Sample Doctorate Research Proposal (Agriculture Plant Biotechnology)

Identification of Resistance Genes in Wheat Breeding Lines through Marker Assisted Breeding

¹ Dr. Sana Zulfiqar ² Dr. Muhammad Naeem

Laureate Folks International

ERC, PAKISTAN

https://laureatefolks.blogspot.com

laureatefolks@gmail.com, WhatsApp: +923334446261

 

1.      INTRODUCTION

The development of resilient wheat varieties to biotic and abiotic stresses is the most economical, practical, and sustainable method for attaining sustainable production in wheat. The deployment of conventional and traditional breeding approaches for the selection, development, and breeding of resistant varieties of wheat has been hampered by the complex nature of the genome of wheat, the interaction between genotypes and environment has compromised the reliability of breeding programs. The recent introduction of using molecular markers for the selection of the best wheat plants has made it possible to accelerate the breeding progress by design. Till now, efficient control measures either through the application of the pesticide or by using genetic engineering approaches do not exist. Partial success has been achieved through the application of chemicals. In the present project, the genetic variability for resistance against biotic and abiotic stresses will be identified. These molecular markers will be used in breeding programs for developing the most resistant varieties of wheat which can offer resistance against all types of constraints hampering wheat production around the globe.

Key Words: Resistance, Molecular Markers, Biotic Stress, Abiotic Stress, Genes, Tritium aestivum

1.1 Research Questions

a.      Identification of resistance genes in wheat

b.      Use of molecular markers or DNA markers for identification of resistance genes

c.       Screening of wheat breeding lines against multiple stresses

d.      Selection of wheat breeding lines resistant against biotic and biotic stresses for use in future crop improvement programs

1.2 Objectives

i.                    Development of molecular markers against resistance genes

ii.                  Screening of breeding lines of wheat with molecular markers

iii.                Identification of resistance genes in wheat breeding lines

iv.                Identification and characterization of resistant breeding lines

v.                  Deployment of resistant wheat breeding lines for making crosses

vi.                Use of these resistance genes for developing resilient wheat lines

1.3 Motivation of the Research:

Agriculture is the backbone of the economy of many countries including South and East Asian countries. Especially the livelihood of poor farming communities in developing countries is dependent upon the agriculture sector. Wheat is the staple crop of many countries around the globe; however, the production of wheat is hampered by many biotic and abiotic stresses. For mitigating these constraints such as diseases, insects, drought, etc. in wheat production, breeding and development of resilient cultivars of wheat are the basic and stable approaches for providing economical as well as user-friendly options for the farming sector around the world. There are several conventional and traditional approaches adopted for mitigating biotic and abiotic stresses such as the use of pesticides, insecticides, chemicals, modification of sowing dates, etc. However, these approaches have some constraints as well. For instance, continuous use of insecticides and pesticides results in insecticide resistance in crops ----further applications of insecticide have no effects on crop, in addition, application of insecticides/pesticides is not an environment-friendly strategy. Moreover, with changing climatic conditions new strains of pests and insects are evolving continuously, a breeding line resistance at one time may become susceptible at a later stage. Hence, there must be some reliable and stable approach for controlling these constraints in wheat yield.

Therefore, the use of molecular markers for the identification of resistance genes and then the deployment of these resistance genes for the development and breeding of resistant wheat cultivars is an effective strategy. The basic theme and idea lying behind the current proposal is the identification of resistance genes by designing molecular markers against known or unknown resistance genes present in the wheat genome or among wheat breeding lines. This study will demonstrate the efficiency of the use of molecular markers for the identification of resistance genes—hence the information generated through identified regions of resistance using molecular markers would be extremely beneficial for the plant breeders at a global scale for breeding resistance wheat cultivars. Furthermore, breeding lines conferring resistance to biotic as well abiotic constraints will be identified. These breeding lines will be shared with farmers, breeders for use in varietal development programs. Thus, the current study will generate genetic knowledge, beneficial breeding and genetic material, and human resource power.

