Map-Based Cloning and its Application in Cloning

Advances in Life Sciences 5(5), Print : ISSN 2278-3849, 1573-1580, 2016

REVIEW PAPER

Map-Based Cloning and its Application in Cloning Plant Disease Resistance Genes AMIT KUMAR SINGH1,2(a), NG. TOMBISANA MEETEI1,2(b), BRIJESH KUMAR SINGH1 AND NIRMAL MANDAL*1 1

Dept.of Agricultural Biotechnology, Faculty of Agriculture, BCKV, Mohanpur, Nadia, W.B. Central Agriculture University, College of Horticulture and Forestry, Pasighat, Arunachal Pradesh. 2(b) Central Agriculture University, College of Post graduate studies, Umiam, Meghalaya email: [email protected] 2(a)

ABSTRACT Map-based cloning is an unique approach that identifies the underlying genetic cause of a variation. This approach has the ability to tap into a nearly unlimited resource of natural and induced genetic variation without prior information of specific genes. Scientist are therefore trying to utilize this technique in cloning disease resistance gene in plants. Diseases caused by different microorganisms are a potential threat to the cultivation of crop plants as plant diseases can cause yield reductions of up to 40% or more. Although timely application of chemicals against such pathogens can provide adequate control, but it increases the production costs and is not safe for the environment. Breeding for resistance is considered as the most effective and efficient strategy for controlling the pathogens, as it does not add up input costs and environmentally safe. Molecular approaches have been found to be most reliable while looking for disease resistance genes. Therefore, more focus is given to molecular mapping and tagging of disease resistance genes and to make such information available to scientific community across the world. This article reviews the principles, requirements of map based cloning and its application in plants. Key words

Crop improvement, Plant disease resistance, molecular genetics, mapping, tagging, cloning

Map-based cloning or positional cloning is the process to recognize the genetic basis of a mutant phenotype with the help of linkage to markers whose physical location in the genome is known. In the early 1950s, the growth of genetics has been exponential with several milestones, with the

determination of DNA as the genetic material in 1944, detection of the double-helix structure of DNA in 1953, the development of electrophoretic assays of isozymes in 1959 and a broad range of molecular markers that can make out differences at the DNA level. Alfred H. Sturtevant presented the first concept of a genetic map in (1913) by ordering five sex-linked characters of Drosophila on the Y chromosome in a linear fashion. The principles of genetic mapping and linkage analyses are used even today in much similar way but with very advanced technologies. Now the whole genomes are being sequenced with greater speed than ever before. With the availability of wholegenome sequences, Map-Based cloning and functional genetic studies in model plant systems have become easier and provide fundamental knowledge for understanding plant growth and environmental response. The time between the conception of the first map and the sequencing of whole genomes was about 100 years leading to the building of physical maps where genes or markers are located at a exact sequence position. A genetic localization research determines the order of linked markers and the distance in percentage recombination or centimorgans (cM). Recombination frequencies differ between different parts of chromosome, physical conditions and sexes. As a result, the ratio between genetic and physical distance is not stable throughout the length of the chromosome. Moreover, genetic distance depends on the used parental combination: lines which are closely related will show an essentially higher recombination frequency than distantly related lines. In the beginning, progress in mapping was delayed due to the lack of sufficient markers

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devoid of Epistatic interactions. Later on for building of denser map important development included the invention of DNA-based marker systems such as restriction fragment length polymorphism (RFLP) analysis (Botstein et al., 1980) and the development of PCR-based markers such as random amplified polymorphic DNA (RAPD by Williams et al., 1990), Simple sequence length polymorphism (SSLP by Bell et al., 1994), amplified fragment length polymorphisms (AFLPs by Vos et al., 1995), Variable number tandem repeat (VNTR) Minisatellites (Jeffreys et al.,1985(a, b), Intersimple sequence repeat (ISSR by Zietkiewicz et al., 1994, Gupta et al., 1994), Cleaved amplified polymorphic sequences (CAPS by Koniecznyand Ausubel, 1993), Derived cleaved amplified polymorphic sequences (dCAPS by Neff et al., 1998) , Allele-specific polymerase chain reaction (AS-PCR by Bottema, and Sommer, 1993, Gu et al., 1995 ), Single-nucleotide amplified polymorphisms (SNAP by Drenkard et al., 2000), Single-strand conformational polymorphism (SSCP by Orita et al., 1989), Arbitrary primed polymerase chain reaction (AP-PCR by Welsh and McClelland 1991), DNA amplification fingerprinting (DAF by Caetano-Anolles et al., 1991), Sequence characterized amplified region (SCAR by Paran and Michelmore, 1993) and Selective amplification of microsatellite polymorphic loci (SAMPL by Witsenboer et al., 1997 ). Hence, the availability of markers is not a limiting factor for any living organism. Furthermore, various sequencing projects facilitate us to allocate marker’s physical position on the map. For instance, a map was developed with more than 1250 AFLP markers on the Arabidopsis sequence by combining experimental and in silico AFLP analysis (Peters et al., 2001). Various numbers of markers and their physical position can be found at the TAIR website. Therefore, it is now possible to focus more precisely on defined region of the genome which contains the gene of interest in sequenced model species.

