Abstract

Media summary

Key Words

Marker-assisted selection (MAS), MFLP, disease resistance, lupin, plant breeding

Introduction

Anthracnose is the most serious disease on lupin crops (Lupinus spp.) worldwide (Yang and Sweetingham 1998). The disease is caused by the fungal pathogen Colletotrichum lupini (Nirenberg et al. 2002), which was formerly classified as C. gloeosporioides (Yang and Sweetingham 1998) or C. acutatum (Sreenivasaprasad et al. 1994). Selection for anthracnose resistance is one of the major objectives in lupin breeding programs.

Marker-assisted selection in plant breeding is superior to traditional glasshouse based selection methods, because it offers the advantages of early generation selection, differentiation of homozygous individuals from heterozygous individuals, selection based on genotype rather than phenotype, multiplex test for simultaneously tracking several traits of interest (Tanksley et al. 1989). An implementable marker, or a “breeder-friendly marker”desirable for practical plant breeding, should meet three requirements: (1) the marker must be linked to an agronomic trait of industry significance; (2) the marker must be simple, and reliable to run; and (3) the marker must be cost-efficient, and amenable to large numbers of samples.

A number of methods are available for development of molecular markers for MAS (Gupta et al. 1999). Recently, Yang et al. (2001) reported the microsatellite-anchored fragment length polymorphism (MFLP) method. MFLP is based on combination of the AFLP concept (Vos et al. 1995) and SSR-primers (Wu et al. 1994) targeting the microsatellite sites in a genome which are highly polymorphic (Chin et al. 1996). Many MFLP polymorphisms are co-dominant, which can easily be converted into simple PCR-based markers desirable for routine marker implementation (Yang et al. 2001, 2002). The MFLP is a versatile system amenable to further modifications. For example, the SSR-primers using in MFLP can be replaced with the nucleotide binding site (NBS) sequence derive primers (NBS-primers) (Collins et al. 1998) to amplify and detect NBS-anchored fragment length polymorphisms (NBS-FLP) associated with plant disease resistance gene analogs (RGAs) (Lopez et al. 2003). The aim of this study is to develop molecular markers linked to an anthracnose resistance gene in lupin using the MFLP and NBS-FLP techniques, and to implement the markers in Australian lupin breeding program for marker-assisted selection.

Methods

Plant materials and disease resistance testing

A cross was made between cultivar Tanjil (resistant to anthracnose disease) and Unicrop (susceptible). The parents, F₁ and the F₂ plants were grown in a 25 cm-diameter plastic pots filled with river sand, and maintained in a 20±2°C glasshouse. When the main stem inflorescences were in full blossom, flowers were carefully excised by cutting through the base of the pedicel with a sharp scalpel blade. The plants were immediately inoculated with anthracnose strain VCG-2 of Colletotrichum lupini (Yang and Sweetingham 1998) by spraying with a conidial suspension (1 × 10⁶ conidia per ml) until run-off. The inoculated plants were enclosed in plastic bags and incubated for 24 h in darkness. Disease on the main stem inflorescence spike was recorded 14 days after inoculation. Plants with small (<5 mm in length) superficial scars were considered as resistant. Plants with collapsed spikes or with large (>10 mm in length) lesions bearing pink conidial masses were regarded as susceptible. The progenies from the same cross were advanced for development of F₈ recombinant inbred lines (RILs). The parents and the F₈ RILs were tested for anthracnose resistance as above.

Search for candidate molecular markers

DNA was extracted from the two parents, and from five resistant and five susceptible progenies. DNA from the 12 plants were subjected to MFLP fingerprinting for searching candidate molecular markers as describe before (Yang et al. 2001). Protocol of the NBS-FLP was the same as MFLP except that the SSR-primers used in MFLP were replace with NBS-primers designed based on Collins et al. (1998). The PCR products in MFLP and NBS-FLP were resolved in a 97-well denaturing sequencing gel. Each gel contained 96 reactions, which consisted of eight sets of MFLP or NBS-FLP reactions each including the 12 plants. Polymorphic DNA bands in the fingerprints showing evidence of correlation to disease resistance or susceptibility were regarded as candidate markers linked to the disease resistance gene, and were subjected to further investigation.