1.4 Contribution of Research

In the current proposed study, genetic resources of wheat such as wheat breeding lines would be explored and exploited for controlling and mitigating the negative impacts on wheat yield resulting due to diseases and insects attack and other abiotic stresses such as drought, temperature, etc. As agriculture is the backbone of the economy of many countries including Turkey, the farming communities are facing heavy losses in production. In the present study wheat breeding lines will be explored for the identification of resistance genes ---these genes will be deployed for developing resilient wheat lines that will reduce the negative impacts on yield and production. By using these resistant breeding lines, farmers and plant breeders can overcome production losses and ultimately strengthen their economy by harvesting high yields in wheat. Hence, the target beneficiaries will be the wheat cultivating farmers, wheat producers, especially those facing extremely high yield losses due to the exposure of wheat crops to many biotic and abiotic constraints.

The resistant wheat breeding lines identified in this study will be bred into locally cultivated and adapted lines of wheat. Then after stabilization and selection of crosses, the stable lines will be multiplied by conducting multi-location field trials all over Turkey. Afterward, the seed of these lines will be provided to the local farmers and breeders. Moreover, molecular markers used in the present project will further be exploited for the identification of resistance genes in other genetic resources including wild germplasm, cultivated varieties, mutant liens, and crosses. Thus, the present research project will contribute to the development of future crop improvement and breeding strategies for mitigating biotic as well as biotic constraints---will pave the way for developing high-yielding varieties to benefit the agriculture sector, farming community as well as the economy in Turkey.

The use of molecular markers for the identification of resistance genes is the most effective approach. The resistant regions identified through the exploitation of molecular markers will be exploited for utilization in crop improvement and breeding programs. Furthermore, these regions would be explored for genetic variations present among wheat germplasm via high throughput sequencing platforms and cutting-edge technologies. Sequencing of resistance regions identified through molecular markers will pave the way for exploring the basic genetic variation by designing single nucleotide polymorphism (SNPs) or competitive allele-specific markers (KASP). Hence, the present study will provide insight into the genetic variability present among wheat breeding lines.

2.      LITERATURE REVIEW

Wheat (Triticum aestivum), stands among the essential cereal crops around the world. It is grown in every geological region from the equator to arctic regions and 35% population of the world use it as a staple crop, which fulfills the basic dietary and nutrition requirements of the whole population (Tehseen et al., 2021). Wheat is a prime source of starch, it is also enriched with vitamins, protein, and dietary fibers which are required for keeping the nutritional level of human beings at a normal pace (Shewry and Hey, 2015; Joye, 2020). The current and future growth of the population demands enhancements in the production of wheat due to the devastating effects resulting because of changing climate (Mansour et al., 2017; Hickey et al., 2019; Khadka et al., 2020). Thus, for feeding a huge number of people with nutritive food at a sustainable rate, there is a dire need to improve and enhance wheat production by deploying effective strategies (Adrees et al., 2020; Nawaz and Chung, 2020).

Sustainable supply of food is a prime agenda for both developing as well as developed countries in the world. Although, many of the nations have struggled for attaining independence and self-sufficiency in staple crops including wheat, however, there are still many challenges that are hampering the production and productivity of agriculture. Changing climate (extreme temperature, water scarcity, epidemics of disease) has imposed severe effects constituting several biotic and abiotic stresses on agriculture in many regions of the world. Among all the challenges farmers are facing, biotic stresses like disease epidemic in plants are a continuous menace and are expected to increase with changing climate conditions posing detrimental effects---ultimately leading to the reduction in the food supply as well as a significant increase in malnutrition (Gregory et al., 2009). In the case of the wheat crop, losses resulting due to pathogen attacks are estimated to be 5 million tons per annum basis.

Continuous efforts are being done at the global scale for the genetic improvement of wheat, however, production of wheat is still stagnant as further improvements are hampered by several biotic (smut, rust, powdery mildew, etc.) as well as abiotic constraints (heat, water deficit, extreme cold during the cropping season, etc.). There are various causal organisms for biotic as well as abiotic stresses. Several plant pathogens damage plants and cause severe disease epidemics---among these rust epidemics are most challenging around the world (Beddow et al., 2015). There are three kinds of rust disease such as black rust, brown rust, and yellow rust. The rust disease resulting due to several pathogens can easily be controlled by adopting chemical treatments, but the fungicide application is expensive and also causes environmental hazards. Thus, the deployment of resistant genes combinations is an effective approach for controlling rust epidemics.

