633.11:632.4:632.938.1

ABOUT DNA-MARKERS AS SOLE CRITERIA FOR POSTULATION OF Lr-GENES OF THE Triticum aestivum L. RESISTANCE TO Puccinia triticina Erikss.: CRITICAL ESSAY

L.G. Tyryshkin

By the use of phytopathological test the author investigated the presence of the Lr1, Lr10, Lr20, Lr26 Lr34 genes of resistance to leaf rust in 44 variants of soft wheat, in which early the presence of this genes was postulated according to STS- or SSR-analysis. It was shown, that the absence of listed genes in 6, 18, 2 and 3 varieties denote the weak genetic linkage between DNA-markers and genes of resistance to disease. The analysis the data of literature is showing that the Lr9, Lr19 Lr24 — effective genes of juvenile resistance — also not always are close linked with ob-tained to them STS markers.

Key words: wheat, leaf rust, genes for resistance, DNA markers.

 

Brown (or leaf) rust caused by Puccinia triticina Erikss. [Syn.: P. recondita Roberge: Desm. f. sp. tritici (Erikss.) C.O. Johnston] is one of the main diseases of wheat (Triticum aestivum L.) in most of cultivation regions. The most cost-effective and environmentally safe method of combating the disease is creation of resistant varieties. However, the reserve of effective resistance genes is extremely small: in the World Collection of N.I. Vavilov All-Russian SRI of Plant Industry (VIR), all the samples highly resistant at juvenile stage are known to be protected only by the genes Lr9, Lr19, Lr24 and Lr41 (1).

One way to extend the "useful life" of a resistance gene is “building pyramids of genes " – providing a genotype with several effective genes or one effective combined with several less effective. Using DNA-markers is considered as a powerful method of breeding wheat varieties possessing multiple genes for resistance to leaf rust, because it is impossible to distinguish only by phenotype  the lines carrying one efficient gene from the lines having more than one gene.

At present time, it has been developed the DNA markers for 30 Lr-genes. However, the experimental data suggest that the genes originally described as strictly specific to a particular gene are not always closely linked with it (2-5). Despite this fact, in the domestic scientific literature, there are the current publications postulating Lr-genes solely upon the presence of a certain DNA-marker (6-10). It is obvious, that using this approach with no close linkage between DNA-marker and Lr-gene may lead to erroneous gene identification of the studied sample.

The purpose of this work - experimental verification of the resistance genes Lr1, Lr10, Lr20, Lr26 and Lr34 presence in wheat varieties presumed as possessing these genes identified solely upon the basis of molecular markers amplification (5-10).

Methods. P. triticina monopustular isolates were selected from the pathogen population collected in the North-West region of Russia. The segments 0,8-1,0 cm long  were cut from the 1st leaf of wheat plants the variety Thatcher, and from its near-isogenic lines (ThLr) carrying the leaf rust resistance genes Lr1, Lr20 and Lr26. The leaf segments were placed in a cuvette on cotton soaked water, and infected with the monopustular fungi isolates; after that, the cuvettes were covered with glass and placed in darkness for 12 h, then transferred into a climate chamber (21°C, constant light). 

Reaction type to the infection was recorded in 6-7 days following the inoculation, according to Mines-Jackson scale  (11): 0 - no symptoms, 0 - necrosis without pustules, 1 - very small pustules surrounded with necrosis, 2 – pustules of medium size surrounded with necrosis or chlorosis, 3 - pustules of medium and large size without necrosis. To obtain the pathogen isolates for testing the genes Lr10 and Lr34, the 1st leaf segments 0,5 cm long were cut from the Thatcher variety plants and its near-isogenic lines carrying these genes, and placed in a cuvette on the cotton wool soaked with benzimidazole (100 mg / l aqueous solution) and infected with P. triticina isolates; the cuvettes were  transferred into a climate chamber (25 °C, constant light) (12). The reaction types were estimated by the above scale on the 7-8th day after inoculation.

From the soft wheat samples of VIR world collection, known as carrying the resistance genes Lr1, Lr10, Lr20, Lr26 and Lr34 upon the presence of molecular markers, the leaf segments (5 from a plant) were taken and placed in cuvettes on a cotton wool soaked with water or benzimidazole aqueous solution, and then infected with rust pathogen isolates avirulent to corresponding resistance genes. The reaction types were accounted on the 6-7th day after inoculation.

Since the interaction in a system “wheat-leaf rust pathogen” is subject to the postulate of H.H. Flor “gene-for-gene" (13), there’s the way to prove the absence of a functional allele of the particular resistance gene in a studied sample upon its susceptibility to pathogen isolates avirulent for Thatcher lines carrying the particular resistance gene. As a resistant control, the authors used the leaf segments from the varieties in which the listed genes were detected by hybridological analysis: the gene Lr1 - Dirkwin, Lr10 - Mayo 52, Lr20 - Thew, Lr26 - Kavkaz and Lr34 - Glenlea (14).

