Deciphering the genetic basis of stripe rust resistance of exotic winter wheat cultivars and their utilization in pre-breeding

Genetic basis of stripe rust resistance was investigated in two exotic winter wheat cultivars namely, Spaldings prolific and China84-40022. Inheritance was studied with the most virulent stripe rust race 46S1119 (avirulent on Yr1, 5, 10, 15, 27, SP, Su, CV and virulent on Yr2, 3, 4, 6, 7, 8, 9, 22, 23, 25). The stripe rust resistance of Spaldings prolific and China84-40022 were governed by single dominant gene. Test of allelism demonstrated that the genes imparting resistance against the pathotype 46S119 in Spaldings prolific, China84-40022, Mega and CappelleDesprez are different. The rust resistance genes of these cultivars were incorporated in to the spring wheat. Three genetic stocks namely, UP2338* 3 /China84-40022 (FLW3), UP2338* 3 /Mega (FLW12) and UP2338* 3 /CappelleDesprez (FLW40) were developed for their subsequent utilization in resistance breeding.


INTRODUCTION
Stripe (yellow) rust (Puccinia striiformis West end.f. sp.tritici) of wheat, is an important cereal disease in many wheat growing regions of the world, especially, in areas with cool and wet environmental conditions (Roelfs et al., 1992).Cultivation of resistant varieties is the most effective, eco-friendly, adopted worldwide and an economically viable method of combating the rust disease (Line and Chen, 1995).Understanding the genetic basis of resistance is of prime importance for their use in breeding programmes.It not only generates information about the nature and number of genes in the donor parents but also helps in formulating efficient strategy for the incorporation of rust resistance.Generally, resistance conferred by single effective seedling resistance genes is neutralized within few years of its commercial cultivation (Line and Chen, 1996;Chen et al., 2002).
There are only a few varieties with narrow resistance base that are occupying large area in India (Punjab, Haryana and West-Uttar Pradesh) making this region more vulnerable to rust epidemics as these are susceptible to the newly evolved races of stripe rust.It is a cause of concern that none of the release varieties or pre-released/advanced lines of wheat are resistant to these races of stripe rust (Nayaret al., 2001;Prashar, 2005).Therefore, it is imperative that the diversity of rust resistance is enhanced through exploitation of new sources of resistance from exotic materials.As these sources, winter wheat in particular, tend to generate large number of undesirable segregants, efforts were made to develop pre-breeding lines carrying diverse resistance genes with desirable agronomic characteristics.An elaborate pre-breeding programme was initiated for the development of superior and agronomically desirable genetic stocks with yet unexploited resistance genes from exotic winter wheat.This was the first step towards diversifying the genetic base of resistance and subsequently pyramiding them.The present investigation was initiated with the dual objectives of understanding the genetic basis of stripe rust resistance of some of the very important winter wheat cultivars including CappelleDesprez and the variety known for durable resistance to stripe rust (Johnson, 1984), and development of resistant lines that carry resistance genes from the donor winter wheat as well as the recipient parent.

Seedling resistance test
The F2 and F3 seedlings were raised in the aluminum trays with the 7 th row of each tray as a susceptible check (Agra Local).Fully expanded primary leaves were inoculated with uredospores suspended in non-phytotoxic isoparaffinic oil (Soltrol 170, Philips Chevron).The inoculated seedlings were kept in a humid glass chamber for 48 h and then transferred to the temperature glass house benches at 16°C.Infection types (IT's) were recorded 14 days after inoculation and were classified as follows: IT's 0; (zero fleck), ; (fleck) and ;2 (fleck two) were classified as the resistant reactions whereas, IT's 3 (three) and 3+ (three plus) were designated as susceptible reactions.Race 46S119 (avirulent on Yr1,5,10,15,27,SP,Su,CV and virulent on Yr2,3,4,6,7,8,9,22,23 and 25) was used for inheritance study.Chi squared tests were applied to check the compliance of postulated and observed genetic ratios.

