Development of molecular markers using MFLP linked to a gene conferring resistance to Diaporthe toxica in narrow-leafed lupin (Lupinus angustifolius L.)

Abstract

Phomopsis stem blight (PSB) caused by Diaporthe toxica is a major disease in narrow-leafed lupin ( Lupinus angustifolius L.). The F(2) progeny and the parental plants from a cross between a breeding line 75A:258 (containing a single dominant resistance gene Phr1 against the disease) and a commercial cultivar Unicrop (susceptible to the disease) were used for development of molecular markers linked to the disease resistance gene. Two pairs of co-dominant DNA polymorphisms were detected using the microsatellite-anchored fragment length polymorphism (MFLP) technique. Both pairs of polymorphisms were isolated from the MFLP gels, re-amplified by PCR, sequenced, and converted into co-dominant, sequence-specific and PCR-based markers. Linkage analysis by MAPMAKER suggested that one marker (Ph258M2) was 5.7 centiMorgans (cM) from Phr1, and the other marker (Ph258M1) was 2.1 cM from Ph258M2 but further away from Phr1. These markers are suitable for marker-assisted selection (MAS) in lupin breeding.

Biotechnology approaches to overcome biotic and abiotic stress constraints in legumes

“…There are some exceptions where MAS has already facilitated breeding efforts for several legume crops against important biotic stress. For instance MAS was successfully used for the breeding of resistant soybean to cyst nematode (Diers, 2004), of resistant pinto bean to common bacterial blight (Mutlu et al, 2005) and of resistant narrow-leafed lupin (Lupinus angustifolius L.) to phomopsis stem blight (Yang et al, 2002) and anthracnose . Moreover, when resistance is conferred by single genes and/or easily overcome by new pathogen races, the gene pyramiding strategy facilitated by MAS can be an efficient method.…”

Evaluation of resistance to Phomopsis stem blight (caused by Diaporthe toxica) in Lupinus albus

“…In each of the experiments it was susceptible to Phomopsis stem and pod blight when challenged with the Tarcutta isolate of D. toxica. This line has been used extensively in breeding for Phomopsis resistant L. angustifolius cultivars and has been used to develop molecular markers for disease resistance using microsatellite-anchored fragment length polymorphism (MFLP) (Yang et al 2002). 75A:258 contains a single, dominant gene (named Phr1) for strong resistance to Phomopsis and may be subject to unlinked modifier genes .…”

“…Glasshouse screening for resistance to D. toxica Experiment 2 Forty-two genotypes of L. albus (11 cultivars, 21 breeding genotypes, and 10 landraces) and three L. angustifolius genotypes were sown in 175 mm diameter pots containing sandy-loam in a randomised glasshouse trial in Wagga Wagga, NSW, Australia with four replicates to assess latent stem infection to D. toxica. The L. angustifolius genotypes were included as standards: 75A:258 is the major source of Phomopsis stem blight resistance in modern L. angustifolius varieties (Yang et al 2002), while…”

Development of a co-dominant DNA marker tightly linked to gene tardus conferring reduced pod shattering in narrow-leafed lupin (Lupinus angustifolius L.)

“…DNA extraction and MFLP tests followed the protocol of Yang et al (2001). MFLP markers showing banding patterns corresponding to the tardus/Tardus phenotypes of the 16 F 8 RIL population were considered as candidate markers (Yang et al 2002;Yang et al 2004). The candidate marker which showed best correlation between phenotypes and marker genotypes on both four domesticated cultivars and four wild types, respectively, was selected for further study to convert into a simple PCR marker for implementation for MAS (Yang et al 2008).…”

“…A pair of sequence-specific primers was designed to flank the DNA variation sites, which were used in PCR to convert the marker into a sequence-specific PCR marker. Detailed methods of DNA cloning and marker conversion were described elsewhere (Yang et al 2002).…”