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PHENOTYPIC CHARACTERIZATION OF WHEAT SELECTED LINES FOR RESISTANCE AGAINST LEAF STRIPE AND STEM RUST THROUGH PHENOTYPIC MARKERS APPLICATION.

Byline: M. Hussain, M. A. Khan, M. Hussain, N. Javed and I. Khaliq

Abstract

Leaf, stripe and stem rust are considered one of the most important diseases of wheat in Pakistan. The use of rust resistant varieties is the most economical and environment friendly way to reduce losses due to rust diseases. For this purpose a study was planned to combine genes for enhancing rust resistance and high yield potential in wheat.The experiments for identification of durable rust resistance genes i.e. Sr2/Yr30, Lr34/Yr18, Lr46/Yr29 on the basis of phenotypic markers like pseudo black chaff (Pbc) and leaf tip necrosis (Ltn1 and Ltn2) were conducted in augmented design during the years 2010-11 and 2011-12 at Wheat Research Institute, Faisalabad and Summar Agricultural Research Station, Kaghan. Total 750 head rows were selected from F6 generation and planted at SARS, Kaghan.

The outstanding 345 lines were selected and planted for further evaluation and on the basis of rust reactions, phenotypic uniformity and high yield under the field conditions, 220 lines were selected for further testing. The results indicate that among 220 entries, 67 entries showed Lr34/Yr18, 23 entries showed Lr46/Yr29 and 62 entries showed Sr2/Yr30 genes combination. While in yield testing, among test entries, 99 entries were found to be high yielding ranging from 3973-4786 kg/ha. From the study, it is concluded that fourteen test entries i.e. V-11211,V-11212, V-11218, V-11227, V-11262, V-11288, V-11296, V-11304, V-11308, V-11319, V-11338, V-11353, V-11365 and V-11396 are the most prominent lines because these lines showed the combination of all three slow rusting genes as well as high yield potential. These selected genotypes will be very useful for the wheat breeders and pathologist of the country in planning of future hybridization program.

Key words: Breeding; resistance; slow rusting genes; phenotypic markers; Triticum aestivum; leaf and stripe rust.

INTRODUCTION

Wheat crop is hit by many biotic and a-biotic maladies which engendered to reduce its produce (Jellis, 2009). Leaf rusts also called as brown rust, stripe rust also called as yellow rust and stem rust also called as black rust, smuts, bunts and aphids as biotic stresses (Hussain et al.,2006) and terminal heat, drought, salinity, winds, hailstorms, fogs and excessive cloudy weather during crop season are the salient a-biotic stresses (Hussain et al., 2011). Rusts are important worldwide due to the ability of rust pathogens to mutate and multiply rapidly, and to use their air-borne dispersal mechanism from one field to another and even over long distances.(Hussain et al.,2006) Rustsare currently the most important diseases of wheat worldwide, which threaten global food security (Hovmoller et al., 2010).

Major wheat growing areas of the world are facing repeated severe yellow rust epidemics since 2000, when two highly aggressive and high temperature tolerant Pst strains appeared (Hovmoller et al., 2008). In Pakistan, rusts have been a constant threat to sustainable wheat production. The reason for the early collapse of varieties is linked to the evolution of new rust races, rendering resistance in the varieties ineffective based on major genes. The latest and current trend of genetic resistance in wheat is "the resistance based on the additive effects of minor genes accumulation" (Singh et al., 1998). The durable resistance to leaf and stripe rusts of several cultivars is based on the slow rusting genes having additive effects (Singh et al., 2005). The most efficient and economical management of wheat rusts is the evolution of rust resistant varieties and their on-farm cultivation (Chaudhary et al., 1998; Kalappanavar et al., 2008).

In the present era of scientific advancement, the wheat research is focused to achieve durable rust resistance through incorporation of multiple minor genes or adult plant resistance genes (Singh et al., 2005). The worst yellow rust epidemic in 2005 has wiped out almost all the commercial wheat varieties of Pakistan (Khan et al., 2005). Yellow rust can reduce wheat yields by as much as 84% (Murray et al., 1995). Rusted plots yielded 4% less crops, compared to fungicide-protected plots for cultivar with hypersensitive resistance (Singh and Rajaram, 1991). Yield and kernel weight average of rusted plots are 8% less for cultivars with partial resistance, but depending on cultivar, varied from 2 to 20% less. At present country is facing critical shortage of appropriate wheat varieties having both features of high yield and rust resistance to (Hussain et al., 2006) leaf, yellow and stem rust.

