1. Chromosome structure, function and implications for inheritance: segregation, independent assortment and linkage
ATGGGCACTGCCACT R r
Source: plant-genetics.kais.kyoto-u.ac.jp

2. A chromosome is a single DNA molecule complexed with histone proteins
DNA is highly compacted: DNA + histone proteins = chromatin
Nucleosome structure
Source: abcam.com

3. Chromosome numbering/naming
Numbering starts with the “largest” chromosome  
Source: ucl.ac.uk

4. Chromosome landmarks
Centromere(s)
Source: nature.com

5. Chromosome landmarks Telomere(s) Source: conciergemedicinela.com
Source: jyi.org

6. Heterochromatin and euchromatin
The degree of compactness of DNA - implications for degree of gene expression
Duggan, N. M. & Tang, Z. I. (2010) The Formation of Heterochromatin. 
Nature Education 3(9):5

7. Heterochromatin and euchromatin
Heterochromatin: highly compact, inaccessible for transcription, and less likely to contain highly expressed genes
Constitutive:
~ Always compact
Facultative:
varying degrees of compactness depending on individual, developmental stage
Implications for epigenetics
Euchromatin: relaxed, accessible, and more likely to contain expressed genes

8. Mitosis and Meiosis Mitosis: 2n 2n Meiosis 2n n
Mitosis: 2 identical daughter cells
Meiosis: 4 daughter cells, which may be genetically different
Mitosis: zygote until death
Meiosis: specialized cells
V v V V
V v
V v v v
V v
Mitosis
Meiosis

9. The cell cycle G1: RNA and protein synthesis, but no DNA replication
S: DNA synthesis. The total DNA content goes from 2n to 4n
G2: After DNA synthesis and before mitosis. At this point a diploid cell contains two complete diploid sets of chromosomes
M: Mitosis 

10. Mitosis

11. Mitosis: Example: Maize; 2n=2x=20 Prophase: Chromatin contracts
Mitosis: Example: Maize; 2n=2x=20 Prophase: Chromatin contracts. Each chromosome consists of two sister chromatids. One is "original" chromosome, other is copy made during S phase. Chromatids held together at centromere. At late Prophase, attachment of spindle fibers to centromeric region and dissolution of nuclear membrane. Continued contraction of paired chromatids. 20 sets of 2 sister chromatids = 40 chromatids.
Metaphase: Orientation of paired chromatids on Metaphase "plate". 20 sets of sister chromatids aligned on plate = 40 chromatids.

12. Mitosis: Example: Maize; 2n=2x=20 Anaphase: Sister chromatids migrate to opposite poles. Separation of sister chromatids accomplishes the equal distribution of chromosomal material. 20 chromatids migrate to each pole. 
Telophase: Single chromatids, or at this point, chromosomes, return to the relaxed Interphase state. Cytokinesis divides original cell into two daughter cells. Two cells, each with 10 pairs of chromosomes = 20 chromosomes per cell.  Maize :  2n=2x=20

13. Telomere shortening during mitosis
Telomeres: Repetitive DNA sequences at ends of chromosomes. Each time the cell divides, the telomere shortens.  Eventually, the chromosomes become "so frayed that the cell senesces". In some cells- eggs, sperm, and cancer cells - an enzyme known as telomerase allows for "reconstruction" of the telomere, thus prolonging cell life.   

14. Development of the male gametophyte
Pollen mother cell (PMC) 
PMC undergoes meiosis
Meiosis gives a tetrad of microspores
Note, this is different than ♀

15. Development of the female gametophyte
Megaspore mother cell (MMC) 
MMC undergoes meiosis
Of 4 megaspores produced 1 survives (most species)
Three post-meiotic mitoses 1 2 3

16. Meiosis 1 Prophase (5 stages)
Leptonema: Longitudinal duality of chromosomes not discernible.
Zygonema: Pairing of homologous chromosomes. Formation of synaptonemal complex and zygotene DNA synthesis.
Pachynema: Pairing persists: synaptonemal complex + crossovers. Bivalent = 2 homologous chromosomes = 2 sets of 2 chromatids. Crossing over occurs (chiasma; chiasmata).
Diplonema: Synaptonemal complex dissolves; visualize longitudinal duality.
Diakinesis: Continued bivalent contraction. 

