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Development and use of different mapping population in brinjal

Basavaraj Panjagal, profile picture
Basavaraj Panjagal

The document discusses the development and use of different mapping populations in brinjal (eggplant) breeding. It describes various mapping populations including F2, F2:3, backcross populations, doubled haploids (DHs), and recombinant inbred lines (RILs). It provides details on their properties, advantages, disadvantages and applications for gene mapping and quantitative trait locus (QTL) analysis. Examples of studies using F2 and backcross populations for mapping parthenocarpy and Fusarium resistance QTLs in eggplant are also summarized.

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6/13/2021 1 Dept.of VSC
6/13/2021 Dept.of VSC 2
Components of molecular mapping Polymorphic markers Suitable software mapping population 6/13/2021 3 Dept.of VSC
University of horticultural sciences , bagalkot k. R. C. college of horticulture , arabhavi department of vegetable science Development and use of different mapping population in brinjal breeding Basavaraj S Panjagal Ph.D in Vegetable Science Seminar – III 6/13/2021 4 Dept.of VSC
Introduction History Types of mapping population Mortal population Immortal population Applications Case studies Achievements Future thrust Conclusion Topic Division 6/13/2021 5 Dept.of VSC
• Brinjal or eggplant (Solanum melongena L.) belongs to the family Solanaceae is an important vegetable crop of sub-tropics and tropics. • In India, it is one of the most common, popular and principal vegetable crops grown throughout the country. (NHB, 2019) Singh et al., 2017 Area (MHa) Production (MT) Productivity (t/ha) India 68.00 13.26 18.68 Karnataka 16.50 0.40 24.67 INTRODUCTION 6/13/2021 6 Dept.of VSC
6/13/2021 Dept.of VSC 7 • Narrow genetic base • Lack of experimental populations • Lack of linkage maps
History • 1865 Mendel’s laws of inheritance – hypothetical factors • 1902 – Sutton and bovery- chromosomal theory of inheritance • 1910 – Morgan- first experimentalevidence for chromosomal theory of inheritance • 1911- Morgan- linkage between genes. • 1913 – Sturtevant- first linkage map of Drosophila sp. • 1914- Vilmorin and Bateson described the first linkage is pea.  Morphological markers  Enzyme based markers  DNA based markers 8 Katharina., 2016 6/13/2021 Dept.of VSC
Mapping population ? • A population used for gene mapping is commonly called a mapping population • Produced by controlled crosses and handled in definite pattern Mapping population used to know  Distance between two loci on chromosome  Linked markers to particular locus  Basic tools in QTL mapping and estimation of its effect on phenotype 9 Singh et al., 2016 6/13/2021 Dept.of VSC Requirements 1.Parents 2.Mating design 3.Marker system
Parents • Success of mapping experiment depends on choice of the parents • Homozygous conditions, preferably double haploids • The sufficient variation for the traits of interest at both the DNA sequence and the phenotype level. • It is desirable to ascertain the polymorphism between parents • Interspecies and intraspecies • Reduced pairing and reduced estimates of distance in wide crosses • Wide cross- large polymorphism, map must be collinear. Singh et al., 2016 6/13/2021 10 Dept.of VSC
Types of mapping populations 1. Primary mapping population: (Biparental) 2. Secondary mapping population: Immortalized F2 A) Mortal population (Temporary/Segregants):  Genetic constitution is not fixed  Used for single experiments Examples: F2, F2:3 , Back cross population B) Immortal population- (Permanent populations)  Genetic constitution is fixed  Used for multiple experiments Examples : DHs, RILs, Nils, BILs Singh et al., 2016 6/13/2021 11 Dept.of VSC
F2 Population • Produced by selfing or sibmating of the F1 individuals • F2 individuals will be heterozygous for the loci to which their parents are differing 6/13/2021 12 Dept.of VSC Katharina, 2015
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Properties of F2 population  Each genotype is represented only once  Genetic composition – heterozygote's  Only one round of recombination between any two loci  Capture recombination from both male and female parents  The segregation ratios 1:2:1 and 3:1 Applications • Best suited for preliminary mapping of oligogenes • Mapping of heterotic QTLs • Provides an estimates of additive, dominance and epistatic component of genetics variance • Requires only two generations • Minimum effort • Replications are not possible • F2 populations are ephemeral • Limited used for fine mapping Advantages Disadvantages 6/13/2021 14 Dept.of VSC Katharina, 2015
Development of selective markers linked to a major QTL for parthenocarpy in eggplant (Solanum melongena L.) Objective:  To identify the loci controlling parthenocarpy in eggplant.  To construct linkage map using F2 population  Development of selective marker Theor. Appl. Genet. Koji et al., 2012 6/13/2021 15 Dept.of VSC