Results. The leaf rust pathogen clones avirulent to the line ThLr1, were found to be virulent for six of the eight studied wheat varieties previously established as having  STS-markers (sequence tagged site markers) for Lr1 (6, 9) – the varieties Doublecrop, Khazarka, Omega, Hope, Prokhorovka and Rodina, which proves that they can’t have this resistance gene. In the other two varieties - Roblin and Pasqua, the presence of Lr1 was possible.

Two of the three VIR collection samples known as carrying the amplification of STS-marker for Lr20 (6) - Zarya and Planet – revealed their susceptibility to P. triticina clone avirulent to this gene, ie they do not possess the resistance gene Lr20. The leaf segments from the variety Omega and the line ThL20 demonstrated identical reaction to the infection with clones, therefore, it’s most likely that this variety is protected by the gene Lr20.

The results of molecular analysis (5, 6) suggested that four of the studied varieties have the resistance gene Lr26, while our phytopathological test showed its absence in genotypes of the varieties Prospect and Equinox (Table 1).

The leaf segments from 18 of the 22 varieties, previously known as carrying the amplification of STS-marker linked with Lr10 (5, 6, 8-10) demonstrated their susceptibility to at least one of the four Lr10- avirulent rust pathogen clones used for the inoculation (Table 2).

1. Types of reaction in the soft wheat varieties carrying the resistance gene Lr26 postulated upon DNA-labeling and infected with Lr26-avirulent clones of Puccinia triticina during a phytopatological test

Variety, sample,
DNA-labeling method

Reference

Clone

26-1

26-2

26-3

26-4

26-5

26-6

STS-analysis:

 

 

 

 

 

 

 

Equinox  

(5)

3

0

0

3

3

3

Khazarka

(6)

0

0

0

0

0

0

Prospect

(6)

0

3

3

0

0

0

Prokhorovra

(6)

0

0

0

0

0

0

Thatcher*

 

3

3

3

3

3

3

ThLr26**

 

0

0

0

0

0

0

Kavkaz***

(14)

0

0

0

0

0

0

Note: STS — sequence tagged site.
*, **, ** - control samples: * - the variety Thatcher, ** - the Thatcher line carrying corresponding resistance gene, *** - the variety for which this gene was revealed by hybridological test.

 

Upon the results of phytopathological test, only four samples (Prospect, AC Domain, Prokhorovka and Roblin) did not refute the hypothesis presuming Lr10 presence in these samples.

2.  Types of reaction in the soft wheat varieties carrying the resistance gene Lr10 postulated upon DNA-labeling and infected with Lr10-avirulent clones of Puccinia triticina during a phytopatological test

Variety, sample,
 DNA-labeling method

Reference

Clone

10-1

10-2

10-3

10-4

STS-analysis:

 

 

 

 

 

Piko

(5)

3

3

3

3

Brigadier 

(5)

0

3

0

3

Vance

(6)

0

0

3

3

Lerma Rojo 64

(6)

0

3

3

0

Rendezvous

(6)

0

2

3

3

Prospect

(6)

0

0

0

0

AC Domain

(6)

0

0

0

0

Leguan

(6)

0

3

3

3

Hope

(6)

0

3

3

3

Prokhorovka

(6)

0

0

0

0

Sarrubra

(6)

0

0

0

3

Roblin

(6)

0

0

0

0

Kharkovskaya 26

(6)

2

3

3

3

Manna 2

(8, 10)

3

3

3

3

Angelina

(8, 10)

3

3

3

3

Mil’trum 63

(8, 10)

3

3

3

3

Eskada 70

(8, 10)

0

3

0

3

Marinka

(8, 10)

0

3

3

3

Mal’tsevskaya 110

(8, 10)

0

0

3

0

Esther

(8, 10)

3

3

0

3

Voronezhskaya 16

(8, 10)

3

3

3

3

Rodina

(7)

3

3

3

3

Thatcher*

 

3

3

3

3

ThLr10**

 

0

0

0

0

Mayo 52***

(14)

0

0

0

0

Note: the same as in Table 1.

 

The line ThLr34 exhibited “0” type of reaction to the infection with four clones of P. triticina, as well four of the seven varieties previously described as carriers of Lr34 gene upon the results of SSR-analysis (simple sequence repeat) (6) (Table 3). The results of inoculation with test-clones contradicted the idea of this gene presence in the varieties Lerma Rojo 64, Zarya and Doublecrop.

The obtained results indicate a high frequency of false identification of Lr1, Lr10, Lr20, Lr26 and Lr34 – the genes for resistance to wheat leaf rust - upon the presence of DNA-markers as a sole postulation criterion.

