ACCUMULATIONS OF GENES FOR DURABLE RESISTANCE TO WHEAT LEAF RUST PATHOGEN

The individual use of single race-specific resistance genes with major phenotypic effects has rarely provided lasting resistance. However, breeding and combining or pyramiding of resistance genes into individual cultivars has had considerable success, particularly in situation where the pathogen does not reproduce sexually, as in the case of wheat leaf rust pathogen. Within international leaf rust of wheat investigations it was necessary, to create by breeding new resistant wheat lines to Puccinia recondita tritici for differentiation of pathogen population, as well as for sources of resistance in European-Mediterranean regions. In the beginning 18 donors of resistance had been selected after an extensive screening test of several International Rust Nurseries, to be crosses with recurrent parents varieties Princ and Starke. These tests proved that in those lines were present new resistant genes. Eighth genetically different hybrids of the first backcross had been selected and tested in the seedling stage with three international pathogen cultures (YU-13-19-1; H-13-9-1 and C2-13-Ar-3). Considerable influence of recurrent parent to the number of resistant genes in donors used was demonstrated. On the other side, it was established considerable influence of the pathogen culture to the number of resistant genes in donors used. The same crossing combinations tested with one pathogen culture results in presence of two resistance genes, but with another culture three or one resistant gene. In order to enhancement resistance and pyramiding genes in these hybrids, eight selected the most interesting lines have been crossed with only effective isogenic containing the strong genes Lr9, Lr19 and Lr24.The genetic analysis of twenty two crossing combinations have been realized by testing with three pathotypes of Puccinia recondita tritici ( Bg.s. 12/89; Is.w 8/89 and Chl.w. 14/89). On the base of different segregation ratios of all crossing combinations it was proved that no one of the resistant donors contained the strong resistant genes used. It means that our hybrid lines contained resistant genes from the donors and in addition three strong resistant genes Lr9, Lr19 and Lr24.


I n t r o d u c t i o n
Leaf rust of wheat caused by Puccinia recondita Rob.ex Desm.f. sp.tritici Eriks.has become an extremely serious disease worldwide and accounts for the greatest loss among cereal rusts over the long term.Thirty years ago, the international Center for leaf rust pathogen was established in Novi Sad (Yugoslavia), dealing primarily with pathogenicity surveys of P, recondita tritici in European-Mediterranean regions and sources of resistance (B o s k o v i c , 1966).It was necessary to have a sufficient number of effective resistance genes for differentiation of parasitic populations, as well as for usable sources of resistance.However, the use of standard differential wheat varieties, and later on differential Lr single genes lines in international surveys had not shown satisfactory results (Boskovic, 1980; B o s k o v i c and B r o w d e r , 1976).Narrow effective genetic base within Lr lines, particularly for breeding for resistance, has stimulated breeding for new efficient differential genetic combinations transferred into one wheat background.It had become clear that these regions needed new, more efficient differential genes and sources of resistance.A large breeding program had been started in that time transferring new resistant genes in one wheat background.
It is known that individual us of single race-specific resistance genes with major phenotypic effects has rarely provided lasting resistance.However, in a way analogous to partial resistance breeding, the combining or pyramiding of resistance genes into individual cultivars has had success for lasting resistance, particularly in situations where the pathogen does not reproduce sexually, as in the case of wheat leaf rust pathogen.Considerable arguments where reported for the durability of cultivars with pyramided race-specific resistance genes (K o l m e r et al, 1991; M u n d t ,1991).In this paper are presented some results of the first part of breeding program, and the second part with pyramiding of resistance genes to Puccinia recondita tritici.(IWWRN 98/1974).Sixteen hybrid combinations with these donors in recurrent parents Princ and Starke were tested with three international cultures.(YU-13-19-1; H-13-9-1 and CZ-13-AR-3).In order to enhancement resistance and pyramiding genes in these hybrids, eight selected the most interesting ones have been crossed with only effective isogenic lines in that time containing the strong genes Lr9, Lr19 and Lr24.These genes have shown in the past the largest spectrum of resistance compared to all other Lr lines (Cliford, 1980;Dontchev, 1979;Reddy and Rao, 1979;Rizvi, 1984;Saadoui, 1985;Statler, 1985;Stuchlikova, 1992).The genetic analysis of twenty crossing combinations has been realized by testing with three pathotypes of Puccinia recondita tritici (Bg.s. 12/89; Is.w.8/89 and Chl.w.14/89).In this paper are presented inheritance of resistance in F 1 b 1 and F 2 generations.Selections the progenies of F 1 b 1 and F 2 generations have been performed in field trials.The number of tested F 2 seedling plants ranged from 144 to 237, the number of F 1 b 1 plants from 24 to 40 per progeny.The X 2 test was used to establish the degree of coincidence between the expected and the obtained segregation ratios.For greenhouse seedling tests individual cultures have been multiplied on susceptible variety NS-1439.The segregation into resistant and susceptible genotypes was also calculated as the frequency of resistant plants (f (R) -number of resistant plants/ number of tested plants per combination).In case of different segregation ratios when testing progenies of individual F1 plants (i.e. the F 2 generation), the higher frequency of resistant genotypes f(R) was taken for the correct result.

