Marker Assisted Foreground Selection of Back Cross Genotypes for Leaf Rust Resistance in Wheat (Triticum aestivum L.)

The investigation was undertaken to identify desirable wheat genotypes at BC1 generation carrying leaf rust resistance alleles using molecular markers. The F1 was generated from cross NI5643 (lacking Lr34) × NIAW34 (carrying Lr34). The F1 was confirmed by using simple sequence repeats (SSR) marker gwm389. This F1 was used for crossing with recurrent parent NI5643 (lacking Lr34) and BC1 plants were produced. Twenty five BC1 plants were screened for the presence of Lr34, by using SSR primers. For this purpose markers from chromosome 7D i.e. cssfr1, cssfr2, KUDS, barc352 were used to screen parental genotypes. All these markers did not show polymorphism among parental genotypes. Therefore these were not used for foreground selection. Another marker from chromosome 7D, wms130 was used to screen BC1 plants for the presence of Lr34 (foreground selection). As per the SSR profile generated by primer wms130, the plants 1, 3, 4, 6, 10, 12, 13, 14, 15, 16, 18, 22 and 25 were found positive for Lr34. Another 10 markers i.e. gwm389, wmc313, wmc468, gwm610, wmc707, gwm60, wmc525, barc137, wmc419 and barc62 were used for background selection. The plants with heterozygous amplification pattern were selected for developing BC2 plants. BC1 plants that are heterozygous or positive for Lr34 using all the markers are 1, 3, 4, 6, 10, 12, 13, 14, 15, 16, 18, 20, 22, 23 and 25. Based on foreground selection and background selection, a total of 15 plants were found carrying desirable alleles and were used for developing BC2. Highlights m Present study was thus carried out with the objective to identify desirable wheat genotypes at BC1 generation containing leaf rust resistance alleles using molecular markers.

Wheat (Triticum aestivum L.) is a major cereal crop in India and it is cultivated worldwide. It belongs to the tribe Triticeae (= Hordeae) in the grass family Poaceae (Gramineae) (Briggle and Reitz 1963). It is widely cultivated as cash crop because it produces a good yield per unit area.
Wheat production is subjected to many yield limiting biotic and abiotic stresses globally. Among biotic stresses, three rust diseases of wheat have been the most devastating throughout the world including Asia (Singh et al. 2004). According to Singh et al. (2004) stripe and leaf rust could adversely affect wheat production in Asia by 46% and 63%, respectively if susceptible wheat cultivars are grown.
Leaf (or brown) rust caused by Puccinia triticina Erikss, is an important and most widely distributed foliar disease of wheat world over. It has a potential of causing significant yield losses in India as it occurs in all the wheat growing areas. Depending upon severity and duration of infection, yield losses can reach up to 50% (McIntosh et al. 1995).
Rust can be managed most effectively and economically through cultivation of resistant Print ISSN : 1974ISSN : -1712 Online ISSN : 2230-732X varieties. Breeding resistant varieties is the most successful and economic approach to combat leaf rust. The traditional approach of transferring Lr resistance genes from wheat related species or pyramiding genes in elite breeding lines is time consuming and very laborious.
Molecular markers are used for two purposes in resistance breeding (1) to monitor the incorporation of designated resistance genes or QTLs into elite wheat genotypes.
One of the markers associated with rust resistance gene pair Lr34/Yr18 is leaf tip necrosis (Ltn). It shows complete linkage with rust resistance gene pair Lr34/Yr18 (Singh 1992). Ltn plays major role in selecting genotypes with multipathogen resistance in wheat breeding programs. However, appearance of Ltn under field conditions is time consuming. For rapid identification of gene Lr34, use of quick screening methods like use of molecular markers is needed.
Variety NIAW34 was developed at Agricultural Research Station Niphad. Its yield potential is 40q/ ha and average yield is 38-40q/ha. It is tolerant to aphids and resistant to black and brown rust. It is good for chapati and its protein content is high. It is recommended for late sown conditions. Variety NI5643 is also developed at Agricultural Research Station Niphad. Its yield potential is 25q/ ha and average yield is 23-25q/ha. It is tolerant to aphids but susceptible to leaf rust. Though its yield potential is low, it is tolerant to aphids. For this purpose this variety was selected in present study for introgression of leaf rust resistant gene Lr34, from the variety NIAW34.
Present study was thus carried out with the objective to identify desirable wheat genotypes at BC 1 generation containing leaf rust resistance alleles using molecular markers.

MATERIALS AND METHODS
The present research work entitled "Marker assisted foreground selection of back cross genotypes for leaf rust resistance in wheat (Triticum aestivum L.)" was carried out at State Level Biotechnology Center, Mahatma Phule Krishi Vidyapeeth, Rahuri (MS) during year 2014-15. The details of materials used and methods adopted in present study are mentioned under following subheadings.

Plant material
Leaf tissues from two parental genotypes (NIAW34 and NI5643), derived F 1 and 25 BC 1 plants were collected from Agricultural Research Station, Niphad, District Nashik (MS) and used for present research work. Details of population development is given in the following flowchart.

Molecular Markers
Simple Sequence Repeats (SSR) were used in the present investigation for identification of plants with presence of Lr34 gene. Thirteen such markers were used to determine their efficiency in identifying Lr34 gene in the wheat genotypes. In addition, two gene specific markers were also used.

