Abstract: Molecular markers linked to the specific gene of interest are proving to be handy tool in targeted selection. Rusts are one of the major causes of yield loss in wheat. Several strategies have been proposed to manage this menace. There are three types of rust in wheat viz., Yellow rust (Puccinia striiformis), Brown rust (Puccinia recondita) and Black rust (Puccinia graminis tritici). Similarly Yr, Lr, and Sr denote the resistance genes respectively. There are several number of genes for yellow (37), brown (51) and stem (45) rust resistance documented so far. Their presence or absence in a particular genotype, decides the reaction of a host genotype towards rusts. Until recent past, host-pathogen interaction (HPI) system alone was being used for postulation and detection of specific rust resistance genes in different genotypes. It is noticed that resistance derived from a single gene is not very durable. However, combining different rust resistance genes in a single cultivar may provide medium to long-term control and is called multi race or multi biotype resistance. This is because of selection pressure and evolution that new and virulent races of the fungus appear which increase the need to develop durable resistance. The process of combining several genes in a single cultivar is called Gene Pyramiding. It is not always easy to identify wheat genotype with more than one resistance gene through conventional breeding programme. The advancement in molecular marker technology has provided the plant breeder with a new tool for targeted selection of a particular gene or gene combinations. Genetic enhancement of rust resistance in wheat utilizing linked molecular markers has been targeted in India. We report in this presentation the confirmation of linkage between Sr24 and Lr24 using separate STS markers using more than 140 genotypes. Besides this, marker assisted detection / confirmation of Sr24 and Sr26 genes in Near Isogenic Lines (NILs) developed under different varietal background, provides an opportunity to the breeders towards incorporation of these genes in a single genotypic background. Such genotypes, thus developed, will provide protection against all possible Stem rust pathotypes in India, including the threatening Ug99 pathotypic race reported recently from Uganda, which has a potential of causing substantial yield loss in wheat.

Introduction: Wheat is one of the most widely used cereals and serves as a staple food for millions in the world. The stability in wheat production has been achieved through sustained efforts in bringing about varietal diversity for resistance to both biotic and tolerance to abiotic stresses. The last few decades representing post green revolution era supported “boom and bust cycle” leading to emergence/build up of several new races of rust pathogens as a result of wide spread use of narrow genetic bases in our wheat cultivars. The field life of genotypes is still a limiting factor because of development of matching pathotypes, making them susceptible. The use of gene combinations, irrespective of whether they are major or minor, has been suggested as the best genetic control of leaf rust (Roelfs, 1988). This can be achieved by pyramiding effective resistance genes, which are often difficult to monitor in the field for expression of individual resistance genes against the background of other resistance genes. A gene pyramid involves the use of several genes in a single cultivar to provide a wider base of disease resistance. The use of major resistance genes is the most cost-effective strategy for preventing stem rust epidemics in wheat crops. The long-term success of this strategy is dependent on combining resistance genes that are effective against all predominant races of the pathogen, a task greatly assisted by the use of molecular markers linked to individual resistance genes.

Stem rust disease is generally prevalent in warm wheat growing areas such as the peninsular and central India, hills of Nilgiris, summer wheat crop areas of Himachal Pradesh and Jammu and Kashmir. However, under favourable environment it may occur in other parts of the country also. The damage caused by stem rust is quite significant. Therefore, near-isogenic lines and various genetic stocks carrying genes for stem rust resistance from allied and alien sources are quite useful, once confirmed possessing effective rust resistance genes.

Materials and Methods: For the amplified product using the primers for linked markers to the genes of leaf rust Lr24, stem rust Sr24 and Sr26 and stripe rust Yr10, the amplification programme is shown in the table below.

Plant materials used for the experiments: Darf*6/ 3Ag3/ Kite

HW2021 (Kalyansona with Sr26, Sr24 and Lr24)

HW2023 (C306 with Sr24 and Lr24)

HW2026 (NI5439 with Sr26, Sr24 and Lr24)

HW2027 (Sonalika with Sr26, Sr24 and Lr24)

HW2094 (Lok-1 with Sr26 and Lr24)

BC1F1 populations derived from two cross combinations : i) Lok1 (recurrent parent) and HW2005 (donor for Lr24), and

ii) HUW234 (recurrent parent) and Yr10 (Moro) (donor for Yr10).

