doc sem

doctoral Seminar-I : 

doctoral Seminar-I On Binod Kumar Ph.D Scholar Deptt . Of Plant Breeding & Genetics Faculty of Agriculture Rajendra Agricultural University, Pusa “Genome mapping and its role in crop improvement”

PowerPoint Presentation: 

Mapping :- Determining the location of genes/elements within a space, with respect to identifiable landmarks.

GENOME MAPPING: 

GENOME MAPPING Genetic mapping: is based on the use of genetic techniques to construct maps showing the positions of genes and other sequence features on a genome. or The relative position of genes determined the frequency of recombinants of alleles of two genes. Physical mapping: uses molecular biology techniques to examine DNA molecules directly in order to construct maps showing the positions of sequence features, including genes. Or The true position of genes along the DNA in ‘ kbp ’ is called as physical map.

PRINCIPLES OF GENETIC MAPPING: 

PRINCIPLES OF GENETIC MAPPING Genetic mapping is based on the principle that genes (markers or loci) segregate via chromosome recombination during meiosis , thus allowing their analysis in the progeny . During meiosis, chromosomes assort randomly into gametes, such that the segregation of alleles of one gene is independent of alleles of another gene. This is stated in Mendel's second law and is known as the law of independent assortment. The law of independent assortment always holds true for genes that are located on different chromosomes, but it does not always hold true for genes that are on the same chromosome.

PowerPoint Presentation: 

continued………………. When two genes are close together on the same chromosome, they do not assort independently and are said to be linked. Genes that are closer together or tightly-linked will be transmitted together from parent to progeny more frequently than those genes located far apart. The chance of a crossover producing recombination between genes is directly related to the distance between two genes - the lower the frequency of recombination between two markers, the closer they are situated on a chromosome.

REQUIREMENTS FOR GENETIC MAPPING: 

REQUIREMENTS FOR GENETIC MAPPING 1) Develop appropriate mapping population and decide the sample size. 2) Decide the type of molecular markers for genotyping the mapping population. 3) Screen parents for marker polymorphism, and then genotype the mapping population (parents plus all progenies). 4) Perform linkage analyses (calculate pair wise recombination frequencies between markers, establish linkage groups, estimate map distances, and determine map order) using statistical programs.

Mapping population: 

Mapping population The mapping population is selecting two genetically divergent parents, which show clear genetic differences for one or more traits of interest. e.g. the recipient or recurrent parent can be a highly productive and commercially successful cultivar but lacks disease resistance, which is present in another donor parent. In self-pollinating species, mapping populations originate from parents that are both highly homozygous (inbred). In cross pollinating (out-crossing) species, the situation is more complicated since most of these species do not tolerate inbreeding.

continued……: 

continued…… Progenies from the second filial generation (F2), backcross (BC), recombinant inbred lines (RILs), double haploids (DHs), and near isogenic lines (NILs) can be used for genetic mapping in self pollinating species. F2 populations are developed by selfing F1 hybrids derived by crossing the two parents. BC population is produced by crossing F1 back into one of the parents. RILs are developed by single-seed selections from individual plants of an F2 population; such selections continue for six to eight generations. If backcross selection is repeated at least for six generations, more than 99% of the genome from BC6 and above will be derived from recurrent parent. Selfing of selected individuals from BC7F1 will produce BC7F2 lines that are homozygous for the target gene, which is said to be nearly isogenic with the recipient parent (NILs).

Continued……..: 

Continued…….. DH population is produced by doubling the gametes of F1 or F2 population. Plants will be regenerated using tissue culture techniques after induction of chromosome doubling from pollen grains or haploid embryos resulting from species crosses. F2 and BC populations are considered to be temporary populations because they are highly heterozygous and cannot be propagated indefinitely through seeds. RILs, NILs and DHs are permanent populations because they are homozygous or ‘true-breeding’ lines that can be multiplied and reproduced without genetic change occurring.

Continued……: 

Continued…… Using a sample size ranging from 50 to 1000 individuals of F2, BC, RILs and DHs populations have shown that the type and size of mapping populations can exert an influence on the accuracy of genetic maps : I. Populations’ size with the lowest number of individuals provided several fragmented linkage groups and inaccurate locus order. II. More accurate maps were obtained for RIL and F2 population with co-dominant markers, while maps constructed from F2 with dominant marker was less accurate. III. The higher the number of individuals, the more precise the map.