Map-based cloning strategies: balancing the available marker systems Map-based cloning approach utilizes the fact that, as distances between the gene of interest and the analysed markers decrease, so does the frequency of recombination. For instance approximately 12 000 well-positioned markers are

needed to map any locus in the Arabidopsis genome in a 10-kb interval and this is possible with presently available technologies. In Arabidopsis the PCR based simple sequence length polymorphism (SSLP) markers have most commonly been used to map the gene of interest (Lukowitz et al., 2000, Choe et al., 2002, González-Guzmán et al., 2002 and Shirano et al., 2002.). These SSLP markers are abundant and co-dominant. Besides their detection are also easy and economical. In sequenced organisms, this is a comparatively easy task but, in species which are being examined for the first time, SSLP markers need to be developed de novo, which is a time-consuming and costly: first, it is necessary to identify sequence segments having SSLPs; then specific PCR primers have to be developed and tested. The use of random primers overcomes this, but the reproducibility of this approach is not satisfactory. Detection of Single Nucleotide Polymorphisms (SNPs) and Insertion/Deletion (InDel) Polymorphisms are now become very easy due to recent developments in sequencing technology and is relatively simple even in crop species, particularly when many EST sequences are available. The AFLP technique is however highly reproducible and does not require prior sequence information (Vos et al., 1995). Therefore, it can be applied directly to any organism for mapping and cloning of gene. Genomic restriction fragments are ligated to the adaptor and PCR primers are designed based on adaptor and restriction site sequences. Subsets of fragments can then be specially amplified by adding random selective nucleotides at the 3’end of the primers. The number of selective nucleotides (indicated by +) used depends on the size of the genome under investigation.

Map-based cloning (MBC) in Arabidopsis Among other techniques, genome-wide mapping procedures based on SSLP markers (Ponce et al., 1999) and SNPs (Cho et al., 1999) is good to begin MBC of a gene. One can exploit the fact that multiple AFLP markers can be obtained per primer combination to develop an AFLP based genome-wide mapping strategy (Peters et al., 2001, Peters et al., 2004). Similar to all genome-wide mapping strategies, it is helpful at the first steps of the MBC, when no information about the position

SINGH et al., Map-Based Cloning and its Application in Cloning Plant Disease Resistance Genes

of the desired gene is available. Only within 3 days by analysing 20–30 mutant individuals from a segregating population with eight primer combinations we can establish Linkage to a, 6 Mb region that provide a well-dispersed grid of 85 AFLP markers to cover the genome. Further, within 1–2 weeks a region of 200–800 kb can be identified by 120 mutant individuals that will typically exhaust the available Arabidopsis AFLP map (Peters et al., 2004). Apart from linkage, the AFLP-based approach will show non-linkage to the rest of the genome, which is essential for recognizing gross chromosomal re-arrangements. It is important to verify that there are no similar (and cloned) mutants located in the identified region before go for a large screen to select recombinants for fine mapping. Therefore, sequence-based map of Arabidopsis genes with mutant phenotypes (Meinke et al., 2003) is a great help to this end. Allelism Test Crosses should be used to test potential candidates. Fine mapping should be initiated once it is established that an unidentified gene is being dealt with. Since the advantage of AFLP is lost at this point, hence we suggest designing other PCRbased markers as flanking markers. A practical source for designing such PCR markers are the InDel polymorphisms (http:// www.Arabidopsis.org/Cereon/index.html). From a segregating population 1000–2000 individuals are tested with the help of flanking PCR markers. Simultaneously the markers can be analysed, for identifying recombinants in the region of interest easily (Peters et al., 2004). All recombinant plants are selected and F3 seeds collected to find out whether wild-type recombinants are homozygous or heterozygous for the locus. Subsequently the selected recombinants can be used to restrict the area containing the gene of interest to a region of approximately 0.025–0.050 cM, corresponding to, on average, 5.5–11.0 kb. A diagram of the described MBC strategy is presented in (Figure 1).