Materials and methods QTL analysis-CIM- QTL cartographic software Linkage map analysis- MAPMAKER/EXP ver. 3.0b program Map construction – 954 markers (324 SSR, 630SNP) Marker analysis – SSR(1054) and SNP(630) Evaluation of the level of parthenocarpy Non –parthenocarpic – LS1934 (Malaysia), Nakate-Shink-uro (Japan) Parthenocarpic – AE-P03 (Japan) F2 population ALF2 , NAF2 16 Fruits were scored as Normal – LS1934=60mm, Nakate=70mm Malformed = fruit length <normal Malformed = even >normal fruit length and presence of cavity Koji et al., 2012 6/13/2021 Dept.of VSC
Koji et al., 2012 Parents No. of plants Lp (%) Description LS1934 n=28 Lp=0% Malformed, un-seeded fruits, which are smaller than the pollinated seeded fruits Nakate shink-uro n=18 Lp=0% AE-P03 n=35 Lp=94.3% Usually set normal sized fruits even without pollination Fig 1: Shape of emasculated fruits: a AE-P03, b LS1934, c Nakate-Shinkuro, d ALF1 (LS1934 9 AE-P03), e NAF1 (Nakate-Shinkuro 9 AE-P03). Shape of pollinated seeded fruits: f LS1934, g Nakate-Shinkuro. 6/13/2021 17 Dept.of VSC
Fig 2: Frequency distribution of level of parthenocarpy in two F2 populations (white bar ALF2, black bar NAF2). Koji et al., 2012 6/13/2021 18 Dept.of VSC
Fig 3: Comparison of three linkage maps (left ALF2, middle LWA2010, right NAF2). Koji et al., 2012 6/13/2021 19 Dept.of VSC
Fig 4: Comparison of the two linkage maps near the Cop8.1 locus (left ALF2, right NAF2). Underlined names indicate the common markers; their positions are connected with lines. The lines and black bars represent that the QTL regions were drawn Koji et al., 2012 6/13/2021 20 Dept.of VSC
Fig 5: Comparison of the two linkage maps near the Cop3.1 locus (left ALF2, right NAF2). Marker names and QTL regions were drawn Koji et al., 2012 6/13/2021 21 Dept.of VSC
Table 1 : QTLs detected using composite interval mapping Koji et al., 2012 6/13/2021 22 Dept.of VSC
Table 2: Mean levels of parthenocarpy (%) of F2 progenies and genotypes around the two identified QTLs (ALF2) 6/13/2021 23 Dept.of VSC
Table 3: Mean levels of parthenocarpy (%) of F2 progeny and genotypes around the Cop8.1 locus Koji et al., 2012 6/13/2021 24 Dept.of VSC
• Produced by selfing of individual F2 plants • Grown as individual plant progenies as F3 families F2:3 Population AA aa Aa 1AA 2Aa 1aa AA 1AA:2Aa:1aa aa Singh et al., 2016 6/13/2021 25 Dept.of VSC
• Used to map oligogenic traits • Suitable for QTL mapping Applications Advantages The replication of can be done The mean phenotypic value of F3 family represents the mean value of its parent F2 plant The genotype of F2 plant can be determined by corresponding F3 family genotype Disadvantages •Segregation, recombination and inbreeding- heterogenous population 6/13/2021 26 Dept.of VSC Singh et al., 2016
Koji et al., 2016 Objectives.: 1. Precise mapping of FW resistance 2. Study the relationships of resistance loci 3. Study the availability of resistance linked markers for MAS.
Materials and methods QTL analysis- performed using DI values and LM of LW2012, EW2012, AL2010 Linkage map analysis- MAPMAKER/EXP ver. 3.0b program LM of LW2012, EW2012, AL2010 previously reported used DNA extraction and marker analysis (AFLP, SSR, SRAP) Inoculation (30DAS old seedling, 2x107 spore/ml)and disease assessment (disease scale 0-2 ) . Disease index (%) FW resistance test: SFU1267 (Japan), F1, F2, F2:3, BILs poulation WCGR-112-8 & AE P03-suseptible to FW (Used as male) LS1934 & EP-1 – Resistant to FW (Used as female)
Fig 6. Frequency distribution of disease indices in two F2 populations. LWF3 plants were grown in 2008; EWF3 plants were grown in 2012. Triangles show the mean values of the parents (paternal: WCGR112-8; maternal: LS1934 for LWF2, EPL-1 for EWF2) and F1 plants in the respective populations Koji et al., 2016
Table 4: The results of QTLanalyses for Fusarium resistance Koji et al., 2016
Fig. 7 Comparison of the positions of Fusarium resistance loci on chromosome 2. Koji et al., 2016
6/13/2021 Dept.of VSC 32 Table 5 : Disease indices of F2 progenies and genotypes of markers closest to FM1
Table 6: Marker genotypes in the region around Fm1 Koji et al., 2016
Table 7: Novel SSR markers developed on the basis of the sequences of the markers close to Rfo-sa1 Table 8: A novel SNP marker developed on the basis of the sequences of a linkage marker for the Fusarium resistance locus of LS2436 Koji et al., 2016
• Generated by backcrossing the F1 to recessive trait harboring parental plant (test cross) Backcross population 6/13/2021 35 Dept.of VSC Singh et al., 2016
• Used further for marker assisted backcross breeding • Needs one more generation to develop • Need to do extensive crosses • Mortal population Advantages Disadvantages 6/13/2021 36 Dept.of VSC Singh et al., 2016
6/13/2021 37 Dept.of VSC Backcross inbred lines (BILs) Tripodi, 2020
6/13/2021 Dept.of VSC 38 Advantage:. Novel varieties can be directly obtained by a few crosses and minimal step improvement. Optimal for QTL analysis  The population is suitable for fine-mapping studies. Pyramiding of gene/ traits is possible Disadvantage: Time and cost consuming for their development.