DNA Isolation
Genomic DNA was isolated from two parental genotypes, F 1 and 25 BC 1 plants by following CTAB (Cetyl Tri-methyl Ammonium Bromide) extraction method with some modifications as described by Helguera et al. (2005).

RNase treatment
Isolated DNA of wheat genotypes were purified by giving RNase treatment.

DNA amplification
Amplification reaction mixture was prepared in 0.2 ml thin walled flat capped PCR tubes, containing the following components. The total volume of each reaction mixture was 20 µl containing 2 µl of 1X Taq bufferB, 1.2 µl of 1mM MgCl 2, 1.6 µl of 3.2 mM dNTP mix, 1.6 µl of 0.32 picomole/ µl forward and reverse primers, 0.33 µl of 1 U taq-polymerase, 2 µl of template DNA and 9.67 µl of sterile distilled water. The DNA amplification was carried out in a Thermal Cycler (Eppendorf, Master Cycler Gradient, Germany). The temperature profiles set for PCR amplification of different primers are mentioned in Tables 2 to 7.

Agarose gel electrophoresis of amplified PCR products
PCR products were separated by Agarose gel (2%) electrophoresis. Agarose gels stained with Ethidium Bromide were used for DNA profiles visualization

Confirmation of F 1 and detection of Lr34 gene in back cross generation by using molecular markers
Simple sequence repeats (SSR) markers were used for confirmation of F 1 . The confirmed F 1 (carrying leaf rust resistance gene Lr34) was used for crossing with NI5643 (lacking Lr34) and BC 1 plants were developed. BC 1 plants were screened by using molecular markers for presence of Lr34 and heterozygous plants having presence of Lr34 were recommended for developing BC 2 plants.

RESULTS AND DISCUSSION
The long term goal of this investigation is to introgress leaf rust resistance gene Lr34 from a donor genotype NIAW34 to a recipient genotype NI5643 through marker assisted selection. The specific objective of present investigation was to identify desirable wheat genotypes at BC 1 generation containing leaf rust resistance alleles using molecular markers. Results thus obtained are presented under following headings.

Molecular Marker analysis
In this study Simple Sequence Repeat (SSR) markers were used to identify desirable wheat genotypes at BC 1 generation containing leaf rust resistance alleles.
Plate 1: Confirmation of F 1 by primer gwm389

Development of BC 1 plants
The confirmed F 1 was crossed with the recurrent parent NI5643 and BC 1 plants were produced. Total 25 seeds were developed. These seeds were sown and 25 BC 1 plants were raised (Plate 2).

Foreground selection
In foreground selection markers from chromosome 7D were used (as Lr34 is present on 7D) to screen the parental genotypes. The markers cssfr1, cssfr2, KUDS and barc352 are tightly linked to Lr34. Therefore these were used to check polymorphism in parental genotypes. All these markers showed no polymorphism among parental genotypes. Therefore these markers were not used for foreground selection of desirable BC 1 plants. However another marker wms130 was found polymorphic and was used to confirm the heterozygotes (

DISCUSSION
The results obtained in this research work have been described and explained in previous chapter. The discussion based on the results of experiment is mentioned in this chapter.

Confirmation of F 1 and production of BC 1 plants
The F 1 generated from cross NI5643 (lacking Lr34) × NIAW34 (carrying Lr34) was confirmed by using simple sequence repeats (SSR) marker gwm389.
The F 1 showed heterozygous amplification pattern. This F 1 was used for crossing with the recurrent parent NI5643 (lacking Lr34) and BC 1 plants were produced.

Screening of BC 1 plants
Three hundred elite wheat lines were earlier screened by Pawar et al. (2013) for identification of Lr34 gene. Among them 60 lines showed presence of the Lr34 gene. The percent of confirmed plant

Foreground selection using markers from chromosome 7D
Thirty eight wheat genotypes comprising susceptible as well as resistant to leaf rust gene Lr34 were earlier used by Muthe (2015) for validation of known markers linked with the gene Lr34 conferring resistance to leaf rust in wheat. Amplification was carried out using STS marker csLV34, SSR markers (from chromosome 7D) wms130, barc352, gwm389, and KUDS and gene specific markers cssfr1, cssfr2, cssfr5. SSR primers wms130, barc352, gwm389 produced both the Lr34+ and Lr34-alleles by indicating presence and absence of Lr34 gene within selected genotypes.
Similarly in the present study the SSR marker wms130 was used for foreground selection of BC 1 plants. The susceptible parent (NI5643) amplified alleles of size 110, 130 and 170bp and resistant parent (NIAW34) amplified alleles of size 110, 135 and 175bp. Other markers i.e. cssfr1, cssfr2, KUDS, barc352 were used to check polymorphism in parental genotypes. These markers showed no polymorphism among parental genotypes. Therefore these were not used for foreground selection of desirable BC 1 plants. This means that utility of marker is dependent on genotypes used.

CONCLUSION
The SSR primer (wms130) tightly linked to Lr34, can be used in foreground selection of desirable wheat genotypes at BC1 generation carrying leaf rust resistant gene Lr34. SSR primer cssfr1, cssfr2, KUDS, barc352 failed to produce polymorphic amplification pattern in these genotypes hence, may not be used. SSR markers gwm389, wmc313, wmc468, gwm610, wmc707, gwm60, wmc525, barc137, wmc419 and barc62 can be used for background selection of desirable wheat genotypes at BC1 generation carrying leaf rust resistance gene Lr34. Based on foreground and background selection, a total of 15 plants carrying desirable alleles were selected.