM84 RM296

Not polymorphic Polymorphic!

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.

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 polymorphismis also shown (marked out in a box). Numbers at the end of the sequences indicatethe expected size of each PCR fragment

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.

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.

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.

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.

Herbicide Resistance MarkersGenes 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.

Selectable marker genes

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