Euphytica. Liu et al., 2014 Objectives.: 1. Interspecific hybridization followed by backcrossing to introgress resistance 2.Identification of gene specific marker for Ve homolog in PI388846 6/13/2021 39 Dept.of VSC
Material and methods  S. linnaeanum accession PI388846 (USDA)  4 Inbred lines of S.melongena (EP-12, EP-15, EP-21, D-13) – Susceptible to wilt  Interspecific hybridization and backcrossing  Vericillium wilt resistant screening – (10 7 CFU/ml) Scoring 2-3 weeks, scoring 0 -3 ( 0 or 1 = resistant , others susceptible )  Molecular Marker analysis:  primers SL01 and SL 02 ,  DNAMAN software for align sequence  Primer 5.0 – to design primer 40 Liu et al., 2014 6/13/2021 Dept.of VSC
Fig 8. Agronomic traits of PI388846 Fig 9. Agronomic traits of EP15 Liu et al., 2014 6/13/2021 41 Dept.of VSC
Fig.10Agronomic traits of F1 Fig.11Agronomic traits of BC2, BC3, BC4 Liu et al., 2014 6/13/2021 42 Dept.of VSC
Fig 12. Symptoms on plants of PI388846 (Sl), EP15, and their progenies inoculated with Verticillium dahliae Fig 13. Response of BC4 population after 3 weeks of inoculation. Liu et al., 2014 6/13/2021 43 Dept.of VSC
Table 9. Primer sequences and expected size used for marker development Liu et al., 2014 6/13/2021 44 Dept.of VSC
Fig.14. Allele-specific PCR marker analysis. a The SNP pos. 829 between PI388846 and three cultivated eggplant inbred lines, and the underlined sequence was used to design allelespecificPCR primers. Fig. 14 b. PCR results of primer SL01 combined with three allele-specific PCR primers SL181, SL182, and SL183. 1–4 represent PI388846, EP12, EP15, and EP21, respectively. c PCR detection of marker SlVR844 in BC4 population. 1–10 are resistant plants and 11–20 are susceptible plants Liu et al., 2014 6/13/2021 45 Dept.of VSC
• These are generated by the chromosome doubling of haploid plants • The haploid plants can be obtained by • Anther/culture of F1 plants Doubled haploids Heterozygous Completely homozygous Haploid Completely homozygous lines in a single generation Development of doubled-haploids 6/13/2021 46 Dept.of VSC Singh et al., 2016
1.ANDROGENESIS In vitro method • Process of embryo development from the male gametophytes i.e microspores or anthers. common guidelines of the anther culture method 47 6/13/2021 Dept.of VSC
Selection of Flower Extraction of ovary Surface sterilization Slicing of ovary Development of haploid embryo Transferred to regeneration medium Cultured on rooting medium DH Plant In vitro method Ovules transferred to basal medium Transferred to Induction medium Colchicine treatment 2. GYNOGENESIS 48 6/13/2021 Dept.of VSC
Applications • Complete homozygous plants • No residual heterozygocity • Higher recombinants than F2 population • Within two generations • Somaclonal variation • Unavailability of tissue culture technique for all crops and genotypes • Greater technical skill and sophisticated structures are needed •Mapping of both oligogenic & polygenic traits •Estimation of additive and additive × additive genetic variance Advantages Disadvantages 6/13/2021 49 Dept.of VSC Singh et al., 2016
Development and characterization of an eggplant (Solanum melongena) doubled haploid population and a doubled haploid line with high androgenic response Objective:  Development of an eggplant DH segregation population  Selection of lines with high embryogenesis response and heritable and stable performance . Euphytica Alba et al., 2017 6/13/2021 50 Dept.of VSC
Materials and methods Data collection and statistical analysis Morphological characterization of DH population Flow cytometer –to check double haploids Generation of DH population (anther culture) Bandera –Commercial F1 hybrid (Spain) 6/13/2021 Dept.of VSC 51 Alba et al., 2017
Table 10: Morphological and reproductive traits evaluated and their description Alba et al., 2017 6/13/2021 52 Dept.of VSC
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Fig. 15 Morphological variability of the DH population. A-C: Flowers with different petal color including white (A), pinkish (B) and purple (C). These flowers present unusual piece numbers (6-7) and different levels of cohesion in some of their petals. Alba et al., 2017 6/13/2021 54 Dept.of VSC
Fig .16 Morphological variability of the DH population. D: Bandera fruits showing, as expected, a remarkable phenotypical homogeneity. E, F: DH fruits with white background and different levels of dark stripes. G, H: DH fruits with different levels of dark background and white stripes. I, J: DH fruits with different levels of uniformly dark background. 6/13/2021 55 Dept.of VSC
Fig: 17 Examples of leaf surfaces of the Bandera hybrid (A) with no prickles, and of some DH individuals with prickles (white arrows) on the midrib and secondary leaf veins (B), and with large (C) and small (D) leaf blades (black arrows) on the midrib. 6/13/2021 56 Dept.of VSC Alba et al., 2017
Fig 18. Isolated microspore culture dishes of Bandera (A) and DH36 (B). Note the difference between genotypes in terms of androgenic response. C shows a microspore derived callus with shoot organogenic nodules (arrows) developing on its surface. 6/13/2021 57 Dept.of VSC Alba et al., 2017
Table 11: Results of the evaluation of morphological and reproductive traits of the donor Bandera hybrid, the DH population and a DH line (DH36). Alba et al., 2017 6/13/2021 58 Dept.of VSC
Table 12: Androgenic competence in anther culture of DH individuals and the donor hybrid (Bandera). Alba et al., 2017 6/13/2021 59 Dept.of VSC
Table 12: Cont.. Alba et al., 2017 6/13/2021 60 Dept.of VSC
Table 13: Androgenic competence in isolated microspore culture of DHS1 lines derived from DH individuals and the donor hybrid (Bandera). Alba et al., 2017 6/13/2021 61 Dept.of VSC
• Set of homozygous lines derived from continuous selfing of individual F2 plants • Sib mating can also be practiced • Population can be handled by SSD. Recombinant inbred lines (RILs) 6/13/2021 62 Dept.of VSC Tripodi, 2020
Applications • Mapping of both oligogenes and QTLs, Fine mapping • Estimation of additive and additive × additive components of genetic variance • many generations to develop • some regions tend to remain heterozygous • difficult to develop in crops having inbreeding depression •Once Homozygosity – propagated without segregation •Immortal population- can be replicated many years and many locations •Only once genotyping Advantages Disadvantages 6/13/2021 63 Dept.of VSC Tripodi, 2020
High-density genetic linkage map based on arbitrary and microsatellite markers using inter-specific recombinant inbred lines in eggplant (Solanum melongena L.) Journal of Plant Biochemistry and Biotechnology Pallavi et al., 2020 Objective:  Develop a RIL population to construct linkage map  Mapping of QTLs. 6/13/2021 64 Dept.of VSC
Materials and methods Construction of linkage map, Joinmap 3.0 Software PCR reactions, scoring of polymorphic bands DNA based marker assay – RAPD, ISSR, SCoT, microsatellite markers Extraction of DNA -CTAB Ramnagar Giant (UP) – Fruits big, round, soft , pulpy few seeds, Bharta W-4 (S. incanum) – small fruits, lesser yield Both parents and 114RILS (F8 generation) are used in the study Pallavi et al., 2020 6/13/2021 65 Dept.of VSC
Pallavi et al., 2020 Table 14 : Comparative details of amplification produced by different molecular markers 6/13/2021 66 Dept.of VSC
Pallavi et al., 2020 Fig. 19 a Genetic linkage map of S. melongena constructed using RAPD, ISSR, SCoT, and SSR markers. Marker distances (in centiMorgan) are indicated towards the left and marker names towards the right side of each LG. 6/13/2021 67 Dept.of VSC
6/13/2021 Dept.of VSC 68 Fig. 19 a Genetic linkage map of S. melongena constructed using RAPD, ISSR, SCoT, and SSR markers. Marker distances (in centiMorgan) are indicated towards the left and marker names towards the right side of each LG.