Map-based cloning in other systems Although MBC is now comparatively simple in Arabidopsis, these conclusions cannot be applied directly to other systems. However, the AFLP-based genome-wide mapping procedure can confidently be used for the first steps of MBC projects in any organism for which a dense AFLP map is available (e.g. barley (Qi et al., 1998), maize (Vuylsteke et

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al., 1999), tomato (Haanstra et al., 1999), lettuce (Jeuken et al., 2001), Petunia (Strommer et al., 2002)) or can be created. The AFLP procedure can also be used in that case when no AFLP map is available because no prior sequence information is required. For single gene project, an important approach is first to identify linked markers using Bulked Segregant Analysis (BSA) (Michelmore et al., 1991), by using a large set of AFLP primer combinations on DNA pools of mutants and wild types. Instead of AFLP markers, any other efficient marker system can be used in combination with BSA. As a result, one can easily identify closely linked markers, facilitating consequent regional fine mapping, followed by joining of Bacterial Artificial Chromosome (BAC) sequences to develop a physical map. For fine mapping, microsynteny of the species under investigation with Arabidopsis (or rice) has been used successfully in some cases (Oh et al., 2002). An interesting approach for plants to identifying linked markers is linkage disequilibrium mapping in natural populations (Rafalski JA. 2002, Gaut and Long 2003). Although fine mapping in plants with large genomes remains difficult and needs more labour in comparison to Arabidopsis, a rather surprising development is taking place in MBC of genes from organisms with larger genomes. From the data obtained so far, it appears that in large genomes, genes are often positioned in gene-rich regions with a local Arabidopsis-like kb/cM ratio (Fu et al., 2001, Brooks et al., 2002). In tomato, for instance, the average is 700 kb/cM (Tanksley et al., 1992) but, in specific cases, 1cM equalled, 100 kb (Ling et al., 2002), 160 kb (Ling et al., 1999), 280 kb (Ballvora et al., 2001) and even 5 kb (Fridman et al., 2000). Contrary to this, in lower combination regions, the gene density might be equally low, thus restricting candidate gene analysis to one or a few genes per 100 kb. Overall, it might appear that cloning a gene from large genomes requires an effort similar to that of cloning from small genomes, once sequence information and marker availability improves for the species under investigation.

Mapping applications For applications such as Marker-Assisted Selection (MAS) (Dekkers and Hospital 2002) and breeding by design (Peleman and van der Voort

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Fig. 1. Process of map-based cloning. The steps ( left) and material (right) needed to map a mutation in Arabidopsis thaliana is given for each step in the procedure. See Ref. (Peters et al., 2001) for the AFLP map referred to in steps 2 and 3. See Ref. (Meinkeet et al., 2003) for the sequence-based map of Arabidopsis genes with mutant phenotypes referred to in step 4. For the TAIR database, see http://www.Arabidopsis.org/.For further details of InDel polymorphisms and SNPs see http:// www.Arabidopsis.org/Cereon/index.html. Abbreviations: TAIR, The Arabidopsis Information Resource; AFLP, amplified fragment length polymorphism; InDel, insertion/ deletion; SNP, single-nucleotide polymorphism.

2003), one can stop once the segment carrying the gene of interest has been minimized to a satisfactory level. In plants with larger genomes, this application can also be used. Some points should be taken into consideration, if one decides to proceed ultimately

to isolate the gene of interest. A more sensible strategy is to identify the gene of interest is to order the appropriate TILLING lines and/or lines in which the candidate genes are tagged by a T-DNA or transposon. For Arabidopsis, a complete list of

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Table 1. Molecularly tagged disease resistance genes in rice and wheat Crop Rice

