Genetic and molecular analyses of barley for seedling and adult plant resistance against rust diseases

Abstract

Genetic studies were carried out to determine the inheritance of unknown seedling resistance (USR) to leaf rust (caused by Puccinia hordei Otth.) in the barley cultivar Ricardo. In the greenhouse Ricardo/Gus F3 (187 lines) and BC1F2 (130 lines) populations based studies using an array of P. hordei pathotypes (pts), revealed that the USR in Ricardo was conferred by a single dominant gene, which was tentatively named RphRic. Bulk segregant analysis (BSA) of the F3 population using a multiplex-ready PCR technique mapped RphRic on chromosome 4H flanked by markers GBM1220 and GBM1003 at distances of 17.4 cM and 20.4 cM, respectively. Being the first gene for leaf rust resistance mapped on chromosome 4H, RphRic was catalogued as Rph21. Phenotyping of Ricardo/Peruvian (Rph2) F3 populations and genotyping of both parents using the Rph2-linked marker ITS1 confirmed the presence of Rph2 in Ricardo. The Ricardo/Gus F3 and BC1F2 populations segregated for the presence of an additional gene when tested under field conditions using the same pathotype (pt), 5457P+ (used in greenhouse). This uncharacterised adult plant resistance (APR) against P. hordei, found in Ricardo, appeared to be distinct from Rph20 when genotyped using a closely linked marker bPb-0837. Responses of 113 advanced breeding lines and cultivars of barley (Hordeum vulgare L. subsp. vulgare), along with the susceptible control genotype Gus, were assessed against P. hordei pts in the greenhouse at seedling and field at adult plant growth stages. The tests revealed the presence of APR in 68 lines, USR in 23 lines and the seedling resistance gene Rph3 in three lines. Marker bPb-0837 was present in 35 of the 68 lines carrying APR, which suggested that these 35 lines carry APR gene Rph20. The remaining 33 lines, which lacked the Rph20 linked marker, likely carry new sources of APR. Pedigree analysis of the 68 lines found to carry APR revealed that 32 were related to cultivar (cv.) Gull and to H. laevigatum, two were related to cv. Bavaria and one related to cvv. Manchuria and Taganrog. Ancestral pedigree analysis also revealed the common presence of cv. Diamant (X-ray mutant) in the parentage of lines likely carrying Rph20. The remaining 19 lines lacked detectable seedling resistance and were susceptible in the field at adult plant growth stages. Four international barley nurseries comprising 820 lines with 579 unique pedigrees were sourced from the International Centre for Agricultural Research in the Dry Areas (ICARDA) and analysed for resistance against isolates of P. hordei, P. graminis f. sp. tritici (Pgt) and barley grass stripe rust (BGYR). Overall analyses of the responses of 783 lines (excluding 37 missing lines) to P. hordei showed that 728 (93%) carried the major seedling resistance gene Rph3, five (0.65%) carried USR, six (0.75%) carried uncharacterised APR and 44 (5.6%) lines were susceptible at all growth stages. Of the six lines identified with uncharacterised APR, three likely carried Rph20 based on the presence of the Rph20-linked marker bPb-0837. Based on tests with several control genotypes, marker bPb-0837 was found to be more reliable than Ebmag0833 in detecting the presence of Rph20. All lines were resistant to Pgt pt 98-1,2,3,5,6 when tested as seedlings in the greenhouse. Out of the 783 lines tested, 164 produced immune responses, 284 produced resistant (1= to 3) responses and 335 produced mesothetic (X type) responses against pt 98-1,2,3,5,6. All but two 783 lines were highly resistant to BGYR in greenhouse tests, showing immune responses. The usefulness of 148 simple sequence repeats (SSRs) in revealing variability among Australian isolates of P. hordei were assessed. The markers comprised 71 developed for Pgt, 40 developed for P. triticina (Pt) and 37 developed for P. coronata f. sp. avenae (Pca). SSRs were tested across 22 pts of P. hordei from Australasia including one isolate of each of the control pathogens [Pt, Pgt, P. striformis f. sp. tritici (Pst), BGYR and P. graminis f. sp. avenae (Pga)]. Genotyping of P. hordei was also conducted with the PCR-fingerprinting primers M13 and (GACA)4. The SSRs developed from Pgt and Pt showed 100% cross amplification in P. hordei, while only nine Pca SSRs showed amplification in P. hordei. Of the 148 markers tested, only two Pgt SSRs (F4-15 and F7-22) were polymorphic. Both PCR-fingerprinting primers revealed polymorphisms among the isolates, with (GACA)4 generating the most informative fragments. Both SSRs and PCR fingerprinting markers out grouped the control pathogens Pt, Pgt, Pst, BGYR and Pga from the P. hordei pts. Polymorphic information content (PIC) values of SSR markers F4-15 and F7-22 were calculated as 0.50 and 0.55 respectively. Molecular analyses revealed evidence of clonal lineages among the P. hordei pts, supporting the hypothesis that some of the pts arose from mutational changes in the virulence of a founder pt. Sexual recombination within P. hordei in Australia on the alternate host Ornithogalum umbellatum may have resulted in some new pts with different virulence against Rph genes. This is the first study of Australasian pts of P. hordei using PCR-fingerprinting technique and SSR genotyping.