Continued…….: 

Continued……. The genetic information for different types of mapping populations in relation to dominant versus co-dominant markers . Maximum genetic information is obtained from F2 population using a co-dominant marker system. Dominant markers supply as much information as co-dominant markers in RIL, NILs and DHs because all loci are homozygous, or nearly so. BC populations can be useful for mapping dominant markers if all loci in the recurrent parent are homozygous, and the donor and recurrent parent have contrasting polymorphic marker alleles

Hypothetical gel showing segregation patterns for codominant (left- hand side) and dominant (right-hand side) markers for five types of mapping populations.: 

Hypothetical gel showing segregation patterns for codominant (left- hand side) and dominant (right-hand side) markers for five types of mapping populations.

Selection of molecular markers for mapping: 

Selection of molecular markers for mapping Restriction fragment length polymorphisms (RFLPs), Amplified fragment length polymorphisms (AFLPs), Random amplified polymorphic DNA (RAPD), Expressed sequence tags (ESTs), Cleaved amplified polymorphic sequence (CAPS), Simple sequence repeats (SSRs), Inter simple sequence repeat (ISSR), Single nucleotide polymorphism (SNP), Diversity arrays technology (DArT).

Restriction fragment length polymorphisms (RFLPs): 

Restriction fragment length polymorphisms (RFLPs) The major strength of RFLP markers : Co- dominent inheritance. good transferability between laboratories. locus specificity that allows synteny. high reproducibility. Limitation: It requires high quantity and quality of DNA. It depends on the development of specific probe libraries for the species. The level of polymorphism is low. It is time consuming and laborious. It usually requires radioactively labeled probes.

Cleaved amplified polymorphic sequence (CAPS): 

Cleaved amplified polymorphic sequence (CAPS) Advantages: CAPS are much easier and less time-consuming than RFLPs. CAPS primers developed from ESTs are more useful as genetic markers for comparative mapping study than those markers derived from non-functional sequences such as genomic microsatellite markers. CAPS markers are inherited mainly in a co-dominant manner and the development of CAPS markers is only possible where mutations disrupt or create a restriction enzyme recognition site.

Simple sequence repeats (SSRs): 

Simple sequence repeats (SSRs) Advantages : High information content, Co-dominant inheritance, Reproducibility, Locus specificity, Highly transferability, Ease for automation for high throughput screening. Disadvantage: Primer development takes lots of work digest, clone, probe, sequence, design primer Primers not broadly applicable across taxa usually work within genera (plants) Fluorescent primers are expensive

Expressed sequence tags (ESTs) : 

Expressed sequence tags (ESTs) Advantages: EST marker is found to be genetically associated with a trait of interest, it is possible that the mapped gene directly affects the trait. ESTs that share homology to candidate genes or differentially expressed ESTs in a tissue of interest, can be specifically targeted for genetic mapping . EST-based markers are very useful for comparative mapping across different species because they generally have high degree of sequence conservation. If DNA sequence information is lacking for a target species, ESTs derived from other species could be used as the basis for genetic mapping in other species of interest.

Diversity arrays technology (DArT): 

Diversity arrays technology ( DArT ) DArT has recently been used in genetic mapping and fingerprinting studies in wheat, Arabidopsis and barley. DArT is a high-throughput, quick, and highly polymorphic and reproducible method but their dominant inheritance is still a limitation for mapping. DArT markers have a great potential for genetic mapping in a number of ‘orphan’ crops.

SNPs - The mother of all markers: 

SNPs - The mother of all markers Advantages: Reproducibility, Accuracy, Capability of multiplexing for high throughput analysis, Cost effectiveness in terms of initial investment for equipment and cost per data point. 80,127 polymorphic sites distinguish between two cultivated rice subspecies, japonica and indica SNP (single nucleotide polymorphism): a point mutation that can be targeted by molecular techniques and, thus, can be exploited as a molecular marker. SNPs are particularly amenable to high-throughput, automated profiling.