There are two types of resistance mechanisms in plants adopted for combating the pathogen attack. Various strategies have been adopted for achieving durable resistance against rust epidemics. There are two types of resistance in plants----adult plant resistance (effective only at maturity) and all stage resistance which is effective from seedling stage till maturity of the plant. Both types of resistance are combined for attaining durable resistance against pathogens. Different types of resistance genes control these mechanisms (Mundt, 2018). Therefore, it is inevitable to identify and screen these resistance genes so that they may be further deployed in crop improvement programs.

The level of resistance of any plant/cultivar/variety is assessed and monitored at field scale generally. The infection of a pathogen is evaluated based upon the phenotype of the plant which is the result of the pathogen and wheat plant interaction. This plant and pathogen interaction is however influenced by the surrounding environment as well as by the variation present among the population of pathogens. In addition, the expression of a gene controlling a particular pathogen is also influenced by changes in climatic conditions such as extreme temperatures. Hence, changing weather not only influences the pathogen’s population but also disturbs the expression level of resistance genes. The expression of resistance genes could be complex under natural field conditions. Therefore, for the identification of resistance genes, screening with the help of molecular markers is essential (Iqbal et al., 2020).

For carrying out resistance breeding in wheat, the molecular markers are effectively used for screening germplasm and breeding lines. With the advancements in new technologies, molecular markers have become an essential breeding tool for stacking resistance genes. Molecular markers closely associated with resistance genes can be efficiently used to the genotypic constitution of any genotype or plant. The deployment of molecular markers for screening at the seedling stage enables a plant breeder to monitor and analyze the genotypic profiles of the plants before they are involved in further crossing procedures. The implication and utilization of conventional breeding procedures used for stacking resistance genes are limited because the expression of resistance genes active at the adult plant stage is often masked by the genes expressed at seedling and other growth stages. Hence molecular markers allow the identification and detection of the resistance genes without the need for any phenotyping procedures. Furthermore, the identification of resistance genes with the help of molecular markers is cost and labor-effective and is the most reliable approach. The wild relatives of wheat as well as their breeding lines serve as a potential source of resistance genes.

Several DNA markers including restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP), random amplified polymorphic DNA (RAPD), simple sequence repeats (SSRs), single nucleotide polymorphisms (SNPs), etc. have been used extensively in wheat for assessment of genetic variation and identification of resistance genes. However, simple sequence repeats (SSRs) are the most commonly used markers. These SSRs markers are abundant in the whole genome, short tandem repeats, and exhibit a high rate of polymorphism in comparison with any other marker system (Miah et al., 2013). In addition, these are co-dominant and automated. There are various molecular markers (SSRs) designed for carrying out screening of wheat breeding material. Therefore, the deployment of these molecular markers for the screening of wheat germplasm enables plant breeders to identify and breed genetic resistance in wheat germplasm in an effective and precise manner (Ismail et al., 2021).

The current project is aiming at screening breeding lines of wheat against resistance. Various molecular markers will be deployed for screening and identification of resistance genes in wheat breeding lines. The wheat breeding lines will be produced and screened for resistance. The molecular markers of known resistance genes will be designed and used for screening wheat breeding lines.

3.      MATERIAL AND METHODS

3.1 Plant Material

The breeding lines of wheat will be screened for identification and characterization of resistance genes with the help of molecular makers.

3.2 Leaf Sample Collection

Fresh leave samples of wheat breeding lines will be collected from 21-day seedlings of wheat plants by following standard procedures. Then leaf samples will be stored at -80 degrees till further processing.

3.3 Screening based upon Molecular Markers

Firstly, the primers for known resistance genes will be designed by using wheat databases such as Ensemble plants or grain genes. Then these molecular markers will be amplified on wheat breeding lines for the identification of resistance regions in the genes.