Now a days, pathologist and breeders have sought resistance mechanism based on minor genes which is called durable rust resistance (Singh et al., 2000). This type of rust resistance mechanism is more effective for many races rather than a single one and is long lasting (Hussain et al., 1999). A high level of resistance to yellow rust could be achieved by accumulating from 4 to 5 minor genes in a variety (Singh et al., 2005). However, moderate level of resistance can be achieved by accumulating 2-3 minor genes in a line (Singh et al., 2005). In spite of the absence of any effective major gene, the partial resistance of varieties indicated the presence of minor genes (Hussain et al., 2006). Parents having partial resistance are crossed to pyramid genes for rust resistance and yield. This resulted many wheat lines that were better in yield and disease resistance as compared to their parent (Hussain et al., 2007).

This, in addition, will result in diversification of wheat genotypes in terms of their resistance background, necessary to avoid rapid evolution of the rust pathogen to acquire new virulence. Genetic resistance of leaf, yellow and stem rust resistant varieties is being considered the only remedy to prevent the crop from diseases as the long-term strategy. The objective of this study was to develop and screen wheat germplasm against leaf and yellow rust under natural and high stress inoculation conditions, to monitor the lines that may have minor gene based resistance and transfer of this resistance to the susceptible but high yielding varieties through conventional hybridization and phenotypic markers application for the development of wheat cultivars having minor gene based resistance.

MATERIALS AND METHODS

The research work was carried out at the Wheat Research Institute, Faisalabad during the year 2011-12, for the selection and hybridization of wheat breeding material.

Selection of breeding material on the basis of parental genes for durable rust resistance in wheat: The material used for the crossing was selected on the basis of higher grain yield among wheat lines from the gene pool of WRI.Faisalabad. The 6-10 year rust history of the lines was also considered for final selection of the parents. The lines with low terminal rust reactions up to 20MRMS in slow rusting response were selected to make desirable crossing combinations (Table 1)220 advance lines were selected from 750 lines for phenotypic characterization.

Table 1. Detail of parents utilized in the gene combining crosses

###Characteristics

Sr.

###Name of cultivar/ line###Leaf rust###Yellow rust###Acceptable###Maximum

No.