Results and Discussion
The inheritance of resistance of the F 1 b 1 progenies from crosses of eight donors with recurrent parents varieties Princ and Starke and tested with three pathotypes of P. recondita tritici is presented in Table 1.The patho-types are marked, for instance YU-13-19-1 means: YU (country), UN 13 from Lr19, infection type "1".It was supposed that the culture is especially virulent isolated from strong resistant gene Lr19 and resistant reactions type.Segregation ratio with the first culture have shown that resistance in the line NS-66/2 x Princ 2 was controlled by two genes, but in the hybrid NS-66/4 x Starke 2 by three genes.The resistance was dominant with complementary effect of two genes.The next hybrid combinations (NS-77/1 x Princ 2 , NS-77/3 x Starke 2 , NS-32/2 x Princ 2 and NS-32/3 x Starke 2 ) have had only one resistant gene in both recurrent parents.

Tab.1. -Inheritance of resistance in F 1 b 1 progenies from crosses of resistant
donors with varieties Princ and Starke tested with three pathotypes of P. reconditia tritici.
Both of the donors 26 and 94 with Princ have had one resistance gene, but the same donors with Starke two genes.According to the segregation ratios in the lines NS-37/2 x Princ 2 and NS-37/3 x Starke 2 three resistance genes have been established and two in hybrids NS-146/3 x Princ 2 and NS-146/1 x Starke 2 .Again, influence of recurrent parent was exposed in the line NS-46/2 x Princ 2 with resistance genes in the front to the line NS-46/1 x Starke 2 with one resistant gene.
Similar results have been obtained with the second pathotype from Hungary (H-13-9-1).Namely, both recurrent parents with donor 77 have shown the presence of one resistant gene, with donor 37 three genes, and donor 146 two resistant genes.All other hybrid combinations confirmed the same segregation ratios with both pathotypes except the lines NS-32/2 x Princ The first segregated for the presence of one resistant gene and second three genes with complementary effect.Analyzing segregation ratios with the third culture from Czechoslovakia (CZ-13-Ar-3) can be observed very similar results, as with two preliminary used cultures of P. recondita tritici.The difference is only with hybrid NS-32/2 x Princ 2 and NS-32/3 x Starke 2 .With this culture both hybrids have had only one resistant gene, it means the same, as with the first culture.That demonstrated the complexity of genetic parasite: host interactions of these hybrids, which differ from the other hybrid combinations.
As we mentioned, further pyramiding resistant genes was realized by selection of the eight best hybrids with same resistant donors and crossed with only effective isogenic lines containing the strong genes Lr9, Lr19 and Lr24.Twenty progenies of F 2 generation with different crossing combinations have been tested with three different international pathotypes of P. recondita tritici (Bg.s.12/89, Is.w.8/89 and Chl.w.14/89).Differentiation of the pathotypes that used was from International survey of P. recondita tritici on differential Lr single gene lines with different virulence/avirulence formulae from distinct regions.The first culture was from Bulgaria, second from Israel and third was from Chile.Genetic analyses of twenty crossing combinations tested in the seedling stage with the first culture are presented in Table 2. Different segregation ratios of resistant and susceptible plants in all crossing combinations proved that no one of the hybrids used did not posses strong genes Lr9, Lr19 and Lr24.Trigenic control of resistance was present in the hybrids: NS-66/2 x Lr9, NS-66/5 x Lr24 and NS-77/2 x Lr19.In crossing combinations NS-66/4 x Lr19 and NS-26/2 x Lr24 segregation's ratio 13R : 3S showed epistatic dominant gene on one locus and epistatic recessive gene on the other.In F 1 b 1 of the same donors have had two or three resistance genes depending of the pathotype applied.The mentioned hybrid with strong genes proved the accumulation of these genes by progenies segregation to resistance susceptible plants.Incorporations of the strong Lr genes with segregation ratio 9R : 7S and two dominant complementary genes have shown the hybrids: NS-77/1 x Lr9, NS-26/ 1 x Lr9, NS-26/2 x Lr19, NS-46/2 x Lr9, NS-94/2 x Lr9 and NS-94/4 x Lr19, since the majority of these hybrids in F 1 b 1 have had only one resistant gene.Two isoepistaic resistant genes according to ratios 15R : 1S were present in the hybrids: NS-77/3 x Lr24, NS-32/3 x Lr24, NS-37/3 x Lr24, NS-46/3 x Lr19, NS-46/3 x Lr24, NS-94/5 x Lr24, NS-146/1 x Lr9 and NS-146/3 x Lr19.It is typical that these ratios were mostly with Lr24 and again the majority in F 1 b 1 has had only one resistant gene, which confirm accumulation of the strong resistant gene.