 3.4 Extraction of DNA and Quantification of DNA

 DNA will be extracted from the fresh samples of leaves of all wheat breeding lines following the Cetyl trimethyl ammonium bromide (CTAB) method. Fresh samples of leaves of almost 1 to 2 grams will be ground in liquid nitrogen and the CTAB method will be used for extracting genomic DNA. Then the extracted genomic DNA will be dissolved and diluted in 1x TBE buffer and kept at - 20°C for use in a polymerase chain reaction (PCR). The quantity and quality of genomic DNA will be assessed using Nano-dropTM 1000 spectrophotometer as well as by running on 0.8 % agarose gel in a gel electrophoresis system. DNA purity will be estimated from the ratio of absorbance at 260nm/280nm.

3.5 Synthesis of SSR primers and optimization of PCR conditions

The simple sequence repeats (SSRs) primers related to some traits e.g. aphid and rust response will be synthesized using sequence information already available in databases and literature. The optimization of the annealing temperature of primers is very critical and will be done prior to carrying out PCR. The PCR will be performed by deploying the PCR kit of Thermo Scientific. The conditions for PCR will be modified and calibrated using different annealing temperatures, the number of reaction cycles, and the concentration of the genomic DNA as well as primers for attaining appropriate amplification results (Table 1). After attaining the required PCR conditions and amplification, PCR products will be assayed on a high-resolution gel of agarose (2.4 %) using an automated gel electrophoresis system. Staining of the gels will be performed using ethidium bromide, and then the gels will be photographed on a GelDoc-It®2 310 Imager. Scoring of molecular markers (SSRs) will be made according to the presence or absence of bands using the 0 and 1 scoring system.

Sr. #	Reagents	Quantity/reaction
1	Buffer	2 ul
2	Water	4.91 ul
3	dNTP’s	6.4 ul
4	MgCl2	1.6 ul
5	Primer	2 ul
6	Taq polymerase	0.2 ul

Table 1: PCR conditions used to optimize the annealing temperature

3.6 Statistical Analysis

Scoring data of molecular markers will be compiled in a Micro Soft Excel sheet and analyzed with the help of appropriate statistical software like STRUCTURE and TASSEL.

3.7 Expected Results

By carrying out the proposed study we are hoping to identify and characterize many molecular markers linked with resistance genes. The wheat breeding lines expressing resistance expression against any of the biotic as well as abiotic stresses will be identified and shared with plant breeders for use in the wheat improvement and breeding programs. The selected markers linked with resistance could be used for the selection of desirable wheat genotypes. Hence, a plant breeder can lessen the cost and effort required for carrying out extensive screening and testing procedures. It will also result in saving the significant length of time required for developing a wheat variety. Since new molecular markers will be made available to the wheat breeders for developing new wheat varieties. The resultant genetic information about the genetic circuits of resistance as well as molecular markers will be used for developing resilient wheat varieties. The newly developed varieties of wheat will be a source of novel genes that are not present in the currently cultivated varieties of wheat. Thus the newly developed varieties will be much more equipped for combating the stresses in wheat.

4.      DISCUSSION

Marker-assisted selection is efficiently used for developing resistant varieties in many staple crops including wheat. Molecular markers have been used for breeding resistant varieties. In the wheat crop, a very high percentage of resistance genes against both biotic as well as abiotic stresses has been assessed and characterized with the help of molecular makers. Thus, the deployment of molecular markers for screening of resistance genes is an efficient strategy for breeding resistance wheat cultivars. The development of genetic resistance is the most reliable and economic approach for minimizing yield losses resulting due to heavy pest attacks (Draz et al., 2015). Although, the extent of production losses depends upon the relative rate of susceptibility or resistance of a cultivar towards insect pest attack (Herrera-Foessel et al., 2011). Because of heavy pest infestations especially rust disease, yield and yield contributing traits in wheat are badly affected (Abdelbacki et al., 2013; Sallam et al., 2016).  

Molecular markers or DNA markers have been used as an invaluable tool in plant breeding programs, including gene identification and marker-assisted selection. One of the major applications of molecular markers in plant breeding is the identification and determination of resistance genes. By deploying molecular markers, resistance genes are identified and utilized in crop improvement programs. Resistance genes are used in different combinations for imparting resistance in wheat cultivars. For example, the combination of multiple resistance genes such as Lr9+Lr24, Lr19+Lr24, Lr19+Lr28, and Lr9+Lr24+Lr28 in winter wheat gave a high level of resistance, in addition, the combination of seedling resistance genes (e.g. Lr16, Lr19, Lr21) with adult plant resistance genes (Lr34, Lr46) could be a good approach to provide durable resistance against leaf rust (Kolmer 2009; Prabhu et al., 2009; Vanzetti et al., 2011).