Pallavi et al., 2020 Table 15: Distribution of different marker types on each linkage group on an intra- specific genetic map constructed using RIL population S. melongena x S. incanum 6/13/2021 69 Dept.of VSC
• Near isogenic lines are pairs of homozygous lines that are identical in genotype, except for single gene • Also differ for variable length of genomic regions flanking this regions. • Can be produced by the selfing or Backcrossing • NIL is essentially segment substitution version of RP Near Isogenic lines(NILs) 6/13/2021 70 Dept.of VSC Singh et al., 2016
Development of NILs Parent A x Parent B F1 (Selfing) F2 Selection and selfing NILs Hom. Dom: Het. : Hom. Rec. Parent A x Parent B F1 x Parent B BC1: Selection and backcrossing NILs Selfing Backcrossing 6/13/2021 71 Dept.of VSC
Application • Immortal population • Linkage drag • Low level of genetic polymorphism Advantages Disadvantages • Best suited for Fine mapping and precise estimation of QTL effects • Suitable for map based gene cloning • Functional genomics, gene expression profiling •NILs can be used to construct high resolution mapping population 6/13/2021 72 Dept.of VSC Singh et al., 2016
• First time the term immortalized F2 population used by Gardiner et al. 1993. • Hua et al. 2003. developed the first immortalized F2 population by intercrossing the set of RILs 6/13/2021 73 Dept.of VSC Immortalized F2 Population • Immortalized F2 populations can be developed by paired crossing of the randomly chosen RILs derived from a cross in all possible combinations excluding reciprocals. Singh et al., 2016
Parent 1 Parent 2 AAbb X aaBB F1 AaBb F2 population Immortalized F2 population RILs produced from AAbb X aaBB AABB, AAbb aaBB, aabb Six possible RIL combinations Six heterozygous genotypes AABB X AAbb X aaBB X aabb AABb AaBB AaBb AAbb X aaBB X aabb AaBb Aabb aaBB X aabb aaBb AB Ab aB ab AB AABB AABb AaBB AaBb Ab AABb AAbb AaBb Aabb aB AaBB AaBb aaBB aaBb ab AaBb Aabb aaBb aabb F2 6/13/2021 74 Dept.of VSC Immortalized F2 Population
Merits • Supports replications • Lines can be evaluated over different locations and years Demerits • Undertaking large number of crossing is cumbersome 6/13/2021 75 Dept.of VSC • Mapping of Heterotic QTLs • Estimation of Heterosis • Determination of Epistatic interaction • Mapping of QTLs Applications Singh et al., 2016
Choice of mapping population :  The short term MP- F2, backcross developed following the bulk segregant analysis (BSA) approach can be a good starting point in molecular mapping.  Long term MP- RILs, DHs, NILs, Immortalized F2 must be developed and characterized properly with respect to the traits of importance for global mapping projects.  Since RILs, DHs, NILs are homozygous they not suitable for studying dominance and interaction effect , except for additive and additive interaction effect.  Immortalized F2 population consists the benefit of perpetual mapping population and the opportunity for studying the dominance  all interaction effects estimable from F2 populations. 6/13/2021 76 Dept.of VSC Choice of mapping population :  The short term MP- F2, backcross developed following the bulk segregant analysis (BSA) approach can be a good starting point in molecular mapping.  Long term MP- RILs, DHs, NILs, Immortalized F2 must be developed and characterized properly with respect to the traits of importance for global mapping projects.  Since RILs, DHs, NILs are homozygous they not suitable for studying dominance and interaction effect , except for additive and additive interaction effect.  Immortalized F2 population consists the benefit of perpetual mapping population and the opportunity for studying the dominance.  all interaction effects estimable from F2 populations. Singh et al., 2016
6/13/2021 77 Dept.of VSC Size of Mapping Population It has been suggested that for most quantitative traits, the mapping population size should be 500 or more (Bernardo 2008).  while a population of at least 200 individuals should be used for mapping of QTLs. But when the objective is positional cloning of genes, populations of several thousand plants should be used. Singh et al., 2016
Segregation ratio Problems in mapping studies: 1. Low polymorphism: Limited variation at DNA sequence level detectable as allele of molecular markers in the elite germplasm of some crops. 2. Segregation distortion of some molecular markers: A significant deviation of the observed segregation ratio for a marker locus from the expected ratio in mapping population. 6/13/2021 78 Dept.of VSC
Singh, 2015 6/13/2021 79 Dept.of VSC
Achievements 1. Nunome et al., 2001 Genetic mapping of fruit shape and colour 2. Barchi et al., 2012 Genetic and association maps 3. Lebau et al., 2013 Genetic mapping of major dominant gene (Ers 1) resistance to bacterial wilt 4. Hui et al., 2019 Haifeng Changqie 5 - new long eggplant hybrid developed by crossing the double haploid line '08-102' as female parent with the double haploid line '08-103' as male parent. 5. Toppino et al. 2016 Fruit properties QTLs 6. García-Fortea et al. (2019) Introduction of distant genepool for eggplant breeding 6/13/2021 80 Dept.of VSC
Future perspectives  Need to develop innovative breeding designs, with the use of minimum resources and yielding maximum mapping resolution  Standardization of breeding designs for biparental and multi parental mapping populations  Sharing of mapping populations across the laboratories 6/13/2021 81 Dept.of VSC
Conclusion 6/13/2021 82 Dept.of VSC An integral component of molecular mapping programmme  Commonly used based on their utility. The basic tools for understanding the effect of selected genetic factors and the organization of the genome of a species as a whole.  These populations are considered as the backbone of genomic research that aims to understand large, complex genomes at the physical or even sequence level.