Adult plant resistance (APR) test
The parents and checks were analyzed with the highly virulent race 46S119.Each test entry was replicated four times and planted in a two meter row bed.Adult plants were inoculated after the appearance of flag leaf with uredospores suspended in nonphytotoxic isoparaffinic oil (Soltrol,170).APR test for leaves, stems and stripe rusts were done in separate green houses which were kept humid for 48 h with the help of humidifiers.Temperature was maintained at the desired level with the help of sensor attached exhaust fans.Terminal disease severity scores were taken and rust severity was recorded as per the modified Cobb's scale (Peterson et al., 1948).

Test of resistance of pre-breeding lines
Resistance of genetic stocks were based on seedling resistance test (SRT) under temperature controlled glass house conditions with appropriate checks, screening under natural epiphytotic at summer nursery Wellington, the hot spot for leaf rust and stem rust, and evaluation in the main crop season at Karnal by artificial inoculation with virulent races of stripe rust.Resistance genes were Datta et al. 6545 postulated on the basis of differential host-pathogen interaction and substantiated with pedigree.Data on morpho-agronomic traits were based on the replicated trial at Karnal, planted in RBD with three replications.

Selection scheme for the development of rust resistant lines
The F1 generation was backcrossed thrice with the recurrent parent UP2338.Continuous backcrosses were made (BC2 was derived from BC1/UP2338 and BC3 from BC2/UP2338).Selection could not be done for the backcrosses of Mega and CappelleDesprez as they produced only susceptible backcross seedlings.Therefore, BC2 were made on 20 random BC1 plants and backcross seed obtained from each plant was kept in a separate packet.After obtaining the selfed seed (BC1F2) from 20 BC1 plants that were used for backcross, the BC1 plants carrying recessive resistance genes in heterozygous condition were identified through progeny testing.
Only those BC2 seeds were retained that was harvested from BC1 plants carrying resistance gene.Similarly, BC3 that were carrying resistance gene were identified.About one hundred BC3F2 progenies of each of the crosses were tested with stripe rust race 46S119 to identify the resistant seedlings.About one hundred resistant BC3F2 seedlings were transplanted in the field and harvested individually.The BC3F3 progenies were planted at the summer nursery of IARI, Regional Station, Wellington, India for the evaluation of stem rust resistance and agronomic traits.One to five stem rust resistant and agronomically desirable plants were selected from each of the BC3F3.The BC3F4 families were tested at the seedling stage with stripe rust race 46S119 and the homozygous families were identified and transplanted in the field.The BC3F5 progenies were screened at Wellington for resistance and agronomically desirable plants were selected individually.The selected BC3F6 progenies were tested at the seedling stage separately with stripe rust race 46S119, stem rust race 62G29-1 and leaf rust race 109R63 in the temperature controlled glass house.The families that were homozygous resistant to all the pathotypes were transplanted in the field and harvested in bulk.These bulk harvested BC3F7 lines along with checks were planted in RBD with three replications at Karnal for agronomic evaluation and rust resistance test.

RESULTS
The seedling infection of the parents and other lines with known resistance genes (McIntosh et al., 1995, Nayar et al., 2001, McIntosh et al., 2005) 2.

Inheritance of stripe rust resistance to race 46S119 in China84-40022
The F 2 generation of the cross between UP2338 and China84-40022 was segregated for 0; and 3+ one hundred and sixty nine seedlings showed resistance and 68 were susceptible (Table 2).The data were good fit to 3 resistant: 1 susceptible (P = 0.18).Among the one  hundred and nine F 3 families, 59 were segregated, 28 were homozygous susceptible, and 22 were homozygous resistant (Table 2).The F 3 family segregation was in compliance with 1 resistant: 2 segregating: 1 susceptible (P = 0.49).