In addition, molecular markers linked to different leaf rust resistance genes such as Lr1, Lr17a, Lr19, Lr21, Lr24, Lr27, Lr34, Lr35, Lr39, Lr42, Lr46, Lr48, Lr67, etc. have been reported by some earlier studies (McCallum et al., 2012; Herrera-Foessel et al., 2012; Dakouri et al., 2013; Imbaby et al., 2014; Abdelbacki et al., 2015; Afridi et al., 2016; Abouzied et al., 2017). Among all these genes, Lr34 is a slowing rusting gene that exhibited a high level of resistance under field conditions. Moreover, four race-specific adult plant resistance genes such as Lr12, Lr13, Lr35, and Lr37 also gave a good level of resistance against leaf rust in several field experiments. Likewise, for stem rust resistance, resistance genes such as Sr-2, Sr13, Sr21, Sr22, Sr28, Sr33, Sr35, Sr40, Sr42, Sr44, Sr45, Sr55, and Sr56, SrCad were identified with the help of molecular markers (Hiebert et al., 2010; 2011; Kokhmetov et al., 2011).

There are several reports on the use of molecular markers for the identification of resistance genes in wheat. Research work has been carried out on several insects and pests regarding molecular markers. For leaf rust resistance, more than 80 resistance genes are identified on a global scale (Aktar-Uz-Zaman et al., 2017). The majority of these genes confer resistance at the seedling stage only, however, there are also some adult stage resistance genes. Some of these genes including Lr16, Lr22a, Lr14a, Lr34, and Lr21 are also cloned (Krattinger et al., 2009; Thind et al., 2017). Hence, cloning of genes helps in designing molecular markers more efficiently and accurately. Among the above-mentioned genes, two genes named Lr22a and Lr21 show resistance against all types of leaf rusts (McCallum et al., 2016). Likewise, molecular markers have been efficiently utilized for screening against stem rust races in wheat germplasm. Recently three genes, Sr42, SrTmp, and SrCad have been identified (Lopez-Vera et al., 2014; Hiebert et al., 2016).  Moreover, two molecular markers have been designed for identifying resistance against loose smut caused by ustilago tritici in wheat. The SSR markers are available for the Ut6 gene (Kassa et al., 2014) as well as KASP makers are also being developed (Kassa et al., 2015). Both these markers are effective in conferring resistance against two strains of loose smut. There are some molecular markers developed for bunt resistance in wheat (Singh et al., 2016; Zou et al., 2017). In addition, a severe disease-causing huge economic losses by affecting yield and quality components in the wheat crop is fusarium head blight. Several genes have been reported for resistance however, its resistance mechanism is quite complex (Comeau et al., 2011).

As a result of recent advancements in molecular marker and sequencing technologies, the number of mapped resistant genes is increasing very rapidly. Breeding programs aiming at selection and prioritization of genes confront a basic research question---which gene or combination of genes should be utilized for providing durable resistance against rapidly evolving pathogens? Many research scientists argue that an accurate and refined classification of resistance genes is required for answering the above question. . It is validated by many plant breeders and molecular biotechnologists working on wheat that, a combination of many minor (conferring resistance at adult plant stage), as well as major genes (showing resistance at the seedling stage), should be adopted for optimization of both---durability and level of resistance in any wheat variety. By adopting this approach, all types of resistance mechanisms can be explored in a defined fashion. Finally, identified resistance genes via molecular makers can be further introgressed into the locally adapted and cultivated varieties of wheat through conventional breeding tools or by designing and targeting molecular markers. Thus, newly screened wheat breeding lines will pave the way for getting insight into the genetic circuits of several resistance mechanisms. Moreover, identification of genes involved in resistance against multiple biotic as well as abiotic constraints would become more convenient.

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