17. Meiosis 1
Metaphase I:
Bivalents  appear on the Metaphase plate. The random alignment of non-homologous chromosomes is the basis of independent assortment.
Anaphase I:
The physical separation of homologous chromosomes is the basis of segregation.
Telophase I.
Each pole receives one-half of the original chromosome number of the meiocyte, i.e. one set of chromosomes in the case of diploidy.

18. Meiosis 2 Prophase II. Chromatin condensation
Metaphase II. Chromosomes align on Metaphase plate
Anaphase II. Sister chromatids go to opposite poles
Telophase II. Cytokinesis: Tetrad

19. Key differences between mitosis and meiosis
Meiosis: Homologous chromosomes pair and separate at Anaphase I; sister chromatids separate at Anaphase II
Mitosis: No pairing of homologous chromosomes
Meiosis: Crossing over may generate new configurations at alleles at linked loci
Mitosis: A special case
Meiosis: 4 daughter cells, which may be genetically different
Mitosis: 2 identical daughter cells
Meiosis 2n to n
Mitosis is 2n to 2n 

20. Key differences between mitosis and meiosis
Meiosis: occurs in specialized cells (megaspore mother cell (MMC); pollen mother cell (PMC)
Mitosis: occurs from the zygote stage onward through the life of the organism
V v V V
V v
V v v v
V v

21. Linkage
The association of two or more phenotypic characters in inheritance because the genes controlling these characters are located in the same chromosome
The closer the gene locations, the stronger the association
Genes carried by the same chromosome are members of the same linkage group
The number of possible linkage groups (assuming sufficient marker loci and no major blocks of monomorphism) corresponds, therefore, to the “n” number of chromosomes in the organism in question
Plant
2n = _X = _
Arabidposis thaliana
2n = 2x = 10
Fragaria vesca
2n = 2x = 14
Theobroma cacao
2n = 2x = 20
Zea mays

22. Linkage
Complete: Pairs of loci are so close together that crossing over rarely occurs and recombinant types are generally not recovered
Partial: Pairs of loci are sufficiently far apart that some recombinant types are recovered

23. Linkage
Crossing over refers to the physical exchange between homologous chromosomes
Recombination is the genetic result of crossing over and is detected by new combinations of alleles at two or more loci
Genetic variability
New combinations of alleles at linked loci
Intra-genic recombination

24. Meiosis
Prophase I
Leptonema:
Zygonema: Pairing of homologous chromosomes. Formation of synaptonemal complex and zygotene DNA synthesis Pachynema: Pairing persists: synaptonemal complex + crossovers. Bivalent = 2 homologous chromosomes = 2 sets of 2 chromatids. Crossing over occurs (chiasma; chiasmata) Diplonema:
Diakinesis:

25. Linkage
Terms describing the allelic condition at linked loci cis aka coupling
trans aka repulsion
A……..B
a……..b
A……..b
a……..B

26. Keys points in non-sister chromatid exchange:
Linkage
Keys points in non-sister chromatid exchange:
Crossing over does not involve loss or addition of chromatin
Only two chromatids are involved in any single crossover event
There may be multiple crossovers between non-sister chromatids
Any combination of crossover configurations can occur, and the outcome of such configurations can be radically different

27. Linkage Types of Crossovers Single crossover 2-strand double crossover

28. Linkage Factors affecting meiotic crossing over Sex chromosomes
Position on chromosome

29. Linkage OWB Dominant 2-row; normal starch; hulled grain; long awns
VV WW NNLL V N L W
Locus abbreviations and alleles
Vrs1 = V: V, v
GBSS = W: W,w
Nud = N: N,n
LKs2 = L: L,l
OWB Recessive
6-row; waxy starch; naked grain; short awns
vv ww nnlll v n l w