PCR Conditions:

Detection of specific rust resistance genes in Near Isogenic Lines (NILs) and segregating populations using

molecular markersYogesh Kumar1, Santosh Kumar1, Priyamvada1, Pallavi Sinha1, Ashish Kumar Mall1, Vinod

Chhokar2, M. Sivasamy3 and Ratan Tiwari11Directorate of Wheat Research, Karnal-132 001, Haryana, India

2Guru Jambeshwar University, Hisar-125 001, India3IARI Regional Station, Wellington (Tamil Nadu), India

F-5 CAC CCG TGA CAT GCT CGT A 3 R-5 AAC AGG AAA TGA GCA ACG ATG T 3

Sr24#12

(Mago R

et al., 2005)

94C

94C65C72C94C58C

72C20C 4C

2 minutes

30 seconds

30 seconds 1C reducing/cycle

40 seconds for 7 cycles

30 seconds

30 seconds 30 cycles

40 seconds

1 minute

Forever.

F-5 AAT CGT CCA CAT TGG CTT CT 3R-5 CGC AAC AAA ATC ATG CAC TA 3

Sr26#43

(Mago R

et al., 2005)

94C

94C59C72C20C4C

3 minutes

30 seconds

30 seconds 30 cycles

40 seconds

1 minute

Forever.

F-5 TCA AAG ACA TCA AGA GCC GC 3 R-5 TGG CCT ACA TGA ACT CTG GAT 3

Yr10 (Moro)

(own marker, not yet

published)

94C94C55C72C72C4C

3 minutes

1 minute

45 seconds 30 cycles

1 minute

5 minutes

Forever.

F-5 TCG TCC AGA TCA GAA TGT G 3R-5 CTC GTC GAT TAG CAG TGA G 3

Lr24

(Prabhu

et al., 2004)

94C94C55C72C72C4C

2 minutes

1 minute

1 minute 34 cycles

1 minute

7 minutes

Forever.

M 1 2 3 4 5 6 7 8 9 10 11 12

M 1 2 3 4 5 6 7 8 9 10 11 12M 1 2 3 4 5 6 7 8 9 10 11 12

M 1 2 3 4 5 6 7 8 9 10 11 12

Results:A set of parental lines along with their respective Near Isogenic Lines (NILs) having Sr26 and

Sr24 developed at the IARI Regional Station, Wellington, Tamil Nadu were subjected to marker assisted screening with separate markers, linked to Sr24 and Sr26. Out of 5 NILs three were found possessing both the genes where as HW2094 showed only Sr26 and HW2023 possessed Sr24 alone. This was difficult to ascertain using Host pathogen Interaction (HPI) approach. When tested with marker linked to Lr24, results confirmed tight linkage between Lr24 and Sr24 as none of the NILs having Sr24 and other 145 genotypes tested, showed breakage of linkage between these genes. Markers for Lr24 and a stripe rust resistance gene Yr10 were applied to two separate sets of segregating populations in BC1F1

generation. MAS was found quite effective in segregating the genotypes with and without these genes for further selection / backcrossing.

The agarose gel picture below is showing the Near Isogenic Lines (NILs) developed under different varietal background amplified with Sr26, Sr24 and Lr24 with the product size of 207bp, 500bp and 713bp respectively.

Conclusion:

With an objective to prepare Indian wheat programme to fight back the possible menace of Ug99 pathotype of stem rust, the present effort was made to identify lines with more than one effective stem rust resistant gene viz., Sr24 and Sr26 for utilization by the breeders. With present studies it is envisaged that marker assisted gene pyramiding/introgression of effective resistance genes is possible. This will then lead to the development of durable disease resistant varieties having prolonged field life. Delayed evolution of pathotypes because of pyramided resistance genes, will provide a breathing space to the breeders, who in turn will be able to concentrate more on enhancing per unit area productivity.

Acknowledgement:

This presentation is an outcome of project No.DWR/RP/04-5.2 financial support from the Department of Biotechnology, Govt. of India is thankfully acknowledged.

Presented at:Indian Institute of Technology (IIT-Delhi), New Delhi, India

Screening of BC1F1 population with Molecular Markers

Figure 2

Figure 3

Sr26

Sr24

Lr24

Fig 1

M-Mol. wt. ladder

1- Darf

2-K’Sona

3- HW 2021

4-Sonalika

5-HW2027

6-Lok 1

7-HW2094

8-C 306

9- HW 2023

10-NI5439

11-HW2026

12-Water controlFigure 1

Figure 2: Screening of BC1F1 population with Lr24 gene marker

Figure 3: Screening of BC1F1 population with Yr10 gene marker

M 1 2 3 4 5 6 7 8 9 10 11 12

M 1 2 3 4 5 6 7 8 9 10 11 12

500bp

713bp

207bp

713bp

753bp