Tab. 2. -The segregation ratios in the F 2 generation of crosses between eight sources of resistance and
The same hybrid combinations tested with second culture of P. recondita tritici in Table 2.According to segregation ratios have proved successful incorporation of Lr9, Lr19 and Lr24.Comparing segregation ratios with the first pathotype, it is expressed considerable influence of the culture used, which is related to the segregation ratio and number of resistant genes.Thus, the segregation ratios and number of the genes in all crossing combinations were different, except in the hybrids NS-94/2 x Lr9, NS-94/4 x Lr19, NS-94/5 x Lr24, NS-66/4 x Lr19, NS-77/1 x Lr9, NS-32/3 x Lr24 where digenic resistant genes were found with both pathotypes and tregenic combination only in NS-66/5 x Lr24.
Segregation ratios with the third culture (Chl.w.14/89) of P. recondita tritici in Table 2. have confirmed transferring of the strong resistance genes, with corresponding differences related to the pathotype applies, as with the previous two cultures.
As it is presented, segregation ratios in F 1 b 1 of eight resistant donors in the same recurrent parents Princ and Starke tested with three International cultures of P. recondita tritici have shown presence of one, two and three resistance genes (Table 1).Inheritance of resistance was mostly dominant, as it was reported by many authors (A n d e r s o n , 1961; R o e l f s et al, 1992; Jerkovic, 1992).Yet, the first investigations found that inheritance of resistance to P. recondita tritici could be dominant, recessive and intermediate, depending of parents combinations (J e r k o v i c , 1992; S c h a c h e r m a y r et al, 1996; Va n G i n k e l and R a j a r a m , 1993).
Complementary acting resistance genes have been found in the hybrids NS-66/4 x Starke 2 and NS-32/2 x Starke 2 containing three resistance genes, similar with investigations of U p a d y a y a et al (1965).They reported in some crosses three resistance genes and two of them complementary acting.In Analyses of F 2 progenies in "pyramiding genes" and transferring of the strong Lr genes (Lr9, Lr19 and Lr24) tested with three different cultures of P. recondita tritici, it was proved succesful incorporation of these genes (Table 2).That was on the base of essential genetic rule that segregation ratios of all crossing combinations were to the resistant and susceptible plants in the progenies.If all progenies of the corresponding crossing combinations would expressed highly resistant reactions, as in the lines Lr9, Lr19 and Lr24, that would means the identity of resistant genes of the both parents used (Bartos et al, 1969).Inheritance of resistance in F 2 progenies was similar as in F 1 b 1 (Table 1 1998, 1999; 2001a; 2001b).Considerable influence of the pathotype used to different segregation ratios and number of resistant genes in F 2 (Table 2), as well as in F 1 b 1 (Table 1) was reported by some other authors (G u p t a et al, 1995).
Prof. Dr M a c K e y (1980) and his reported strategies for breeding for resistance our European Center for leaf rust of wheat (B o s ko v i c , 1966) were responsible to start large breeding program for European resistant genes to Puccinia recondita tritici transferred in one wheat background.Intensive testing program of International Wheat Rust Nurseries were realized in the seedlings to prevalent European races of the pathogen, and in the adult stage in the field nurseries during several years (B o s k o v i c and M o m c i l o v i c , 1979).Sixteen genetically different resistant donors were selected and backcrossed twice with the recurrent parents P r i n c and S t a r k e (B o s k o v i c and M o m c i l o v i c , 1984).The following eight most interesting donors in these hybrids were selected for further investigations.
identification of resistance genes Lr33 and Lr34 (D y c k and S a m b o r s k i , 1982; D y c k , 1987; D y c k et al, 1987; Singh and Huerta-Espino, 2003) it was found complementary effect of Lr34 with Lr33 or LrT3.It was expressed the influence of recurrent parent to the number resistance genes.Resistant donors 26 and 94 with Princ have had one resistant gene, but with Starke two genes.Similar influence of different recurrent parents was already reported (B o s k o v i c , et al, 2006).
).The dominance of resistance prevailed, as in the papers (B o s k o v i c , et al.1999) and complementary effects (D y c k et al, 1987; J e r k o v i c , 1992; B o s k o v i c et al,