3.  Types of reaction in the soft wheat varieties carrying the resistance gene Lr34 postulated upon DNA-labeling and infected with Lr34-avirulent clones of Puccinia triticina during a phytopatological test

Variety, sample,
 DNA-labeling method

Reference

Clone

34-1

34-2

34-3

34-4

SSR-analysis:

 

 

 

 

 

Lerma Rojo 64

(6)

3

3

3

3

Zarya

(6)

3

3

3

3

Manitou

(6)

0;

0;

0;

0;

Pasqua

(6)

0;

0;

0;

0;

Doublecrop

(6)

3

3

3

3

AC Domain

(6)

0;

0;

0;

0;

Roblin

(6)

0;

0;

0;

0;

Thatcher*

 

3

3

3

3

ThLr34**

 

0;

0;

0;

0;

Glenlea***

(14)

0;

0;

0;

0;

Note: SSR — simple sequence repeat.
*, **, ** - control samples: * - the variety Thatcher, ** - the Thatcher line carrying corresponding resistance gene, *** - the variety for which this gene was revealed by hybridological test.

 

Thus, the data of many studies (15) suggest that STS-marker pTAG621 is not closely linked with Lr1, but the amplification of this marker is considered as the only criterion for this gene presence in modern wheat varieties (6), as well as in the variety Rodina and its numerous derivatives with heterogenous genetic material (7, 9). According to our data obtained in phytopathological test, the gene Lr1 is absent in six of the eight studied samples. Using molecular markers Lrk 10D (16), the gene Lr10 was postulated in many modern wheat varieties (5, 6, 8, 10), whereas it wasn’t found in most of these samples during our phytopathological test: only four of 22 samples previously presumed to carry the amplification Lrk 10D, can be the carriers of this gene (see Table. 2). In four wheat varieties, the literature data reported the amplification of DNA fragment 542 bp length using primers to the marker STS638 (17) - the base for postulation the existence of Lr20 gene in these four varieties (6). We studied 3 of them with phytopathological test, and  it has been shown that two samples can not carry this gene.

 The presence of Lr26 was proved in 11 wheat varieties (6) upon the amplification of IB-267 molecular marker closely linked with this gene (18). VIR collection includes only three such varieties, and the presence of Lr26 gene in one of them (the variety Prospect) was refuted by the results of our phytopathological test (see Table 1). In the variety Equinox, Lr26 was postulated on the basis of STS-analysis (5), but in our tests it appeared to be susceptible to the clones avirulent for ThLr26 and, therefore, Equinox can’t carry Lr26.

And finally, the gene Lr34 was postulated in 18 soft wheat varieties known to possess the SSR amplification of Xgdwm295 loci (6). We studied seven of them, and it has been proved the absence of Lr34 in three varieties (see Table. 3).

Analysis of literature data shows that the genes of effective juvenile resistance are not always closely linked with newly developed molecular markers for these genes. For example, the marker J 13 (19), according to the results of many studies, is closely linked with the gene Lr9. At the same time, the amplification of this marker was shown for four wheat lines carrying the genetic material from Aegilops speltoides and Triticum kiharae (7). The publication reported that the study did not involve using Lr9-virulent rust pathogen pathotypes, but these lines were affected by some of rust clones, and, therefore they can’t carry the functionally active allele of this resistance gene. Thus, Lr9 can not be considered as closely linked with the marker J 13 (at least in the wheat varieties obtained introgressive hybridization).

The STS-marker Gb (20), was believed to be closely linked with Lr19. This contradicts to the fact: one of the near-isogenic lines (maternal form - the variety Rodina, father - Ae. triuncialis) was supposed to carry the gene Lr19 postulated upon STS-analysis using primers to Gb marker (9), but this marker was not found to be amplified in both parental forms.

Despite the reports about a close linkage between the marker STS-J 09 and Lr24 gene (21), there are at least two facts indicating that this linkage is not that close. First, in nearly-izogenic lines of the variety Thatcher, this marker was amplified in line carrying Lr24, as well as in line carrying Lr25 (22). Secondly, the amplification was observed in two lines of the variety Rodina with genetic material from Ae. triuncialis and T. kiharae (9), but these lines can’t possess the functionally active allele of Lr24: our study has revealed the clones of P. recondita virulent for these two lines and non-virulent to the resistance gene Lr24.

Thus, the experimental data and literature analysis have proved that DNA-markers developed for the wheat leaf rust resistance genes - Lr1, Lr9, Lr10, Lr19, Lr20, Lr24, Lr26 and Lr34 - are not always closely linked with these genes. It is obvious, that the selection of lines with multiple resistance genes requires preliminary verifying the linkage between a marker and the resistant gene from each donor using DNA-labeling (marker assisted selection).

 

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N.I.Vavilov Research and Development Institute of Plant Growing,
St.Petersburg 190000, Russia,
e-mail: tyryshkinlev@rambler.ru

Received November 24, 2009

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