Wheat

Name of trait / gene tagged Resistance genes for bacterial blight xa1, xa2, Xa3, Xa4, xa5, xa8, xa13 and Xa21 Resistance genes for blast Pi2(t), Pi4(t), Pi10(t) Resistance for rice tungro spherical virus (RTSV) Resistance genes for powdery mildew Pm1, Pm2, Pm8, Pm12 Resistance genes for stem rust Sr21, Sr33 Resistance genes for leaf rust Lr9, Lr10, Lr21, Lr24, Lr37, Lr34, Lr46

sequence tagged lines can be found at the website of the Nottingham Arabidopsis Stock Centre (http:/ /nasc.life. nott.ac.uk/); for obtaining TILLING lines, one should consult the Arabidopsis TILLING project website (http://tilling.fhcrc.org:9366/). Phenotypes study of such lines and by performing allelism tests might direct you to the desired gene. Transformation of the mutant with subclones of the bacterial artificial chromosome (BAC(s)) containing the wild-type version of the gene of interest is another sound approach to test whether the clone used for transformation can complement the mutant of interest (Tao et al., 2002, Chang et al., 2003). Finally when the gene is pinpointed, sequencing the mutant allele(s) and comparing these to the wild-type sequence will certainly spot the desired gene.

Cloning of disease resistance genes Cloning can be done after identification and mapping of disease resistance genes. Basically cloning means increasing the copy number of genes. Due to two main reasons, there is a need to have many copies of resistance gene fragment: 1 Disease resistance gene after identification from the source variety, it is needed to transfer the genes to various varieties of the crop, where resistance is needed from similar pathogens. Hence the scientist wanted to keep many copies of genes for exploitation or intentionally spread copies of disease resistance genes to species and genera infected by same group of pathogens. 2. Various disease resistance genes have been identified which are able to recognize more than one type of avr proteins. These genes have been well characterized from crops like wheat (Lr34, Sr2), maize (RP1), tomato (Pto), barley (Mla), pepper (Bs2), Arabidopsis (Npr). Due to increased copy number the level of expression of certain

resistance genes were changed where resistance is associated with constitutive defence response. These types of process for producing broad spectrum resistance are strong because it is independent of an interaction with a specific Avr gene (Hulbert et al., 2001). Various disease resistance genes have been cloned by transposon tagging and map-based cloning techniques (Tab. 1). Transposon tagging is difficult job because it necessitates to search for a susceptible plant where the transposon has been inserted into a resistance gene to inactivate it. During the use of transposon specific sequences we have to amplify the flanking sequences with inverse PCR. After that these flanking sequences can be utilized for isolation and copying of disease resistance gene. However a lot of resistance genes have been cloned by means of transposon tagging but still map-based cloning has advantages over it and is considered as main choice. Crucial requirements for map-based cloning are the availability of complete genomic libraries in yeast artificial chromosome vectors and chromosome maps of high-density RFLP or RAPD. The gene of interest is ultimately isolated by cloning of the DNA between closely flanking markers, detection of open reading frames and complementation analyses.

Chromosomal location, molecular tagging and mapping of disease resistance genes When the source of disease resistance is identified, the recognized resistant cultivars are crossed to the susceptible ones to study the Mendalian inheritance of the resistance genes. The inheritance analysis of resistance genes in segregating generations will clear about the quality and number of genes controlling the trait. Aneuploid studies which is the conventional approaches to

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search the chromosomal location of disease resistance genes were found to be inappropriate and confusing. Therefore, such conventional approaches were substituted by the modern molecular approaches. The analysis depends on these molecular techniques is thought to be reliable. Now several disease resistance genes have been identified on their respective chromosomes, molecularly tagged and mapped. For instance, a gene Lr34, responsible for resistance against leaf rust pathogen is situated on chromosome 7Ds and strongly linked to the molecular marker Xgwm295. DNA markers tightly linked to quantitative resistance loci controlling quantitative disease resistance can be used for marker assisted selection (MAS) to incorporate these valuable traits (St Clair DA. 2010.). Steps in identifying and tagging of a disease resistance gene are:

Conventional aspect 1) Crossing between resistant and susceptible cultivar; 2) Development of F1, F2 and F3 Populations; 3) Analysis of response to the pathogen infection at parent, F1, F2 and F3 level and recording the data on their infection type or disease severity.

Molecular aspect 1) Use of molecular markers for parental polymorphism study (preferentially chromosome specific markers); 2) Parents differentiation at molecular level with molecular markers using DNA from the F2 resistant and susceptible bulks; 3) Markers differentiate these bulks to screen segregating (F2) population without any error; 4) Compare the phenotypic and molecular data for linkage analysis by using computer programmes like QTL Cartographer and MAPMAKER etc.

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Accepted on 20-02-2016