Molecular Plant Breeding 2016, Vol.7, No.33, 1
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15
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climatological conditions, land property and a rich crop pattern. The main rice growing regions in Turkey are the
North western (Marmara-Thrace) and the northern parts of the country (Black sea region). Blast disease is one of
the most serious problems in temperate rice cultivation of Turkey. A heavy blast epidemic occurred in some
growing areas at northwest of Turkey in 1995 and 1997 which caused 25 percent yield loss in 25,000 hectares of
rice growing areas in the region. Therefore, varietal improvement for blast disease resistance is imperative toward
development of high yielding varieties (Beşer and Sürek, 2012).
Osmancık-97 is a semi-dwarf elite Turkish cultivar released in 1997. It has all desirable agronomic and grain
quality traits with a high milling yield potential but susceptible to blast disease (Manners, 2013). Halilbey is
another elite variety with high yield potential but highly susceptible to leaf and panicle blast. In some years, rice
production and rice quality is reduced due to severity of the blast disease. Therefore, the development of a
blast-resistant cultivar has still been the most effective and economical breeding strategy to control the spread of
the disease (Jeung et al., 2007). Resistance of rice cultivars to blast disease are developed by conventional
breeding using the sources of resistance from land races or diverse rice varieties. However, the resistance breaks
down in two to three years (Miah et al., 2013). Novel sources of blast resistance genes have been identified in the
introgression lines derived from wild
Oryza
species (Jena and Kim, 2010). Of the 100 blast resistance genes, the
Pi40
gene has been identified in an indica introgression line that inherited the resistance gene from a wild species
O. australiensis
and has durable broad-spectrum of blast resistance
(Jeung et al., 2007; Sharma et al., 2012; Suh et
al., 2009). The
Pi40
gene confers blast resistance at seedling as well as reproductive stages. It has shown
resistance to emerging virulent blast strains in several countries and several provinces of China (Wang, 2012,).
Marker-assisted backcross breeding is a powerful tool to incorporate novel resistance genes into susceptible
cultivars and develop improved breeding lines in a short period of time (Jena and Mackill, 2008).
The objective of this study is to incorporate the
Pi40
gene into two Turkish elite cultivars, Osmancık-97 and
Halilbey, through marker-assisted backcross breeding and develop improved blast resistant cultivars for Turkey.
This study will lead to the production of promising breeding lines with blast resistance in a short period of time
using resistance gene specific molecular marker analysis tools. Background genome analysis of progenies will
show conversion of recurrent parent genome with the
Pi40
gene-derived blast resistance introgression.
1 Results and Analysis
1.1 Development and molecular analysis of ABLs of Osmancık-97and Halilbey
Five F
1
plants from the crosses of Osmancık-97 and Halilbey with donor parent (DP) were produced. Hybridity of
the F
1
plants were confirmed and were further backcrossed to recurrent parent (RP). The presence of the
Pi40
gene
was tracked in backcross (BC) progenies by marker-assisted selection (MAS). Several BC progenies from
Osmancık-97 and Halilbey crosses were selected and validated by MAS (Figure 1a). Selected plants having the
Pi40
gene were advanced until BC
3
F
5.6
and BC
4
F
5
.
6
and the progenies having phenotypic traits similar to the respective
RP were selected (Supplementary Figure 1).
The usability of
Pi40
gene-specific marker in high throughput genotyping technologies was checked using
Fragment Analyzer. Capillary electrophoresis of polymerase chain reaction (PCR) amplicons of the susceptible
recurrent parents detected three susceptible allele-specific bands, 128 bp, 140 bp, and 415 bp for Halilbey, and 128
bp, 140 bp, and 420 bp for Osmancik-97. On the other hand, four bands were detected in the resistant genotype,
donor parent. Among the four products, two amplicons (resistance specific-alleles) were unique to the donor parents
which were 194
bp and 230 bp. Eleven advanced backcross lines (ABLs) were also analyzed and all showed the
presence of the resistance specific-alleles (Figure 1b). The designed DNA marker linked to the
Pi40
gene required
the digestion of the PCR products with
MluC1
which produced two unique bands for the resistant allele while the
susceptible allele showed two common bands. Our results from the Fragment Analyzer were in congruence with
these as it was able to detect the same profile of bands in susceptible and resistant genotypes (Figure 1b).