###resistance status###resistance status###yield kg/ha###yield kg/ha

1###Shafaq-06###Partially resistant###Partially resistant###4100###6400

2###Waxwing###Slow rusting###Susceptible###4000###6150

3###MH-97###Partially resistant###Partial resistant###4500###6750

4###Kiritati###Resistant###Resistant###4050###6050

5###Juchi-F2000###Resistant###Resistant###2450###3550

6###Khawaki###Resistant###Resistant###3900###4800

7###Dollarbird###Resistant###Susceptible###4100###6700

8###Kingbird###Slow rusting###Resistant###4000###6450

9###Yecora-73###Susceptible###Susceptible###3860###6125

10###Lasani-08###Resistant###Resistant###4100###6500

11###V03007###Resistant###Resistant###4050###6450

12###V04179###Resistant###Resistant###4250###6600

13###Seher-06###Susceptible###Resistant###4500###6800

14###V04178###Resistant###Resistant###4400###6750

15###V02192###Resistant###Resistant###4050###6200

16###Lr19/V02192###Resistant###Resistant###3300###4900

17###Iqbal-2000###Resistant###Resistant###3750###5500

18###FRT/SA42//PRL/SA42###Resistant###Resistant###3700###4800

###Pfau/Seri.1B//Ammad/3/Wax

19###Resistant###Resistant###3800###4900

###wing

20###Wblli2*/Brambling###Resistant###Resistant###4250###6850

###Wattan/2*ERA/2/pak-

21###Resistant###Resistant###4300###6950

###81/2*Wattan/3/Shafaq-06

22###Wattan/Fsd-08###Resistant###Resistant###4150###7000

23###Pak-81 2*/Wattan//2*Shafaq-06###Resistant###Resistant###4100###6750

24###Kambi/2*Khawaki###Resistant###Resistant###4550###6041

25###Luan/Kohistan//Pak81###Resistant###Resistant###4000###6400

26###SH88/90A204//MH97###Resistant###Resistant###3950###6300

27###PRL/2*Pastor###Resistant###Resistant###3800###6450

28###Wattan/2*ERA###Resistant###Resistant###3500###5500

29###Shafaq-06/ Luan###Resistant###Resistant###4100###6300

###Uqab-2000/

30###Resistant###Resistant###3900###5300

###Wattan/Lr28//Yecora-70

31###SH88/ Pak-81// MH97###Resistant###Resistant###4200###6750

32###Wattan/ Lr28//Yecora###Resistant###Resistant###4000###6200

Data recording of leaf and yellow rust: Rusts data were recorded at 10 days interval. The rust severity and field response were recorded according to modified Cobb's scale described by Peterson et al. (1948). Severity was recorded on the basis of percentage of leaf area infected and field responses. Severity ratings were based upon visual observations recorded at 10 days intervals as Trace 5, 10, 20, 40, 60 and 100 percent infection, and field response as immune, resistant, moderately resistant, intermediate, moderately susceptible and susceptible by the scale given in (Table 2). At least three observations regarding rust severity were recorded before the physical maturity of the crop. Three hundred and forty five desirable lines were selected on the basis of rust reaction. The other parameters like Plant height, Grain yield assessment ha-1, 1000 grain weight and protein percentage were also recorded.

Table 2. Disease rating scale used to record rust severity and level of resistance/susceptibility of wheat varieties

###Field Response###Symptoms

0###Immune###No visible infection.

R###Resistant###Visible chlorosis or necrosis, no uredia are present.

MR Moderately Resistant###Small uredia are present and surrounded by either chlorotic or necrotic areas.

M###Intermediate (Mixed)###Variable sized uredia are present some with chlorosis, necrosis or both

MS Moderately susceptible###Medium sized uredia are present and possibly surrounded by some chlorotic areas.

S###Susceptible###Large uredia are present, generally with little or no chlorosis or necrosis.

(Peterson et al., 1948).

The 345 lines selected from Kaghan Disease Screening Nursery along with Morocco as a rust spreader were planted at WRI, Faisalabad. The trial was sown by hand drill in augmented design having plot size 1.2m x 2.5m during 1st week of December, 2010-11 in order to evaluate their yield and rust resistance basis. The Morocco (used as spreader) was inoculated artificially using a hypodermic needle injection method (Rao et al.,1989) as well as spraying and dusting methods, twice in the month of January and February, until a heavy inoculum developed (Roelfs et al.,1992). During the growing period the applied rust inoculums consisted of mixture of leaf rust (TKTRN, TKTPR, KSR/JS, PGRTB and PHTTL) and Yellow rust (80E85) races collected from Faisalabad, Murree and Kaghan. This facilitated the development of epidemic rust conditions and led to reliable evaluation of rust resistance of wheat lines under study.

Rust severity (percentage) and responses of plants were assessed for three consecutive observations with 10 days intervals when Morocco was about 40-50% rust intensity. The rust severity and field response were recorded according to modified Cobb's and Roelfs scale as mentioned in (Table 2). From 345 advance lines; two hundred and twenty lines were selected for high yield and rust resistance performance.

Phenotypic characterization and yield testing of 220 selected lines through markers applications: For phenotypic characterization of selected material for rust resistance was done through three phenotypic markers i.e. leaf tip necrosis-1 (Ltn-1), leaf tip necrosis-2 (Ltn-2) and pseudo black chaff. This research work was conducted at the Wheat Research Institute, Faisalabad, Ayub Agricultural Research Institute Faisalabad during the year 2011-12.

