Construction Of Genetic Linkage Map And Development Of Molecular Marker Linked To Resistance Gene Of Gray Leaf Spot Disease In Lolium Multiflorum

Italian ryegrass (Lolium multiflorum Lam) is one of the most important temperate forage grasses and is widely cultivated in temperate areas of the world and also in the southern of China. Disease is one of the main factors, which affects its forage yield and quality of Italian ryegrass. Ryegrass blast, also called gray leaf spot, is caused by the fungus Pyricularia sp. It is one of the most serious diseases for Italian ryegrass in China and other temperate regions. With construction of genetic linkage map and development of molecular marker based on DNA, it would facilitate genetic research and breeding of Italian ryegrass. The present study was designed according to the NBS-LRR motif of disease resistance gene by isolating the resistance gene analogs from Italian ryegrass using PCR amplification, multiple combinations of degenerate primers and construct a high-density molecular linkage map of Italian ryegrass, one pseudo-testcross full-sib F₁ population consisting of 124 individuals was used to analyze four types of marker: (1) amplified fragment length polymorphism (AFLP) markers, (2)simple sequence repeat (SSR) markers, (3)expressed sequence tag cleaved amplification polymorphism sequence (EST-CAPS) markers and (4)resistance gene analog CAPS (RGA-CAPS) markers. An F₁ population, consisted 162 individuals, composed from a cross between resistant and susceptible cultivars, was used to screen closely linked markers to resistance gene of gray leaf spot disease by AFLP, EST-CAPS and RGA-CAPS. The main results are as follows:1. RGA isolated from the genome of Italian ryegrassThe large-scale cloning of resistance gene analogs (RGAs) was determined in Italian ryegrass. The degenerate primer designed from conserved motifs of known plant resistance gene products were used to amplify genomic DNA sequences by nested PCR amplification. Total 24000 PCR clones were selected and subjected to one pass sequencing for the sequence similarity search with public database. As a result, 9354 clones showed significantly similar levels of identity compared with known resistance genes or RGAs derived from other species. All these clones were clustered and grouped into 185 clusters based on the nucleotide sequence similarity. The representative clone was selected from each cluster and then was defined as Italian ryegrass RGAs. 150 representative clones, which contains continuous open reading frame, they were selected to covert RGAs to STS markers, 113 specific sequence tagged site (STS) primer sets were designed successfully from these RGA sequences.2. Genetic linkage map of Italian ryegrassOne pseudo-testcross full-sib F₁ population generated for the genetic analysis of cytoplasmic male sterility (CMS) analysis, consisting of 124 individuals, was used to construct a high-density linkage map of Italian ryegrass. Male and female molecular-marker linkage maps were developed using SSR, AFLP, EST-CAPS, RGA-CAPS markers, respectively. In the linkage map of female parent, a total of 362 marker loci, which was consisted of 240 SSR markers, 84 AFLP markers, 24 EST-CAPS markers and 14 RGA-CAPS markers, they were divided into seven linkage groups and covered a total length of 776.4cM. The size of the linkage groups ranged from 85.8 to 135.0 cM with an average of 2.14 cM among markers. The male linkage map was consisted of 376 marker loci, 252 SSR markers, 82 AFLP markers, 29 EST-CAPS markers and 13 RGA-CAPS markers among seven groups, which covered the map distance 710.0 cM. The longest and shortest linkage groups were LG6 and LG7, 113.7 cM and 80.5 cM, respectivcly. The average distance is 1.89 cM among markers. These linkage maps were compared with those constructed from perennial ryegrass, the map constructed in this research showed very high-density of markers, moderately in length, and markers distributed evenly.3. Distribution of RGA in the genome of Italian ryegrassCMS F₁ population (consisting 124 individuals) and 113 pairs of RGA primers were used for RGA mapping. Eighty pairs of RGA primers were amplified with one clear anticipated STS (300-600bp), and then were selected. Using of the 80 pairs of primers to amplify the genomic DNA of two parents and individuals of F₁ population, PCR products digested with endonucleases Aluâ… , Afaâ… , Mboâ… , Mspâ… , Mseâ… , Hinfâ… , Sau96â… , Hindâ…¢, SerFâ… , HpyCH4â…¢, HpyCH4â…£, Nlaâ…£, Hhaâ… , Hpy188â… , Hpy188â…¢, Haeâ…¢; Ddeâ… and Fnu4Hâ… , respectively. Twenty-five polymorphisms were detected and 23 RGA-CAPS markers were mapped to all the seven genetic linkage groups of Italian ryegrass. RGA-CAPS markers distribute all the genome, however, clustered in some loci. Two threemarkers and one two-markers were clustered on the genetic map. Marker RG124E11-2, RG001E01-2 and RG088C11-1 clustered on LG2 within 8.5 cM, marker RG192G09-2, RG073A04-3 and RG119F 12-1 clustered on LG7 within 10.2 cM, marker RG021 F04-2 and RG227D4-1 were mapped on LG3 within 2.0 cM. The region clustered RGA-CAPS markers maybe existed disease resistance locus or loci.4. Screening molecular markers linked to resistance gene of gray leaf spot in Italian ryegrassAn F: population with a cross species from a resistant (Sachiaoba) and a susceptible (Minamiaoba) cultivar was analyzed. The disease severity distribution in the F₁ population suggested that the disease resistance was controlled by a major gene (LmPil). Analysis of amplified fragment length polymorphisms (AFLP) with bulked segregant analysis identified several markers tightly linked to LmPil. To identify other markers linked to LmPil, expressed sequence tag cleaved amplified polymorphic sequence (EST-CAPS) markers and resistance gene analog CAPS (RGA-CAPS) markers were also screened and analyzed. One EST-CAPS marker, p56, and one EST-CAPS marker, RG036G04-1, were also found to be closely linked to LmPil locus. Twenty-seven markers were mapped to a closely linked group with 19.6 cM in length. Furthermore, twelve markers including one ESI-CAPS marker, p56, and other eleven AFLP markers clustered in one locus. QTL analysis indicated that the region clustered markers supported 1-LOD interval, and the result also indicated one major gene controlled the disease resistance. The restriction pattern of p56 amplification showed a unique fragment corresponding to the resistant allele at the LmPi1 locus. A nkage map constructed from the reference population showed that the LmPi1 locus was located in linkage group 5 (LG5) of Italian rycgrass. Genotype results obtained from resistant and susceptible cultivars indicate that the marker p56 is useful for introduction of the LmPi1 gene into susceptible germplasm in order to develop Italian ryegrass cultivars with enhanced resistance to Italian ryegrass gray leaf spot disease. The cloning of RGAs, construction of high density genetic linkage map and development of molecular markers closely linked to gray leaf spot disease resistance gene will provide for gene targeting, quantitative trait loci mapping, and marker-assisted selection in Italian ryegrass.