Quantitative Trait Loci

1. Mapping of QTL Controlling
Powdery Mildew Resistance
in Mungbean.
Presented By :
Vishwas Acharya M.Sc. (Scholar)
Genetics and Plant Breeding

2. What is Quantitative Trait Loci Analysis ?
 Quantitative trait locus (QTL) analysis is a statistical method that links
two types of information phenotypic data (trait measurements) and
genotypic data (usually molecular markers) in an attempt to explain
the genetic basis of variation in complex traits.
 QTL analysis allows researchers to link certain complex phenotypes to
specific regions of chromosomes.
 The goal of this process is to identify the action, interaction, number,
and precise location of these regions.

3. QTL Mapping
a) Quantitative trait locus (QTL) mapping requires parental
strains that differ genetically for the trait.
b) The parental lines are crossed to create mapping population
like F2, RIL, AIL, etc.. Phenotyping and Genotyping.
c) Statistical techniques such as CIM evaluate the probability
that a marker or an interval between two markers is
associated with a QTL affecting the trait, while
simultaneously controlling for the effects of other markers on
the trait. The results of such an analysis are presented as a
plot of the test statistic against the chromosomal map
position, in recombination units (cM).

4. Case Study
 Two quantitative trait loci (QTLs) controlling resistance to the Powdery Mildew disease in a RIL
population of 190 F7 lines.
 The population was developed from the cross between a susceptible cultivar, “Kamphaeng Saen 1”
and a resistant line, “VC6468-11-1A”.
 Results from analysis of variance revealed that 15 SSR loci on three linkage groups (LG) associated
with the resistance.
 Composite interval mapping consistently identified two QTLs on two LGs, qPMR-1 and qPMR-2,
conferring the resistance. qPMR-1 and qPMR-2 accounted for 20.10 and 57.81% of the total
variation for plant response to the disease, respectively.
 The SSR markers flanking and closely linked to qPMR-1(CEDG282 and CEDG191) and qPMR-2
(MB-SSR238 and CEDG166) are useful for marker-assisted selection for mung bean resistance to
powdery mildew.

5. Materials and Methods
Plant Materials
 The cross between a susceptible cultivar, “Kamphaeng Saen 1”
(Female) and a resistant line, “VC6468-11-1A” (Male).
 Finally, 190 F7 recombinant inbred lines (RILs) were obtained and
used in this study.
 Genomic DNA extraction by CTAB method.

6.  Evolution of PM Resistance
 The RILs and their parents were assayed in RCBD with three replications.
The susceptible cultivar, KPS1 was grown surrounding the evaluating plots
as disease spreading rows.
 Plants were scored in rows based on percentage of leaf area covered by the
disease at 7-day interval from the first appearance of the disease until
maturity.
 1 (0.1 - 10% leaf coverage), 2 (10.1 - 25% leaf coverage), 3 (25.1 - 50% leaf
coverage), 4 (50.1 - 75% leaf coverage), and 5 (75.1 - 100% leaf coverage).
 Disease index (DI) was calculated. In addition, to compare the progress of
powdery mildew development among the RILs, the area under the disease
progress curve (AUDPC) was computed.
 For greenhouse evolution same manner as field evolution describe above.

7.  DNA marker analysis
 Total of 412 SSR primer pairs from various legume crops were used to detect
polymorphism between the parents.
 PCR amplification was performed by GeneAmp® PCR System 9700 thermocycler.
 Then, 1.5 - 2 μl of the final product was loaded onto 5% polyacrylamide gel and
visualized by silver staining.
 To find out whether the putative QTL(s) found in our study is possibly the same as
those reported earlier by Chaitieng et al. (2002), two sequence tagged site (STS)
markers were developed from DNA sequences of AFLP bands reportedly associated
with the resistance.
 Polymorphism among the parents was used to identify the location of the QTLs from
both studies. SSR and STS markers developed by Zhang et al. (2008) closely linked to
the major QTL identified by Humphry et al. (2003) were also used to compare our
QTL location to these previous results.

8.  Linkage map
 A genetic linkage map was constructed by MAPMAKER/EXP 3.0.
 A minimum LOD threshold of 4.0 and maximum distance of 50 cM were used for
construction of linkage groups.
 Map distance in centimorgan (cM) values was calculated using Haldane mapping
function.
 The linkage map was drawn using MapChart software.
QTL analysis
 For QTL analysis, one-way ANOVA was analyzed to determine significant association
between powdery mildew resistance and a marker at P = 0.001.
 The software program WinQTL Cartographer version 2.5 was used to identify QTLs
associated with the disease resistance by composite interval mapping (CIM).

9. Results
Disease variation in RIL population
 In this study, we used DI to indicate disease coverage on leaves, and AUDPC to
indicate progress of disease development.

10.  Linkage map
 Among 412 SSR primer pairs used to test for polymorphism between the
parents, only 38 (9.2 %) showed polymorphic bands.
 These 38 SSR markers were used to genotype the RILs and a partial genetic
map was constructed.
 Thirty SSR markers could be assigned into five linkage groups (LG) and eight
markers unlinked.
 The total length of the map was 236 cM. The length among the 5 LGs varied
between 1.2 to 78.9 cM with an average of 47.2 cM.

11. QTL map
 Preliminary identification for QTL controlling powdery mildew resistance by one-way
ANOVA revealed that 15 markers on 3 LGs showed significant association (P < 0.001) with
DI and AUDPC under field and greenhouse conditions.
 For QTL mapping, the significant LOD thresholds computed for DI and AUDPC under field
conditions were 10.60 and 10.70.
 Composite interval mapping consistently identified two QTLs [one minor (qPMR-1) and
one major (qPMR-2)] on different linkage groups (LG I and LG II) involved in powdery
mildew resistance.
 The qPMR-2 was located on LG II between markers MB-SSR238 and CEDG166. The
percentage of phenotypic variance explained (PVE) by this locus for DI and AUDPC was
between 38.53 to 57.81%. The qPMR-1 was localized on LG I between markers CEDG282
and CEDG191.
 ThePVE for DI and AUDPC at this QTL was between 14.64 to20.10%. At both loci, alleles
from VC6468A decreased DI and AUDPC values, i.e. increased the resistance.

12. QTL confering powdery mildew resistance in the RIL population as
detected by composite interval mapping

13. Discussion
 In our study, the QTL mapping using SSR markers revealed one major and one minor
locus associated with resistance to powdery mildew disease.
 while ours is the first study of gene mapping using SSR markers in this crop.
Although due to a lack of common markers available, it is difficult to compare QTL
location found in this study with the previously identified QTLs.
 Currently, SSR markers flanking and closely linkedto qPMR-1 (CEDG282 and
CEDG191) and to qPMR-2 (MBSSR238 and CEDG166) can be used to aid the
selection for mungbean resistance genotypes.
 In conclusion, resistance to powdery mildew disease in VC6468-11-1A has high
heritability. QTL mapping revealed major and minor QTLs controlling the resistance.

14. Discussion
 Location of QTL conferring resistance
to powdery mildew disease on an SSR
partial linkage map of mungbean RIL
population derived from the cross
between KPS1 x VC6468A.
 Dotted line with hash mark indicates
unlinked markers.

15. Thank You !
Vishwas Acharya M.Sc. 2nd Sem.
(Genetics and Plant Breeding)
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