Identification of durable resistance genes through phenotypic markers: For identification of durable rust resistant genes on the basis of phenotypic expressions on adult plant stage i.e. pseudo black chaff for Sr2/Yr30, leaf tip necrosis (Ltn-1) for Lr34/Yr18. Leaf tip necrosis are closely associated with this gene (Singh, 1992) and is being used as its phenotypic marker and leaf tip necrosis (Ltn-2) for Lr46/Yr29.The planted material comprises of 220 selected lines along with Morocco as a rust spreader around each plot were planted at WRI, Faisalabad as mentioned earlier. The rust data for both types i.e. LR and YR were recorded keeping in view the phenotypic expressions. The genotypes showing symptoms of pseudo black chaff (Fig.1) and leaf tip necrosis (Ltn-1) for Lr34/Yr18 as well as leaf tip necrosis (Ltn-2) for Lr46/Yr29

RESULTS

The present study was planned to achieve durable rust resistance through combining designated slow rusting genes with high yield potential of wheat genotypes. The material was selected from 750 heads rows of 25 crosses planted at Kaghan during June, 2010, only 345 wheat lines were selected on the basis of rust reactions and grain characteristics as shown in (Table 3).

Phenotypic characterization and yield testing of 220 selected lines through markers applications: The outstanding 345 lines were evaluated for their rust response on the basis of rust reactions, phenotypic uniformity and high yield under the field conditions at experimental area of Wheat Research Institute, Faisalabad 220 lines were selected. In further testing, it was found that among 220 entries under yield, 99 entries were found to be high yielding ranging from 3973-4786 kg/ha, 71 entries showed height ranging from 100 to 120 cm, 34 entries showed higher 1000 grain weight ranging from 42 to 47 grams and 50 entries had higher protein % which ranged from 12-14 percent and their rust response on the basis of AUDPC as compared to check varieties. These genotypes would be a good source for future wheat hybridization program in the country to achieve higher yield and high resistance. Such type of work has been reported by research workers (Singh et al., 1991; McIntosh, 1992).

Among tested entries, 67 entries showed Lr34/Yr18, 23 entries showed Lr46/Yr29 and 62 entries showed Sr2/Yr30 the presence. The results for identification of durable rust resistant genes were inferred on the basis of phenotypic expressions (Table 4).

Phenotypic characterization of 220 selected lines through markers applications: Among tested entries, 67 entries showed leaf tip necrosis-1 (Ltn-1), 23 entries showed leaf tip necrosis-2 (Ltn-2) and 62 entries showed pseudo black chaff (Table 4).

Table 3. Selection of single headlines from F6 generation crosses

Detail of Selected entries from 750 progeny lines of 25 crosses planted at Kaghan during 2010