30. Linkage Alleles at all loci: segregation
V on a different chromosome than
W, N, and L: therefore alleles at
V show independent
assortment with those at W, N, and L
W, N, L on same chromosome:
Therefore potential for linkage
W “far enough” from N and L
to show no linkage
(independent assortment)
N and L close enough to show
linkage N L W n l w V v
OWB Dominant
2-row; normal starch; hulled grain; long awns
VV WW NNLL
OWB Recessive
6-row; waxy starch; naked grain; short awns
vv ww nnlll

31. Independent assortment and recombination could lead to an entirely new combination of traits: 2-row; waxy starch; hulled grain; short awn
OWB Dominant
2-row; normal starch; hulled grain; long awns
VVWWNNLL V x N L W v n l w
OWB Recessive
6-row; waxy starch; naked grain; short awns
vvwwnnll v V F1 n l N L w W

32. Meiosis 1 & Crossing Over – Cell 1w v W V N L
Pairing and
crossing over n l w v W V N L
Anaphase I v V v v V V
Pre-meiotic
Heterozygous parent
After
DNA replication w W w w W W n N n n N N l L l l L L

33. Meiosis 2 & Crossing Over – Cell 1
Anaphase II N L w v W V V W n l v w n l N L W V
Non-Recombinant
2row, non-waxy, hulled, long awn N L w v
Recombinant
6row, waxy, hulled, long awn n l W V
Recombinant
2row, non-waxy, naked, short awn n l w v
Non-Recombinant
6row, waxy, naked, short awn

34. Meiosis 1 & Crossing Over – Cell 2w W N L v V
Pairing and
crossing over n l w W N L v V
Anaphase I
Pre-meiotic
Heterozygous parent
After
DNA replication w W w w W W n N n n N N l L l l L L

35. Doubled Haploid Phenotypes
2row, non-waxy, hulled, long awn N L W V
Non-Recombinant N L W V
6row, waxy, hulled, long awn N L w v
Recombinant N L w v
2row, non-waxy, naked, short awn n l W V
Recombinant n l W V
6row, waxy, naked, short awn n l w v
Non-Recombinant n l w v
2row, waxy, naked, short awn n l w V
Non-Recombinant n l w V
6row, non-waxy, naked, short awn n l W v
Recombinant n l W v
2row, waxy, hulled, short awn N L w V
Recombinant N L w V
6row, non-waxy, hulled, long awn
Non-Recombinant N L W v N L W v

36. Dihybrid Ratio for Doubled Haploids (2/4 loci)
Gametes VW Vw vW vw DH
VVWW
VVww
vvWW
vvww
Class
Non-recombinant
Recombinant
Non-Recombinant 1
0.25
Note: The “recombinant “ types in this case (between the V and W loci) are not due to
crossovers. They are due to random alignment of paired homologous chromosomes at
Metaphase I

37. Linkage Test – V and W
Gametes VW Vw vW vw DH
VVWW
VVww
vvWW
vvww
Non-recombinant
Recombinant
Non-Recombinant
0.25
Expected
20.5
Observed 20 15 25 22
(O-E)2/E
0.012
1.476
0.988
0.110
2[3] = 2.586; P=0.460; Null hypothesis of no linkage accepted
Independent Assortment : Loci are on different chromosomes
Random alignment of chromosomes at Metaphase 1

38. Linkage Test – W and N
Gametes WN Wn wN DH
WWNN
WWnn
wwNN
wwnn
Non-recombinant
Recombinant
Non-Recombinant
0.25
Expected
20.5
Observed 27 18 13 24
(O-E)2/E
2.061
0.305
2.744
0.598
2[3] = 5.707; P=0.128; Null hypothesis of no linkage accepted
Independent Assortment:
Sufficient crossovers between loci far enough apart on same chromosome

39. Linkage Test – W and L
Gametes WL Wl wL wl DH
WWLL
WWll
wwLL
wwll
Non-recombinant
Recombinant
Non-Recombinant
0.25
Expected
20.5
Observed 26 19 20 17
(O-E)2/E
1.476
0.110
0.012
0.598
2[3] = 2.195; P=0.533; Null hypothesis of no linkage accepted
Independent Assortment:
Sufficient crossovers between loci far enough apart on same chromosome