Thank you… 83

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Development and use of different mapping population in brinjal

  • 3. Components of molecular mapping Polymorphic markers Suitable software mapping population 6/13/2021 3 Dept.of VSC
  • 4. University of horticultural sciences , bagalkot k. R. C. college of horticulture , arabhavi department of vegetable science Development and use of different mapping population in brinjal breeding Basavaraj S Panjagal Ph.D in Vegetable Science Seminar – III 6/13/2021 4 Dept.of VSC
  • 5. Introduction History Types of mapping population Mortal population Immortal population Applications Case studies Achievements Future thrust Conclusion Topic Division 6/13/2021 5 Dept.of VSC
  • 6. • Brinjal or eggplant (Solanum melongena L.) belongs to the family Solanaceae is an important vegetable crop of sub-tropics and tropics. • In India, it is one of the most common, popular and principal vegetable crops grown throughout the country. (NHB, 2019) Singh et al., 2017 Area (MHa) Production (MT) Productivity (t/ha) India 68.00 13.26 18.68 Karnataka 16.50 0.40 24.67 INTRODUCTION 6/13/2021 6 Dept.of VSC
  • 7. 6/13/2021 Dept.of VSC 7 • Narrow genetic base • Lack of experimental populations • Lack of linkage maps
  • 8. History • 1865 Mendel’s laws of inheritance – hypothetical factors • 1902 – Sutton and bovery- chromosomal theory of inheritance • 1910 – Morgan- first experimentalevidence for chromosomal theory of inheritance • 1911- Morgan- linkage between genes. • 1913 – Sturtevant- first linkage map of Drosophila sp. • 1914- Vilmorin and Bateson described the first linkage is pea.  Morphological markers  Enzyme based markers  DNA based markers 8 Katharina., 2016 6/13/2021 Dept.of VSC
  • 9. Mapping population ? • A population used for gene mapping is commonly called a mapping population • Produced by controlled crosses and handled in definite pattern Mapping population used to know  Distance between two loci on chromosome  Linked markers to particular locus  Basic tools in QTL mapping and estimation of its effect on phenotype 9 Singh et al., 2016 6/13/2021 Dept.of VSC Requirements 1.Parents 2.Mating design 3.Marker system
  • 10. Parents • Success of mapping experiment depends on choice of the parents • Homozygous conditions, preferably double haploids • The sufficient variation for the traits of interest at both the DNA sequence and the phenotype level. • It is desirable to ascertain the polymorphism between parents • Interspecies and intraspecies • Reduced pairing and reduced estimates of distance in wide crosses • Wide cross- large polymorphism, map must be collinear. Singh et al., 2016 6/13/2021 10 Dept.of VSC
  • 11. Types of mapping populations 1. Primary mapping population: (Biparental) 2. Secondary mapping population: Immortalized F2 A) Mortal population (Temporary/Segregants):  Genetic constitution is not fixed  Used for single experiments Examples: F2, F2:3 , Back cross population B) Immortal population- (Permanent populations)  Genetic constitution is fixed  Used for multiple experiments Examples : DHs, RILs, Nils, BILs Singh et al., 2016 6/13/2021 11 Dept.of VSC
  • 12. F2 Population • Produced by selfing or sibmating of the F1 individuals • F2 individuals will be heterozygous for the loci to which their parents are differing 6/13/2021 12 Dept.of VSC Katharina, 2015
  • 14. Properties of F2 population  Each genotype is represented only once  Genetic composition – heterozygote's  Only one round of recombination between any two loci  Capture recombination from both male and female parents  The segregation ratios 1:2:1 and 3:1 Applications • Best suited for preliminary mapping of oligogenes • Mapping of heterotic QTLs • Provides an estimates of additive, dominance and epistatic component of genetics variance • Requires only two generations • Minimum effort • Replications are not possible • F2 populations are ephemeral • Limited used for fine mapping Advantages Disadvantages 6/13/2021 14 Dept.of VSC Katharina, 2015
  • 15. Development of selective markers linked to a major QTL for parthenocarpy in eggplant (Solanum melongena L.) Objective:  To identify the loci controlling parthenocarpy in eggplant.  To construct linkage map using F2 population  Development of selective marker Theor. Appl. Genet. Koji et al., 2012 6/13/2021 15 Dept.of VSC
  • 16. Materials and methods QTL analysis-CIM- QTL cartographic software Linkage map analysis- MAPMAKER/EXP ver. 3.0b program Map construction – 954 markers (324 SSR, 630SNP) Marker analysis – SSR(1054) and SNP(630) Evaluation of the level of parthenocarpy Non –parthenocarpic – LS1934 (Malaysia), Nakate-Shink-uro (Japan) Parthenocarpic – AE-P03 (Japan) F2 population ALF2 , NAF2 16 Fruits were scored as Normal – LS1934=60mm, Nakate=70mm Malformed = fruit length <normal Malformed = even >normal fruit length and presence of cavity Koji et al., 2012 6/13/2021 Dept.of VSC
  • 17. Koji et al., 2012 Parents No. of plants Lp (%) Description LS1934 n=28 Lp=0% Malformed, un-seeded fruits, which are smaller than the pollinated seeded fruits Nakate shink-uro n=18 Lp=0% AE-P03 n=35 Lp=94.3% Usually set normal sized fruits even without pollination Fig 1: Shape of emasculated fruits: a AE-P03, b LS1934, c Nakate-Shinkuro, d ALF1 (LS1934 9 AE-P03), e NAF1 (Nakate-Shinkuro 9 AE-P03). Shape of pollinated seeded fruits: f LS1934, g Nakate-Shinkuro. 6/13/2021 17 Dept.of VSC
  • 18. Fig 2: Frequency distribution of level of parthenocarpy in two F2 populations (white bar ALF2, black bar NAF2). Koji et al., 2012 6/13/2021 18 Dept.of VSC
  • 19. Fig 3: Comparison of three linkage maps (left ALF2, middle LWA2010, right NAF2). Koji et al., 2012 6/13/2021 19 Dept.of VSC
  • 20. Fig 4: Comparison of the two linkage maps near the Cop8.1 locus (left ALF2, right NAF2). Underlined names indicate the common markers; their positions are connected with lines. The lines and black bars represent that the QTL regions were drawn Koji et al., 2012 6/13/2021 20 Dept.of VSC
  • 21. Fig 5: Comparison of the two linkage maps near the Cop3.1 locus (left ALF2, right NAF2). Marker names and QTL regions were drawn Koji et al., 2012 6/13/2021 21 Dept.of VSC
  • 22. Table 1 : QTLs detected using composite interval mapping Koji et al., 2012 6/13/2021 22 Dept.of VSC
  • 23. Table 2: Mean levels of parthenocarpy (%) of F2 progenies and genotypes around the two identified QTLs (ALF2) 6/13/2021 23 Dept.of VSC
  • 24. Table 3: Mean levels of parthenocarpy (%) of F2 progeny and genotypes around the Cop8.1 locus Koji et al., 2012 6/13/2021 24 Dept.of VSC
  • 25. • Produced by selfing of individual F2 plants • Grown as individual plant progenies as F3 families F2:3 Population AA aa Aa 1AA 2Aa 1aa AA 1AA:2Aa:1aa aa Singh et al., 2016 6/13/2021 25 Dept.of VSC
  • 26. • Used to map oligogenic traits • Suitable for QTL mapping Applications Advantages The replication of can be done The mean phenotypic value of F3 family represents the mean value of its parent F2 plant The genotype of F2 plant can be determined by corresponding F3 family genotype Disadvantages •Segregation, recombination and inbreeding- heterogenous population 6/13/2021 26 Dept.of VSC Singh et al., 2016
  • 27. Koji et al., 2016 Objectives.: 1. Precise mapping of FW resistance 2. Study the relationships of resistance loci 3. Study the availability of resistance linked markers for MAS.