Sr. No Name of the cross###Tested entries###Selected entries

1###FRT/SA42//PRL/SA42/4/Pfau/SERI.1B//Ammad/3/Waxwing###20###12

2###FRT/SA42//PRL/SA42/3/Wblli2*/Brambling###22###13

3###FRT/SA42//PRL/SA42/3/Kiritati###24###16

4###Wattan/2*ERA/2/Pak-81/2*Wattan/3/Shafaq-06/4/Brambling###37###24

5###Wattan/2*ERA/2/Pak-81/2* Wattan /3/Shafaq-06/3/Wblli/ Brambling###21###12

6###Wattan /Fsd-08//Kiritati###29###12

7###Pak-81 2*/Wattan//2*Shafaq-06/3/Kiritati###35###16

8###Pak-81 2*/Wattan//2*Shafaq-06/3/Juchi F2000###21###7

9###Pak-81 2*/Wattan//2*Shafaq-06/3/Dollarbird###24###9

10###Pak-81 2*/Wattan//2*Shafaq-06/3/Kambi/2*Khawaki###23###6

11###Luan/Kohistan/Pak81/3/Kiritati###42###24

12###Wattan/2*ERA/2/Pak-81/2*Wattan/3/Shafaq-06/4/Kiritati###26###14

13###Wattan/2*ERA/2/Pak-81/2*Wattan/3/Shafaq-06/4/Kingbird###22###6

14###SH88/90A204//MH-97/3//PRL/2*Pastor###20###9

15###Wattan/2*ERA//Lasani-08###22###12

16###Shafaq-06/ Luan// MH-97###34###19

17###Uqab-2000/ Wattan/Lr28//Yecora-70###37###22

18###SH88/ Pak-81// MH97/3/ Shafaq-06###27###16

19###Wattan/ 2*ERA// V04178###35###17

20###Wattan/ 2*ERA// V03007###42###14

21###Wattan/ 2*ERA// V04179###25###6

22###Wattan/ 2*ERA// Wattan/ Lr28//Yecora-70###28###6

23###Lasani-08/Seher-06###38###10

24###Lasani-08/Iqbal-2000###27###14

25###Lr19/V02192// Shafaq-06###56###27

###Total###750###345

From the study, it was concluded that out of 220 genotypes, only nine genotypes V-11211,V-11227, V-11288, V-11296, 11304, V-11308, V-11319, V-11353 and V-11396 showed the combination of three designated slow rusting genes, along with high yield , 1000-grain weight, protein % and plant height ranging from (100 to120cm). This is very important combination, as it provides protection against all three types of rusts (LR, YR and SR), while 15 genotypes including V-11203, V11212, V-11218, V-11223, V-11245, V-11248, V-11250, V-11262, V-11267, V-11289, V-11321, V-11232, V-11338, V-11365 and V-11359 showed the combination of Sr2/Yr30 and Lr34/Yr18. Similarly, the combination of Sr2/Yr30 and Lr46/Yr29 was found in 2 genotypes including V-11190, V-11193 and the combination of Lr46/Yr29 and Lr34/Yr18 was found in 6 genotypes including V-11276, V-11247, V-11313, V-11345, V-11376 and V-11380 (Table 5).

Table 4.Genotypes showing slow rusting linkage.

###Units of

Genotypes###Phenotypic Markers###AUDPC

###ranging

###Lr34/Yr18###Lr46/Yr29###Sr2/Yr30

V-Codes###Total###Lr###Yr

###(Ltn-1)###(Ltn-2)###(Pbc)

V-11195, V-11196, V11211, V11222, V-

11227, V-11230, V-11231,V-11288, V-

###14###+###+###+###0-200###0-175

11296, 11304, V-11308, V-11319 V-

11353andV-11396

V-11194, V11198, V11200, V-11203,

V11207 ,V-11208,V11209, V11212,

V11215, V11216, V-11218, V-11219, V-

11221,V-11223,V11224, V11225, V-

11226, V11228,V-11229,V-11232,V-

11244, V -11245, V-11248, V-11250, V-

###46###+###-###+###0-325###0-225

11262, V-11263, V-11267,V-11270,V-

11280,V11282, V-11289,V-11307, V-

11321,V-11328, V-11232,V-11329, V-

11333, V-11337,V-11338,V-11340,V-

11356,V-11359,V-11367,V-11375, V-

11390, and V-11392

V-11190 and V-11193###25-

###2###-###+###+###25-200

###100

V-11276, V-11247,V-11290,V-11313, V-

###7###+###+###-###0-325###0-275

11345,V-11376 and V-11380

Table 5. Elite lines with combination of three designated slow rusting/durable resistant genes, high in yield, grain weight, proteins percentage and height

Sr. No###Selection###V-Code###Lr34/Yr18###Lr46/Yr29###Sr2/Yr30###Kg/ha###1000-###height in###Protein