40. Linkage - Test N and L
Gametes NL Nl nL nl DH
NNLL
NNll
nnLL
nnll
Non-recombinant
Recombinant
Non-Recombinant
0.25
Expected
20.5
Observed 37 3 9 33
(O-E)2/E
13.280
14.939
6.451
7.622
2[3] = ; P=<0.001; Null hypothesis of no linkage rejected
Not Independent Assortment = linkage
Few crossovers between loci close together on same chromosome

41. Recombination percentageDH
NNLL
NNll
nnLL
nnll
Non-recombinant
Recombinant
Non-Recombinant
Observed 37 3 9 33
Parentals (Non-recombinants) >> Recombinants = linkage
Linkage is calculated as the total recombinant types/ total population size
(9+3)/( ) = 12/82 = = 14.6%
“The association of two or more phenotypic characters in inheritance because the genes controlling these characters are located in the same chromosome”
N L
14.6

42. N L
14.6

43. Recombination, double crossovers and map distances
% recombination values are biased due to double crossovers
Double crossovers can give parental combinations of alleles and therefore underestimate the % recombination at values of ~ 10% recombination and higher
At values of ~ 10% recombination and lower double crossovers occur less often than expected
% recombination values are not additive: the maximum of recombination is 50%
Therefore the centiMorgan (cM) – a computed value derived from the observed % recombination

44. CentiMorgans (cM) Distance (cM) Haldane =-0.5*LN(1-2*r)
Distance (cM) Kosambi=0.25*LN((1+2*r)/(1-2*r))
The centiMorgan is a unit of distance without a physical basis: the ratio of cM to base pairs can vary enormously across the genome

45. CentiMorgans (cM) Distance (cM) Haldane =-0.5*LN(1-2*r)
Distance (cM) Kosambi=0.25*LN((1+2*r)/(1-2*r))

46. Three Point Linkage Test
rWN=0.39
rNL=0.14 N L W
rWL=0.48
The recombination frequency over the interval W – L (rWL) is greater than either rWN or rNL therefore W and L are the two loci furthest apart and N is likely to lie between them. N is likely to be closer to L as rWN > rNL.

47. Making Linkage Maps
Linkage analysis is now an "automated" procedure: data on thousands, tens of thousands of loci
Key issues in linkage map construction are locus order and distance
The likelihood odds (LOD) score is a test statistic that is used to test the hypothesis that there is no linkage
LOD of 3.00 is approximately equal to P ~ .001
LOD > 3 then one concludes that two loci are indeed linked.

48. Value of Genetic Linkage Maps within-species
Determine order of loci in the chromosome
Determine how likely it is that non-parental combinations of alleles at linked loci will be obtained
Determine if trait relationships are due to linkage or pleiotropy
Create complete linkage maps (average ~ 100 cM/chromosome) where # chromosomes (n level) = number of linkage groups
Build platforms for
Cloning genes
Anchoring whole genome sequences
Identifying genes controlling quantitative traits

49. Value of linkage maps within-species Graphical Genotypes
Key points
Alleles at all loci coded as to parental origin
No heterozygotes in this data set; if there were, they would be coded AB
Crossovers are only detected in regions where that individual is polymorphic; crossovers in monomorphic regions not detectable
Double crossovers between loci closer than 10cM may be due to errors in allele calling – therefore may be replace by “-” indicting missing data

50. Anchoring the genome sequence to the genetic map
Value of linkage maps
Anchoring the genome sequence to the genetic map
94% of scaffolds anchored to the diploid Fragaria reference linkage map using 390 genetic markers
Pseudochromosomes ~ linkage groups

51. DNA (molecular) markers Linkage mapping, quantitative trait locus (QTL) mapping, anchoring genome sequences

52. Applications of Markers
Are trait associations due to linkage or pleiotropy?
Identify markers that can be used in marker assisted selection
Locate genes for qualitative and quantitative traits
A starting point for map-based cloning strategies