  • 28. Materials and methods QTL analysis- performed using DI values and LM of LW2012, EW2012, AL2010 Linkage map analysis- MAPMAKER/EXP ver. 3.0b program LM of LW2012, EW2012, AL2010 previously reported used DNA extraction and marker analysis (AFLP, SSR, SRAP) Inoculation (30DAS old seedling, 2x107 spore/ml)and disease assessment (disease scale 0-2 ) . Disease index (%) FW resistance test: SFU1267 (Japan), F1, F2, F2:3, BILs poulation WCGR-112-8 & AE P03-suseptible to FW (Used as male) LS1934 & EP-1 – Resistant to FW (Used as female)
  • 29. Fig 6. Frequency distribution of disease indices in two F2 populations. LWF3 plants were grown in 2008; EWF3 plants were grown in 2012. Triangles show the mean values of the parents (paternal: WCGR112-8; maternal: LS1934 for LWF2, EPL-1 for EWF2) and F1 plants in the respective populations Koji et al., 2016
  • 30. Table 4: The results of QTLanalyses for Fusarium resistance Koji et al., 2016
  • 31. Fig. 7 Comparison of the positions of Fusarium resistance loci on chromosome 2. Koji et al., 2016
  • 32. 6/13/2021 Dept.of VSC 32 Table 5 : Disease indices of F2 progenies and genotypes of markers closest to FM1
  • 33. Table 6: Marker genotypes in the region around Fm1 Koji et al., 2016
  • 34. Table 7: Novel SSR markers developed on the basis of the sequences of the markers close to Rfo-sa1 Table 8: A novel SNP marker developed on the basis of the sequences of a linkage marker for the Fusarium resistance locus of LS2436 Koji et al., 2016
  • 35. • Generated by backcrossing the F1 to recessive trait harboring parental plant (test cross) Backcross population 6/13/2021 35 Dept.of VSC Singh et al., 2016
  • 36. • Used further for marker assisted backcross breeding • Needs one more generation to develop • Need to do extensive crosses • Mortal population Advantages Disadvantages 6/13/2021 36 Dept.of VSC Singh et al., 2016
  • 37. 6/13/2021 37 Dept.of VSC Backcross inbred lines (BILs) Tripodi, 2020
  • 38. 6/13/2021 Dept.of VSC 38 Advantage:. Novel varieties can be directly obtained by a few crosses and minimal step improvement. Optimal for QTL analysis  The population is suitable for fine-mapping studies. Pyramiding of gene/ traits is possible Disadvantage: Time and cost consuming for their development.
  • 39. Euphytica. Liu et al., 2014 Objectives.: 1. Interspecific hybridization followed by backcrossing to introgress resistance 2.Identification of gene specific marker for Ve homolog in PI388846 6/13/2021 39 Dept.of VSC
  • 40. Material and methods  S. linnaeanum accession PI388846 (USDA)  4 Inbred lines of S.melongena (EP-12, EP-15, EP-21, D-13) – Susceptible to wilt  Interspecific hybridization and backcrossing  Vericillium wilt resistant screening – (10 7 CFU/ml) Scoring 2-3 weeks, scoring 0 -3 ( 0 or 1 = resistant , others susceptible )  Molecular Marker analysis:  primers SL01 and SL 02 ,  DNAMAN software for align sequence  Primer 5.0 – to design primer 40 Liu et al., 2014 6/13/2021 Dept.of VSC
  • 41. Fig 8. Agronomic traits of PI388846 Fig 9. Agronomic traits of EP15 Liu et al., 2014 6/13/2021 41 Dept.of VSC
  • 42. Fig.10Agronomic traits of F1 Fig.11Agronomic traits of BC2, BC3, BC4 Liu et al., 2014 6/13/2021 42 Dept.of VSC
  • 43. Fig 12. Symptoms on plants of PI388846 (Sl), EP15, and their progenies inoculated with Verticillium dahliae Fig 13. Response of BC4 population after 3 weeks of inoculation. Liu et al., 2014 6/13/2021 43 Dept.of VSC
  • 44. Table 9. Primer sequences and expected size used for marker development Liu et al., 2014 6/13/2021 44 Dept.of VSC
  • 45. Fig.14. Allele-specific PCR marker analysis. a The SNP pos. 829 between PI388846 and three cultivated eggplant inbred lines, and the underlined sequence was used to design allelespecificPCR primers. Fig. 14 b. PCR results of primer SL01 combined with three allele-specific PCR primers SL181, SL182, and SL183. 1–4 represent PI388846, EP12, EP15, and EP21, respectively. c PCR detection of marker SlVR844 in BC4 population. 1–10 are resistant plants and 11–20 are susceptible plants Liu et al., 2014 6/13/2021 45 Dept.of VSC
  • 46. • These are generated by the chromosome doubling of haploid plants • The haploid plants can be obtained by • Anther/culture of F1 plants Doubled haploids Heterozygous Completely homozygous Haploid Completely homozygous lines in a single generation Development of doubled-haploids 6/13/2021 46 Dept.of VSC Singh et al., 2016
  • 47. 1.ANDROGENESIS In vitro method • Process of embryo development from the male gametophytes i.e microspores or anthers. common guidelines of the anther culture method 47 6/13/2021 Dept.of VSC
  • 48. Selection of Flower Extraction of ovary Surface sterilization Slicing of ovary Development of haploid embryo Transferred to regeneration medium Cultured on rooting medium DH Plant In vitro method Ovules transferred to basal medium Transferred to Induction medium Colchicine treatment 2. GYNOGENESIS 48 6/13/2021 Dept.of VSC