Conversion of candidate markers into sequence-specific PCR markers

DNA fragments were isolated from dried sequencing gel, re-amplified in PCR, then cloned into E. coli and sequenced according to standard procedures (Ausubel et al. 1988). Methods of designing sequence-specific primers to convert the candidate markers into simple PCR based markers were described before (Yang et al. 2001, 2002). The converted PCR based markers were tested on 221 F₈ RILs. The marker score data and the disease score data on the F₈ RILs were merged for linkage analysis using computer program MapMaker (Lander et al. 1987).

Implementation of molecular markers in lupin breeding

DNA for marker implementation was extracted using a rapid and inexpensive procedure. Two leaflets from a 10-day old lupin seedling were homogenised in DNA extraction buffer in a 96-deep well plate on a Mixer Mill (Model MM300, Qiagen Pty Ltd, Victoria 3068, Australia), heated at 90°C in a water bath for 20 min. After cooling down and centrifugation, the upper aqueous phase was transferred into a new plate, and was mixed with 0.5x 6 M sodium acetate. DNA was precipitated with 0.8x isopropanol, and dissolved in 100 μl TE buffer. PCR was conducted using 96-well plate as describe before (Yang et al. 2002). The marker bands were resolved on 97-well denaturing sequencing gel. Lupin plant with homozygous resistance bands were selected, and handed to breeders for further testing in the breeding cycle.

Results

Genetics of disease resistance in Tanjil

In glasshouse tests, the parent Tanjil and F₁ plants were all resistant, and the parent Unicrop were susceptible. Among 167 F₂, 127 plants were resistant to anthracnose, 40 plants were susceptible, which fitted the 3:1 ratio (χ² = 0.098, P = 0.754). With 221 F₈ RILs tested, 106 lines were resistant, and 115 lines were susceptible, which fitted the 1:1 ratio (χ² = 0.367, P = 0.549). It is concluded that the anthracnose resistance in Tanjil of L. angustifolius is controlled by one dominant gene, designated as Lanr1.

Marker development

One set of co-dominant candidate molecular marker was detected in a NBS-FLP fingerprints generated by NBS primer GLPL-A (5’-GAGIGCIAGIGGIARICC-3’) with AFLP primer MseI-AA (Figure 1). The second set co-dominant candidate marker was detected in a MFLP fingerprints generated by SSR-primer MF201 (5’-CCCATTGTTGTTGTTG-3’) in combination with AFLP primer MseI-CAA.

Figure 1. An section of NBS-FLP fingerprint showing a co-dominant marker (arrowed) linked to anthracnose resistance gene Lanr1 in L. angustifolius. First three and the last three plants were resistant to the disease. The six plants in middle were susceptible.	Figure 2. Genetic linkage of the two molecular markers and the anthracnose resistance gene Lanr1 in Lupinus angustifolius based on 221 F8 recombinant inbred lines (RILs) using computer program MapMaker.

The cloning and sequencing on DNA fragments of candidate markers revealed that both of the two sets of co-dominant markers originated from variation of insertion/deletion. Sequence-specific primers were designed flanking the variation sites, which successfully converted the candidate markers into co-dominant, simple PCR based markers (Figure 3).

Linkage analysis using the marker score data and the disease score data on the F₈ RILs suggested the two markers were flanking the anthracnose resistance gene Lanr1. One marker (AntjM1) is 3.5 centiMorgans (cM) from the gene Lanr1, and the other marker (AntjNBS) is 2.1 cM from the gene Lanr1(Figure 2).

Figure 3. Example of marker screening on F₂ plants of L. angustifolius using molecular marker AntjNBS in Australian lupin breeding program in 2003. In each cross, 94 plants were tested. The marker bands from two crosses were loaded in 15 min interval and resolved in one gel. Plants with homozygous resistance marker band (showing top band only, marked with red dot above) were selected, and handed to breeders for further testing in the breeding cycle. Plants showing heterozygous resistance marker pattern (showing two bands) or homozygous susceptible marker (showing bottom band only) were discarded.

Implementation of anthracnose resistance marker in lupin breeding

The anthracnose resistance markers were implemented in the Australian Collaborated Lupin Improvement Program. In 2003, a total of 7321 breeding materials were screened with anthracnose markers. F₂ plants showing homozygous resistance marker bands were selected (Figure 3), and handed to breeders for further testing in the breeding cycle.

Disease tests were conducted in glasshouse on F3 plants derived from the F2 plants selected based on marker screening. 98% of these plants showed resistance to the disease.

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