Polymorphism Screening and Genotyping of the Mapping Population: 

Polymorphism Screening and Genotyping of the Mapping Population The third step in the construction of a linkage map is to identify sufficient number of markers that reveal differences between parents (i.e., polymorphic markers). The cross pollinating species possess higher levels of polymorphism compared to inbreeding species. Once sufficient numbers of polymorphic markers have been identified between parents, they must be used to genotype the entire mapping population. A genetic map is as good as the data that were used to construct it. Researchers construct a linkage map assuming no errors present in the data and then look for improbable genotypes, such as those originating from double recombination. The data must be critically checked for all possible errors, such as typographical error, missing data, genotype coding error, order of genotypes along all loci, etc.

Continued……..: 

Continued…….. This is increasingly the case as the marker density increases and the proportion of true recombination between neighboring markers falls. The presence of missing values in the marker data means that information about the number of true recombination that has taken place along the chromosome is lost.

Linkage Analyses: 

Linkage Analyses Linkage analyses and mapping are computerized. Different computer packages are presently available for genetic linkage mapping but the most widely used are JoinMap (Stam, 1993a), MAPMAKER/EXP (Lander et al., 1987), GMENDEL (Echt et al., 1992), LINKAGE (Suiter et al.,1983), Map Manager QTX (Manly et al., 2001).

LIMITATIONS: 

LIMITATIONS A map generated by genetic techniques is rarely sufficient for directing the sequencing phase of a genome project. This is for two reasons: 1) The resolution of a genetic map depends on the number of crossovers that have been scored . Genes that are several tens of kb apart may appear at the same position on the genetic map. 2) Genetic maps have limited accuracy . Presence of recombination hotspots means that crossovers are more likely to occur at some points rather than at others. physical mapping techniques has been developed to address this problem.

Prospects of Genetic Mapping: 

Prospects of Genetic Mapping The development of genetic maps based on markers that are simple to generate, highly reproducible, co-dominant and specific for known linkage groups are highly desirable for their application in breeding. The transferability of maps constructed using AFLP, RAPD and ISSR is limited between population and pedigrees within a species because each marker is primarily defined by its length (i.e. sequence information may be limited). Genetic map based on DNA markers are available for several economically important plants, including Arabidopsis, maize, rice, wheat, barley, tomato, potato, sunflower, pea, bean, rye, millet, cotton, soybean, sorghum, cowpea, tobacco, turnip, rape, carrot, sugarcane, sugarbeet , coffee, and rape

PHYSICAL MAPPING: 

PHYSICAL MAPPING Restriction mapping: which locates the relative positions on a DNA molecule of the recognition sequences for restriction endonucleases. Fluorescent in situ hybridization (FISH): in which marker locations are mapped by hybridizing a probe containing the marker to intact chromosomes. Sequence tagged site (STS) mapping: in which the positions of short sequences are mapped by PCR and/or hybridization analysis of genome fragments.

The basic methodology for restriction mapping: 

The basic methodology for restriction mapping

Fluorescent in situ hybridization (FISH): 

Fluorescent in situ hybridization (FISH) FISH enables the position of a marker on a chromosome or extended DNA molecule to be directly visualized. In FISH, the marker is a DNA sequence that is visualized by hybridization with a fluorescent probe. In situ hybridization intact chromosome is examined by probing it with a labeled DNA molecule.

In situ hybridization with radioactive or fluorescent probes: 

In situ hybridization with radioactive or fluorescent probes The position on the chromosome at which hybridization occurs provides information about the map location of the DNA sequence used as the probe. DNA in the chromosome is made single stranded (‘denatured’). The standard method for denaturing chromosomal DNA without destroying the morphology of the chromosome is to dry the preparation onto a glass microscope slide and then treat with formamide.

FISH: 

FISH

Sequence tagged site (STS) mapping: 

Sequence tagged site (STS) mapping A sequence tagged site or STS is simply a short DNA sequence, generally between 100 and 500 bp in length, that is easily recognizable and occurs only once in the chromosome or genome being studied. To map a set of STSs, a collection of overlapping DNA fragments from a single chromosome or from the entire genome is needed.