  • 49. Applications • Complete homozygous plants • No residual heterozygocity • Higher recombinants than F2 population • Within two generations • Somaclonal variation • Unavailability of tissue culture technique for all crops and genotypes • Greater technical skill and sophisticated structures are needed •Mapping of both oligogenic & polygenic traits •Estimation of additive and additive × additive genetic variance Advantages Disadvantages 6/13/2021 49 Dept.of VSC Singh et al., 2016
  • 50. Development and characterization of an eggplant (Solanum melongena) doubled haploid population and a doubled haploid line with high androgenic response Objective:  Development of an eggplant DH segregation population  Selection of lines with high embryogenesis response and heritable and stable performance . Euphytica Alba et al., 2017 6/13/2021 50 Dept.of VSC
  • 51. Materials and methods Data collection and statistical analysis Morphological characterization of DH population Flow cytometer –to check double haploids Generation of DH population (anther culture) Bandera –Commercial F1 hybrid (Spain) 6/13/2021 Dept.of VSC 51 Alba et al., 2017
  • 52. Table 10: Morphological and reproductive traits evaluated and their description Alba et al., 2017 6/13/2021 52 Dept.of VSC
  • 54. Fig. 15 Morphological variability of the DH population. A-C: Flowers with different petal color including white (A), pinkish (B) and purple (C). These flowers present unusual piece numbers (6-7) and different levels of cohesion in some of their petals. Alba et al., 2017 6/13/2021 54 Dept.of VSC
  • 55. Fig .16 Morphological variability of the DH population. D: Bandera fruits showing, as expected, a remarkable phenotypical homogeneity. E, F: DH fruits with white background and different levels of dark stripes. G, H: DH fruits with different levels of dark background and white stripes. I, J: DH fruits with different levels of uniformly dark background. 6/13/2021 55 Dept.of VSC
  • 56. Fig: 17 Examples of leaf surfaces of the Bandera hybrid (A) with no prickles, and of some DH individuals with prickles (white arrows) on the midrib and secondary leaf veins (B), and with large (C) and small (D) leaf blades (black arrows) on the midrib. 6/13/2021 56 Dept.of VSC Alba et al., 2017
  • 57. Fig 18. Isolated microspore culture dishes of Bandera (A) and DH36 (B). Note the difference between genotypes in terms of androgenic response. C shows a microspore derived callus with shoot organogenic nodules (arrows) developing on its surface. 6/13/2021 57 Dept.of VSC Alba et al., 2017
  • 58. Table 11: Results of the evaluation of morphological and reproductive traits of the donor Bandera hybrid, the DH population and a DH line (DH36). Alba et al., 2017 6/13/2021 58 Dept.of VSC
  • 59. Table 12: Androgenic competence in anther culture of DH individuals and the donor hybrid (Bandera). Alba et al., 2017 6/13/2021 59 Dept.of VSC
  • 60. Table 12: Cont.. Alba et al., 2017 6/13/2021 60 Dept.of VSC
  • 61. Table 13: Androgenic competence in isolated microspore culture of DHS1 lines derived from DH individuals and the donor hybrid (Bandera). Alba et al., 2017 6/13/2021 61 Dept.of VSC
  • 62. • Set of homozygous lines derived from continuous selfing of individual F2 plants • Sib mating can also be practiced • Population can be handled by SSD. Recombinant inbred lines (RILs) 6/13/2021 62 Dept.of VSC Tripodi, 2020
  • 63. Applications • Mapping of both oligogenes and QTLs, Fine mapping • Estimation of additive and additive × additive components of genetic variance • many generations to develop • some regions tend to remain heterozygous • difficult to develop in crops having inbreeding depression •Once Homozygosity – propagated without segregation •Immortal population- can be replicated many years and many locations •Only once genotyping Advantages Disadvantages 6/13/2021 63 Dept.of VSC Tripodi, 2020
  • 64. High-density genetic linkage map based on arbitrary and microsatellite markers using inter-specific recombinant inbred lines in eggplant (Solanum melongena L.) Journal of Plant Biochemistry and Biotechnology Pallavi et al., 2020 Objective:  Develop a RIL population to construct linkage map  Mapping of QTLs. 6/13/2021 64 Dept.of VSC
  • 65. Materials and methods Construction of linkage map, Joinmap 3.0 Software PCR reactions, scoring of polymorphic bands DNA based marker assay – RAPD, ISSR, SCoT, microsatellite markers Extraction of DNA -CTAB Ramnagar Giant (UP) – Fruits big, round, soft , pulpy few seeds, Bharta W-4 (S. incanum) – small fruits, lesser yield Both parents and 114RILS (F8 generation) are used in the study Pallavi et al., 2020 6/13/2021 65 Dept.of VSC
  • 66. Pallavi et al., 2020 Table 14 : Comparative details of amplification produced by different molecular markers 6/13/2021 66 Dept.of VSC
  • 67. Pallavi et al., 2020 Fig. 19 a Genetic linkage map of S. melongena constructed using RAPD, ISSR, SCoT, and SSR markers. Marker distances (in centiMorgan) are indicated towards the left and marker names towards the right side of each LG. 6/13/2021 67 Dept.of VSC
  • 68. 6/13/2021 Dept.of VSC 68 Fig. 19 a Genetic linkage map of S. melongena constructed using RAPD, ISSR, SCoT, and SSR markers. Marker distances (in centiMorgan) are indicated towards the left and marker names towards the right side of each LG.