53. Markers are based on polymorphisms
Amplified fragment length polymorphism
Restriction fragment length polymorphism
Single nucleotide polymorphism
The polymorphisms become the alleles at marker loci
The marker locus is not necessarily a gene: the polymorphism may be in the dark matter, in a UTR, in an intron, or in an exon
Non-coding regions may be more polymorphic
Molecular markers are abundant

54. Marker polymorphisms are based on
mutations
Silent
*** CTG GGA GAT TAT GGC TTT AAG***
*** CTG GGA GAT TAT GGC TTC AAG*** alignment
Leu Gly Asp Tyr Gly Phe Lys
Leu Gly Asp Tyr Gly Phe Lys translation
Missense
*** CTG GGA GAT TAT GGC TAT AAG*** alignment
Leu Gly Asp Tyr Gly Tyr Lys translation
Nonsense
*** CTG GGA GAT TAG GGC TTT AAG*** alignment
Leu Gly Asp Tyr Gly Phe Lys
Leu Gly Asp STOP translation

55. Markers
Polymorphisms can be visualized at the metabolome, proteome, or transcriptome level but for a number of reasons (both technical and biological) DNA-level polymorphisms are currently the most targeted
Regardless of whether it is a “perfect” or a “linked” DNA marker, there are two key considerations that need to be addressed in order for the researcher/user to visualize the underlying genetic polymorphism

56. DNA Markers
Finding and understanding the genetic basis of the DNA-level polymorphism, which may be as small as a single nucleotide polymorphism (SNP) or as large as an insertion/deletion (INDEL) of thousands of nucleotides
Detecting the polymorphism via a specific assay or "platform". The same DNA polymorphism may be amenable to different detection assays

57. Marker examples: Simple Sequence Repeats (SSRs)
Simple sequence repeats (SSRs) (aka microsatellites) are tandemly repeated mono-, di-, tri-, tetra-, penta-, and hexa-nucleotide motifs
SSR length polymorphisms are caused by differences in the number of repeats
Assayed by PCR amplification using pairs of oligonucleotide primers specific to unique sequences flanking the SSR
Multiple platforms

58. Simple Sequence Repeats (SSRs)
Marker examples
Simple Sequence Repeats (SSRs)
Simple sequence repeats in hazelnut:
Note the differences in repeat length AND the consistent flanking sequences
mind42.com

59. Marker examples Simple Sequence Repeats (SSRs)
Highly polymorphic
Highly abundant and randomly dispersed
Co-dominant
Locus-specific
Amenable to high throughput assays

60. SSR Concept
Individual 1 (AC)x9
Individual 2 (AC)x11
51 bp
55 bp

61. Marker examples: Single Nucleotide Polymorphisms (SNPs)
DNA sequence variations that occur when a single nucleotide (A, T, C, or G) in the genome sequence is altered
Alleles
Single
Nucleotide
Polymorphisms
(SNPs)
…..ATGCTCTTACTGCTAGCGC……
…..ATGCTCTTCCTGCTAGCGC……
…..ATGCTCTTACTGCAAGCGC……
Consensus…..ATGCTCTTNCTGCNAGCGC……

62. Marker examples: SNPs Highly abundant (~ 1 every 200 bp)
Locus-specific
Co-dominant and bi-allelic
Basis for high-throughput and massively parallel genotyping technologies
Connectivity to reference genome sequences

63. SNP Detection Strategies
Locus specific systems
Many samples with few markers
Markers for key target characters
Example: KASP
Genome wide systems
Fewer samples with many markers
Germplasm characterization
Genotyping panels for Genome Wide Association Studies Example: Illumina

64. SNPs on KASP and Illumina 9K

65. Why use markers rather than whole genome sequences?
A way of addressing plant genetics and breeding challenges:
The large number of genes per genome
Huge genome sizes
Often a subset of the total genome is of interest

66. available for every plant !!!!!
Abundant markers are
available for every plant !!!!!

67. Applications of Markers
Establish evolutionary relations: homoeology and synteny

68. Marker applications: Construct linkage maps to find
Quantitative Trait Loci (QTLs)
Genetic determinants of quantitative variation
Loci that determine, or are associated with loci that determine,
variation in a phenotype