###(Ltn-1)###(Ltn-2)###(Pbc)###grain wt###cm###%

1###119###11308###+###+###+###4786###44###109###10

2###23###11212###+###-###+###4780###45###119###10

3###73###11262###+###-###+###4749###34###113###10

4###130###11319###+###+###+###4737###47###115###11

5###115###11304###+###+###+###4663###42###114###10

6###78###11267###+###-###+###4577###37###117###10

7###31###11220###-###-###+###4035###39###113###11

8###38###11227###+###+###+###4558###34###118###13

9###34###11223###+###-###+###4558###37###119###10

10###100###11289###+###-###+###4552###36###124###10

11###176###11365###+###-###+###4550###41###112###10

12###61###11250###+###-###+###4539###38###110###11

13###149###11338###+###-###+###4517###35###119###12

14###107###11296###+###+###+###4515###33###107###10

15###56###11245###+###-###+###4502###40###120###12

16###59###11248###+###-###+###4490###33###115###12

17###207###11396###+###+###+###4488###41###118###11

18###29###11218###+###-###+###4484###37###120###12

19###164###11353###+###+###+###4467###34###110###11

20###58###11247###+###+###-###4465###36###114###10

21###99###11288###+###+###+###4453###37###124###12

22###132###11321###+###-###+###4416###40###137###12

23###211###11400###-###-###-###4414###37###129###12

24###131###11320###+###-###-###4391###37###122###12

25###32###11221###+###-###+###4360###44###113###10

26###43###11232###+###-###+###4360###41###136###12

27###87###11276###+###+###-###4317###46###118###11

28###140###11329###+###-###+###4270###35###120###13

29###170###11359###+###-###+###4270###38###111###13

30###14###11203###+###-###+###4262###38###107###12

31###136###11325###+###-###-###4245###43###119###11

32###201###11390###+###-###+###4241###41###109###10

33###2###11191###+###-###+###4237###33###133###14

34###124###11313###+###+###-###4231###47###129###11

35###37###11226###+###-###+###4225###44###120###12

36###70###11259###-###-###+###4218###33###103###9

37###218###11407###-###+###-###4216###36###101###11

38###28###11217###+###-###+###4212###46###106###9

39###166###11355###-###-###-###4208###43###112###9

40###90###11279###-###+###-###4194###45###120###12

41###93###11282###+###-###+###4194###45###120###12

42###4###11193###-###+###+###4175###31###110###12

43###220###11409###-###-###-###4167###30###108###10

44###167###11356###+###-###+###4159###41###122###12

45###72###11261###-###-###+###4157###34###109###9

46###88###11277###-###-###-###4157###36###110###12

47###186###11375###+###-###+###4118###42###104###12

48###156###11345###+###+###-###4085###40###110###11

49###163###11352###-###-###-###4085###44###100###10

50###135###11324###+###-###-###4072###36###117###12

51###96###11285###-###-###-###4070###39###121###13

52###184###11373###-###-###-###4068###42###112###13

53###122###11311###-###-###-###4058###42###121###11

54###92###11281###-###-###+###4046###44###137###11

55###191###11380###+###+###-###4031###38###114###10

56###1###11190###-###+###+###4027###42###113###12

57###161###11350###-###+###-###4023###42###113###11

58###169###11358###-###-###4023###36###102###10

59###187###11376###+###+###-###4019###38###102###11

60###152###11341###-###+###-###3986###43###111###10

61###39###11228###+###-###+###4311###36###116###10

62###139###11328###+###-###+###4295###35###19###10

63###140###11329###+###-###+###4270###34###120###13

64###22###11211###+###+###+###4484###37###120###11

DISCUSSION

The current study was conducted to characterize selected wheat genotypes of wheat research Institute, AARI, Faisalabad, Pakistan for combining rust resistance genes with high yield potential. The characterization of current study indicates that among the tested genotypes, the advance lines that are V-11211, V-11212, V-11218, V-11227, V-11262, V-11288, V-11296, V-11304, V-11308, V-11319, V-11338, V-11353, V-11365 and V-11396 showed the combination of three designated slow rusting genes. Sr2/Yr30, Lr46/Yr29 and Lr34/Yr18, along with high yield, 1000 grain weight, protein % and plant height (ranging from 100 to 120cm) carried resistance near immunity under the severe leaf and yellow rust severity conditions. A high level of resistance to yellow rust could be achieved by accumulating from 4 to 5 minor genes in a variety (Singh et al., 2005). However, moderate level of resistance can be achieved by accumulating 2-3 minor genes in a line (Singh et al., 2005).

These lines may be a valuable source of rust resistance with amber grain color. The resistance in the derived lines seems to be race non-specific and durable nature. The major genes possessed by the parents were susceptible as the individual line V-87094 had high terminal rust rating up to 80% in the rust screening nurseries and Era exhibited 10-20% rust rating. Combinations form these parents against the prevalent leaf and yellow rust races showed fairly very low rust intensity in the country (Hussain et al., 2006).