  • 69. Pallavi et al., 2020 Table 15: Distribution of different marker types on each linkage group on an intra- specific genetic map constructed using RIL population S. melongena x S. incanum 6/13/2021 69 Dept.of VSC
  • 70. • Near isogenic lines are pairs of homozygous lines that are identical in genotype, except for single gene • Also differ for variable length of genomic regions flanking this regions. • Can be produced by the selfing or Backcrossing • NIL is essentially segment substitution version of RP Near Isogenic lines(NILs) 6/13/2021 70 Dept.of VSC Singh et al., 2016
  • 71. Development of NILs Parent A x Parent B F1 (Selfing) F2 Selection and selfing NILs Hom. Dom: Het. : Hom. Rec. Parent A x Parent B F1 x Parent B BC1: Selection and backcrossing NILs Selfing Backcrossing 6/13/2021 71 Dept.of VSC
  • 72. Application • Immortal population • Linkage drag • Low level of genetic polymorphism Advantages Disadvantages • Best suited for Fine mapping and precise estimation of QTL effects • Suitable for map based gene cloning • Functional genomics, gene expression profiling •NILs can be used to construct high resolution mapping population 6/13/2021 72 Dept.of VSC Singh et al., 2016
  • 73. • First time the term immortalized F2 population used by Gardiner et al. 1993. • Hua et al. 2003. developed the first immortalized F2 population by intercrossing the set of RILs 6/13/2021 73 Dept.of VSC Immortalized F2 Population • Immortalized F2 populations can be developed by paired crossing of the randomly chosen RILs derived from a cross in all possible combinations excluding reciprocals. Singh et al., 2016
  • 74. Parent 1 Parent 2 AAbb X aaBB F1 AaBb F2 population Immortalized F2 population RILs produced from AAbb X aaBB AABB, AAbb aaBB, aabb Six possible RIL combinations Six heterozygous genotypes AABB X AAbb X aaBB X aabb AABb AaBB AaBb AAbb X aaBB X aabb AaBb Aabb aaBB X aabb aaBb AB Ab aB ab AB AABB AABb AaBB AaBb Ab AABb AAbb AaBb Aabb aB AaBB AaBb aaBB aaBb ab AaBb Aabb aaBb aabb F2 6/13/2021 74 Dept.of VSC Immortalized F2 Population
  • 75. Merits • Supports replications • Lines can be evaluated over different locations and years Demerits • Undertaking large number of crossing is cumbersome 6/13/2021 75 Dept.of VSC • Mapping of Heterotic QTLs • Estimation of Heterosis • Determination of Epistatic interaction • Mapping of QTLs Applications Singh et al., 2016
  • 76. Choice of mapping population :  The short term MP- F2, backcross developed following the bulk segregant analysis (BSA) approach can be a good starting point in molecular mapping.  Long term MP- RILs, DHs, NILs, Immortalized F2 must be developed and characterized properly with respect to the traits of importance for global mapping projects.  Since RILs, DHs, NILs are homozygous they not suitable for studying dominance and interaction effect , except for additive and additive interaction effect.  Immortalized F2 population consists the benefit of perpetual mapping population and the opportunity for studying the dominance  all interaction effects estimable from F2 populations. 6/13/2021 76 Dept.of VSC Choice of mapping population :  The short term MP- F2, backcross developed following the bulk segregant analysis (BSA) approach can be a good starting point in molecular mapping.  Long term MP- RILs, DHs, NILs, Immortalized F2 must be developed and characterized properly with respect to the traits of importance for global mapping projects.  Since RILs, DHs, NILs are homozygous they not suitable for studying dominance and interaction effect , except for additive and additive interaction effect.  Immortalized F2 population consists the benefit of perpetual mapping population and the opportunity for studying the dominance.  all interaction effects estimable from F2 populations. Singh et al., 2016
  • 77. 6/13/2021 77 Dept.of VSC Size of Mapping Population It has been suggested that for most quantitative traits, the mapping population size should be 500 or more (Bernardo 2008).  while a population of at least 200 individuals should be used for mapping of QTLs. But when the objective is positional cloning of genes, populations of several thousand plants should be used. Singh et al., 2016
  • 78. Segregation ratio Problems in mapping studies: 1. Low polymorphism: Limited variation at DNA sequence level detectable as allele of molecular markers in the elite germplasm of some crops. 2. Segregation distortion of some molecular markers: A significant deviation of the observed segregation ratio for a marker locus from the expected ratio in mapping population. 6/13/2021 78 Dept.of VSC
  • 80. Achievements 1. Nunome et al., 2001 Genetic mapping of fruit shape and colour 2. Barchi et al., 2012 Genetic and association maps 3. Lebau et al., 2013 Genetic mapping of major dominant gene (Ers 1) resistance to bacterial wilt 4. Hui et al., 2019 Haifeng Changqie 5 - new long eggplant hybrid developed by crossing the double haploid line '08-102' as female parent with the double haploid line '08-103' as male parent. 5. Toppino et al. 2016 Fruit properties QTLs 6. García-Fortea et al. (2019) Introduction of distant genepool for eggplant breeding 6/13/2021 80 Dept.of VSC
  • 81. Future perspectives  Need to develop innovative breeding designs, with the use of minimum resources and yielding maximum mapping resolution  Standardization of breeding designs for biparental and multi parental mapping populations  Sharing of mapping populations across the laboratories 6/13/2021 81 Dept.of VSC
  • 82. Conclusion 6/13/2021 82 Dept.of VSC An integral component of molecular mapping programmme  Commonly used based on their utility. The basic tools for understanding the effect of selected genetic factors and the organization of the genome of a species as a whole.  These populations are considered as the backbone of genomic research that aims to understand large, complex genomes at the physical or even sequence level.