Inheritance of stripe rust resistance to pathotype 46S119 in Spaldings Prolific
The F 1 generation of Agra Local and Spaldings Prolific were resistant.The F 2 was segregated for two infection types (0; and 3 + ).Two hundred and twenty one seedlings were resistant and 66 showed susceptibility which was in compliance with 3 resistant: 1 susceptible (P = 0.43, Table 2).The F 3 family segregation was a satisfactory fit to single gene segregation (P = 0.52, Table 2).

Test of allelism
The F 2 population of the crosses of CappelleDesprez/  China84-40022,Mega/China84-40022, CappelleDesprez-/Spaldings Prolific, Spaldings Prolific/China84-40022, Spaldings Prolific/Mega and CappelleDesprez/Mega segregated confirmed that different genes were involved in the expression of stripe rust resistance in these cultivars.The infection types of China84-40022 against leaf and stem rust pathotypes indicated the presence of Lr26/Sr31/Yr9 (data not presented).The F 2 seedlings of China84-40022/TcLr26 did not segregate when tested with race 46S103 (avirulent on Yr9) which confirmed presence of Yr9 in China84-40022.Allelism test for Yr5, Yr10 and Yr15 was also done for China84-40022 because its pedigree information was not available and the infection types of lines carrying Yr5, Yr10 and Yr15 were similar to China84-40022.Absence of susceptible seedlings in the test crosses confirmed absence of Yr5, Yr10 and Yr15 in China84-40022.

Development of rust resistant lines
The means of morpho-agronomic traits at Karnal are iven in Table 3.The seedling reactions and field resistance of the pre-breeding lines are presented in Table 4.

FLW3, the line derived from China84-40022
In the BC 3 F 2 population of UP2338/China84-40022, 115 resistant seedlings were selected and transplanted.Sixty four agronomically desirable plants were selected at maturity.One advance line was named FLW3.In addition to 1BL.1RS (Lr26, Sr31 and Yr9) this line also carries at least one more Yr gene from China-84-40022.It is immune to stripe rust and resistant to black rust.FLW3 has test weight of 39.8 g, short stature and matures in about 121 days.

FLW12, the line derived from Mega
In the BC 3 F 2 population of UP2338/Mega, one hundred and twenty resistant seedlings were transplanted.Thirty agronomically desirable plants were selected at maturity and all of them produced homozygous stripe rust resistance to Indian stripe rust pathotypes might be an undesignated gene since all the known genes of Mega namely; Yr3, Yr4, Yr14 (McIntosh et al., 1995) are susceptible to race 46S119 of yellow rust (Table 1).FLW12 has long spikes and test weight of 43 g, average plant height was125 cm and matures in about 125 days.

FLW40, the line derived from CappelleDesprez
In the BC 3 F 2 population of UP2338/CappelleDesprez, ninety six resistant seedlings were transplanted and twenty were selected at maturity.One agronomically desirable advance line (FLW40) was derived from BC 3 F 4 family that exhibited; CN IT's.FLW40 showed immune reaction at seedling as well as, adult plant stage.Based on SRT data and pedigree, FLW40 is expected to carry novel stripe rust seedling resistance genes from CappelleDesprez.FLW40 has small spikes and test weight 40.5 g, average plant height was 85 cm and matures in about 127 days.