As the lines which possessed major genes individually are susceptible in most rust virulences in Pakistan. Although the rust development was slow in case of Lr34, but alone this gene did not give desired protection and terminal rust rating was more than 60%. The better resistance in the derivatives from these crosses was most probably through the pyramiding of additional minor genes in their ancestors. Most of the lines were resistant to moderately resistant to leaf and yellow rust under high leaf and yellow rust inoculums pressure, developed artificially at the WRI, Faisalabad. The spreader rows of susceptible Morocco were full of rust rating 80-100SN and there was no chance of escape. The year 2012 was the worst epidemic year for yellow rust, wiped out most of the wheat cultivars of the country including Seher-2006, MH-97 and Bakhar-2002. Only Faisalabad-2008, Lasani-2008, AARI-11, Millat-11 and Pb-11 were found relatively resistant.

Therefore, a mechanism based on the additive effects of partial resistance/ minor genes and probably differentfrom all the existing wheat varieties of the country Pakistan. This kind of resistance is desirable, as it is long lasting, more durable against changing rust virulence patterns. This is evidently supported by the consistent resistance behavior of Frontana and Era in Pakistan for the last twenty years (Hussain et al., 1999). Hence high economic returns may be achieved from such kind of resistance. Similar findings and ideas have been emphasized, entrusted and floated by Sing et al., (1998). Some new forms of rust virulence are generated as the result of mutations in the nature. New rust virulence is appearing with the introduction of new wheat varieties and many wheat varieties have been banned for commercial cultivation only due to rust susceptibility against new rust virulence (Khan et al., 2002).

Incorporation of more than one gene to cultivars for durable leaf rust resistance has remained the focus of the breeders to cope with the dynamic nature of the pathogen (Roelfs, 1988). To address this issue, gene postulation as well as phenotypic marker approach is being utilized for enhancing rust resistance mainly through identification of durable rust resistance gene. In present investigation, the phenotypic characterization through the application of phenotypic markers i.e pseudo black chaff (Pbc), for Sr2/Yr30, leaf tip necrosis (Ltn-1) for Lr34/Yr18 and leaf tip necrosis (Ltn-2) for Lr46/Yr29 revealed that 67 entries showed presence of Lr34/Yr18; 23 entries showed Lr46/Yr29 while 62 entries showed Sr2/Yr30 linkage therefore it remains successful for longer time, even if the pathogen under goes mutations.

Hence, as per our findings, lines showing low frequency of disease severity with lower AUDPC values could be considered as slow rusting lines carrying durable rust resistance against Lr34, Lr46 and Sr2 virulences, which can be utilized in breeding programs. Determining the presence of Lr34, Lr46 and Sr2 in current cultivars can be helpful to predict the field resistance. The durability of these cultivars aid decisions in selecting parents for future breeding and development of new improved cultivars with improved leaf rust resistance. Therefore, the strategy of incorporating partial resistant minor gene in wheat genotypes through hybridization is the best way to achieve long lasting resistance in the wheat cultivars under the changing pattern of rust races/ virulence in the country.

Conclusion: Among the tested genotypes nine lines including V-11211, V-11227, V-11288, V-11296, V-11304, V-11308, V-11319, V-11353 and V-11396 showed the combination of three designated slow rusting/durable resistant genes (Sr2/Yr30, Lr46/Yr29 and Lr34/Yr18). Fifteen lines including V-11203, V11212, V-11218, V-11223, V-11245, V-11248, V-11250, V-11262, V-11267, V-11289, V-11321, V-11232, V-11338, V-11365, and V-11359 showed the combination of Lr34/Yr18 and Sr2/Yr30. Two lines including V-11190, as well as V-11193 showed the combination of Lr46/Yr29 and Sr2/Yr30. Six genotypes including V-11276, V-11247, V-11313, V-11345, and V-11376 and V-11380 showed combination of Lr46/Yr29 and Lr34/Yr18.

The outstanding lines having highly better level of partial resistance along with lower AUDPC may be used in breeding program to transfer its better partial/ durable resistance character to the adapted wheat varieties of Pakistan (Inqilab-91, MH-97, Wattan, Pb-96, Seher-2006 and Shafaq-2006 etc).

Acknowledgements: The authors dedicated this research manuscript to Dr. Makhdoom Hussain, Director Wheat Research Institute AARI, Faisalabad for providing research facilities at Wheat Research Experimental Area, Faisalabad.

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