STS Mapping: 

STS Mapping

Genetic vs. Physical Distance : 

Genetic vs. Physical Distance Map distances based on recombination frequencies are not a direct measurement of physical distance along a chromosome. Recombination “hot spots” overestimate physical length. Low rates in heterochromatin and centromeres underestimate actual physical length.

Genetic vs. Physical Distance : 

Genetic vs. Physical Distance

Genetic and physical maps may differ in relative distances and even in the position of genes on a chromosome. : 

Genetic and physical maps may differ in relative distances and even in the position of genes on a chromosome.

List of Mapped Crop Plants: 

List of Mapped Crop Plants Organism Type Relevance Genome size Number of genes predicted Organization Year of completion Arabidopsis lyrata - Model plant 207 Mb 32,670 University of Southern California and VIB, Gent 2011 Arabidopsis thaliana Ecotype:Columbia Wild mustard Thale Cress Model plant 119 Mb 25,498 , 27,400, 31,670 Arabidopsis Genome Initiative 2000 Cucumis sativus Cucumber 'Chinese long' inbred line 9930 Vegetable crop 367 Mb 26,682 Chinese Academy of Agricultural Sciences, Beijing 2009 Glycine max Soybean Soyabean Soya Protein and oil crop 1,100 Mb 46,430 Purdue University 2010 Malus domestica Apple "Golden Delicious" Fruit Tree 927 Mb 57,000 International consortium 2010 Oryza sativa ssp indica Rice Crop and model organism 420 Mb 32-50,000 Beijing Genomics Institute, Zhejiang University and the Chinese Academy of Sciences 2002 Solanum tuberosum Potato Crop plant 844Mb 39,031 Multiple institutions 2011 Sorghum bicolor Crop plant 730Mb 27,640 Multiple institutions 2009 Zea mays ssp mays Corn (maize) B73 Cereal crop 2,800 Mb 63,300 NSF 2009

PowerPoint Presentation: 

To monitor the inheritance of gene that is difficult to score. To differentiate the genes of similar phenotype. To link the phenotypic data to biochemical data. To diagnose the plant genes and their regulation. To accelerate breeding programmes through Marker Aided Selection. Benefits of Mapping

PowerPoint Presentation: 

Parental polymorphism for markers may not be sufficient for mapping. Marker identified for a gene in one cross is not usable in other cross. Tagging genes in all the crosses breeders make is not possible. Some of the traits are not easily amenable for phenotyping. Difficulties of Mapping

CONCLUSION: 

CONCLUSION Genetic mapping construction requires the development appropriate mapping population. It also depends on sample size and type of molecular marker used. The construction of fine genetic maps with high levels of genome coverage is a first step for localizing genes or quantitative trait loci (QTL) that are associated with economically important traits. Highly reproducible, high throughput, co-dominant, and transferable molecular markers, especially developed from expressed regions, are sought to increase the utility of genetic maps. Physical maps are extremely useful for the purpose of map-based gene cloning. A complete saturated map has application in Marker assisted selection, comparative mapping between different species.

REFERENCES: : 

REFERENCES: Brown, T.A., (2006) Genomes 3, 3 rd edition. Garland science, Newyork , London. Genome mapping, pp 126-160. Chahal , G.S. and Gosal , S.S.(2008) Principles and procedures of plant breeding, biotechnology and conventional approaches, Narosa publishing house, New Delhi.pp-510-511. K. Semagn , Å. Bjørnstad and M. N. Ndjiondjop (2006) Principles, requirements and prospects of genetic mapping in plants. African Journal of Biotechnology Vol. 5 (25), pp. 2569-2587. K. Semagn , Å. Bjørnstad and M. N. Ndjiondjop (2006) An overview of molecular marker methods for plants. African Journal of Biotechnology Vol. 5 (25), pp. 2540-2568. Lander ES, Green P, Abrahamson J, Barlow A, Daly MJ, Lincoln SE, Newburg L (1987). MAPMAKER: an interactive computer package for constructing primary genetic linkage maps of experimental and natural populations. Genomics 1: 174–181. Stam P (1993a). Construction of integrated genetic linkage maps by means of a new computer package: JoinMap . Plant J. 3: 739-744. http://www.academicjournals.org/AJB