DISCUSSION
The stripe rust resistance of Spaldings prolific and China84-40022 were governed by the single dominant gene.Test of allelism clearly demonstrated that the resistance gene of Spaldings prolific, China84-40022, Mega and CappelleDesprez are different.As of now, there are no reports of seedling resistance genes other than Yr3 and Yr4 in CappelleDesprez and Mega (Chen and Line, 1993;McIntosh et al., 1995).Both of them have shown seedling resistance against highly virulent pathotypes 46S119 (avirulent on Yr1,5,10,15,27,SP,Su,CV and virulent on Yr2,3,4,6,7,8,9,22,23,25) and 78S84 (avirulent on Yr1,5,10,15,25,SP,CV and virulent on Yr2,3,4,6,7,8,9,22,23,27 and A) of stripe rust.Though, the seedling resistance genes of CappelleDesprez (Yr3a, Yr4a and unknown Yr gene) are not effective against many races of P. striiformis in Europe and other places, but in India, it showed that seedling resistance against all the races of stripe rust were detected (Datta et al., 2008).While CappelleDesprez is planted every year in the experimental field at Flowerdale, Shimla, India for the last 17 years, the races 46S119 (46E151+Yr9) and 78S84 (78E16) are quite virulent and existing since 1996 and 2001, respectively (Prashar et al., 2007).There is no incidence of stripe rust on CappelleDesprez both at seedling stage and adult plant stage under laboratory and field condition.This indicated that the resistance genes of CappelleDesprez may be effective against stripe rust in India.The stripe rust resistance of CappelleDesprez was effective for a long time in Europe (Johnson, 1984) probably because of the presence of adult plant Resistance gene (Powell et al., 2008) and it may also be resistant families.One advance line was named FLW12.It was completely resistant to stripe rust.In addition to yellow rust resistance genes from Mega, it also carries Lr26, Sr31 and Yr9 from UP2338.The gene imparting durable in India.CapelleDesprez showed moderate susceptibility to stripe rust at Lincoln, Newzealand which suggested that either Yr16 has become less effective or it conferred only a modest degree of resistance (Chng et al., 2011).The real durability of Yr16 against stripe rust races in India can be judged when line possessing Yr16 is cultivated in substantial area year after year for a long time.Therefore, in India, the role adult plant resistance gene Yr16 on stripe rust can be studied if either the unknown seedling resistance gene (Datta et al., 2008) of CappelleDesprez is knocked out or is rendered susceptible by a new P. striiformis race.
Inheritance studies not only unveiled the genetic makeup of the lines under investigation but also generated valuable information for their use in breeding.The lines derived from the crosses of CappelleDesprez, China84-40022 and Mega were agronomically superior to their winter wheat parents for yield and maturity traits.The line derived from the cross between UP2338 and CappelleDesprez was inferior among the derived lines.The reason for its agronomic inferiority may be due to the fact that only one BC 3 F 3 line was selected for generation advancement which drastically minimized the probability to generate good progenies in the later generations.Though this line is not high yielding but its growth habit is spring type with good tillering capacity and hence, can be used in breeding programmes.The number of plants to be selected in the BC 3 F 2 generation is dependent on the number of genes governing resistance and the agronomic behaviour of the parents used in the crosses.Although, we recovered some agronomically desirable lines from the crosses of UP2338* 3 /China84-40022 and UP2338* 3 /Mega in advanced generations but it is recommended to advance at least 500 resistant BC 3 F 2 plants in order to derive agronomically superior plants from spring/winter wheat crosses in the cases where resistance is governed by a single gene.The number of resistant seedlings must be increased drastically for complementary or multigenic additive resistance as in the case of CappelleDesprez.The derived progenies of the pre-breeding programme are not expected to yield as much as the best checks of the variety trials but they will be good candidates as parents in breeding programmes.FLW3 possess good agronomic traits and high yield potential in addition to excellent rust resistance, and sensing the present threat of yellow rust, it should offer one of the best sources of stripe rust resistance to wheat breeding programme.Techniques should be devised to incorporate adult plant resistance genes like Yr16 component and Yr18 along with seedling resistance genes for better management of rust diseases.
are presented in Table1.

Table 1 .
Rust response of some Yr genes against yellow rust races in the seeding resistance test.
SRT, Seedling resistance test; APR, adult plant resistance; TSA, Triticumspelta album;a, terminal disease severity of flag leaf under polythene house.

Table 2 .
F2 and F3 segregation for rust response and tests of allelism.

Table 4 .
Seedling resistance test (SRT) and field resistance (FR) of genetic stocks against virulent races of rusts.