Principles of Cytogenetics

Teacher

Dr. K. Krishnam Raju

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Core Courses

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Course Name: Principles of Cytogenetics

Code(Credit): MAGP1102 (2-1-0)

Course Objectives

The course provides insight into the structure and number of chromosomes using microscopic analysis. For instance a decrease or an increase in the chromosomal number or translocation of one to another chromosome or even chromosome behavior during mitosis and meiosis. Haploidy and its applications in genetics and plant breeding are studied in detail. Additionally the course provides insight on chromosome mapping approaches in modern genomics, polyploidy and cytogenetic aspects of crop evolution.

Learning Outcomes

Students gain knowledge on

1: Evolution of various chromosomal aberrations (structural and numerical), their applications in alien gene transfer and hybrid seed development.

2: pollen culture in haploid development and development of diploid inbreds or hybrids or doubled isogenic lines from haploids that has got important applications in plant breeding.

3: Cytogenetic tools such as FISH and GISH (Genomic In Situ Hybridization) techniques, that rely on "painted chromosomes" approach, the behavior of individual genomes, individual chromosomes, or chromosomal fragments in natural and artificial hybrids (particularly allopolyploids) can be analyzed.

4: Important application of plant cytogenetics in validation of physical maps and guiding efficient choice of bacterial artificial chromosomes for sequencing of genomes using chromosome walking and chromosome jumping.

Course Syllabus

Theory

UNlT l: Architecture  of  chromosome  in  prokaryotes  and  eukaryotes; Terminology.  Euchromatin  and heterochromatin;  karyotype  and  techniques  for  karyotyping;  Banding  patterns  for  identification  of chromosomes; C  value  paradox-DNA  content  (genome  size)  and  adaptability,  Split  gene;  Special types  of  chromosomes -lamp  brush  chromosomes,  polytene  chromosomes,  B- chromosomes  and  sex chromosomes.

UNIT ll

Mitosis  cell  cycle-significance  of  mitosis;  Meiosis  cell  cycle-significance  of  meiosis,  Difference between  mitosis and meiosis;  Crossing over -mechanisms and theories of crossing over.

UNIT III

Recombination models  and  cytological  basis  of  crossing  over;  Structural  chromosomal  aberrations, Deletions types    of  deletions,  origin    and  occurrence. Meiosis  and  breeding  behaviour  of  deletion heterozygote,  genetics  of  deletions;  Duplications-origin,  types  of  duplications, chromosome pairing  and  crossing    over  at  meiosis  in  duplicate  heterozygotes,  phenotypic  effects  of  duplications; Bridge-breakage-fusion  cycle  in  corn.  Role  of  duplications  in  plant  breeding  and  evolution; lnversions: origin, types of inversions–meiotic pairing in inversions-detection and uses of pericentric and  paracentric  inversions;  Breeding  behaviour  of  inversion  heterozygotes,  Role    of  inversions  in  evolution  and  karyotype;  Breeding  behaviours  of  translocation heterozygote, Permanent  hybrids  in Oenothera, Robertsonian  translocations,  detection  and  uses;  Numerical  chromosomal  aberrations: classification; Euploidy;  Haploidy, Terminology and classification of haploids; Origin,  occurrence and  production  of  haploids  Detection  of  haploids  Phenotypic  effects  of  haploids, Meiosis  and Breeding behaviour of haploids. Use of haploids in plant breeding.

UNIT IV

Polyploidy - autopolyploidy,    Origin    and    types    of    autopolyploids,    Meiotic    behaviour    in autopolyploids - Autotriploids  and  autotetraloids;  Allopolypioidy-segmental  allopolyploidy, Genome analysis of  allopolyploids-Evolution of important polyploid crops Wheat, Tobacco, Brassica and Cotton;  Aneuploidy: hyperploids- trisomics  and  tetrasomics;  primary  trisomics  and  secondary trisomics,  Meiotic  behaviour  in  trisomics  and  uses;  Balanced  tertiary  trisomics  in  hybrid  seed production-Trisomics  in  polyploids;  tetrasomics;  Aneuploidy-hypoploidy; Monosomics  and nullisomics; Method   of   production   of   monosomics; Meiotic   behaviours   of   monosomics; Monosomics  in maize;  Production  of  nullisomics; Meiotic  behaviour  of  nullisomics,  nullisomic analysis,   use   of   nullisomics   in   locating   genes   on   chromosomes;   Aliengene   transfer   through chromosome manipulations-transfer of genome to 4x and 6x wheat; transfer of genome in the genus Arachis.

UNIT V

Transfer  of  individual  whole  chromosome -alien  addition  lines-alien  substitution  lines;  Apomixis-Evolutionary  and  genetic  problems  in  crops  with  apomixis;  Chromosome  painting,  chromosome walking  and  chromosome  jumping;  Artificial  chromosome  construction  and  its  uses;  Reversion  of  autopolyploids to diploids; Genome mapping in polyploids.

Unit VI: Fertilization  barriers  in  crop  plants  at  pre-and  post -fertilization  levels; In  vitro techniques  to overcome  the  fertilization  barriers  in  crops;  chromosome  manipulations  in  wide  hybridization;  case studies-Production and use of haploids, dihaploids and doubled haploids in genetics and breeding.

Practice

S. No

Topics to be covered

1

Learning  the  cytogenetics  laboratory,  various  chemicals    to  be  used  for  fixation,  dehydration, embedding, staining, cleaning etc.

2

Microscopy: various types of microscopes. Observing sections of  specimen  using Electron microscope; Preparing specimen  for observation-Fixative preparation  and fixing  specimen  for  light  microscopy  studies  in  cereals

3

Studies  on  the  course of  mitosis  in  wheat, pearl  millet-Studies  on  the  course  of  mitosis  in  onion  and  Aloe  vera

4

Studies  on  the  course  of meiosis in cereals, millets and pulses-Studies on the course of meiosis in oilseeds and forage crops

5

Using micrometers and studying the pollengrain size in various crops:   Various methods of staining and  preparation  of    temporary  and  permanent  slides. Pollen  germination in vivo and in  vitro

6

Microtomy  and  steps  in  microtomy;  Agents  employed for  the  induction  of  various  ploidy  levels; Solution preparation  and  application  at  seed,  seedling level-Identification  of  polyploids  in  different crops

7

Induction and  identification  of  haploids; Anther  culture  and  Ovule  culture. Morphological observations on synthesized autopolyploids

8

Observations on C-mitosis, learning on the dynamics of spindle  fibre  assembly. Morphological observations  on  alloployploids-Morphological  observations on   an euploids

9

Cytogenetic   analysis of   interspecific   and   intergeneric   crosses-Maintenance   of

10

Polyploidy in ornamental  crops-Fluorescence  in  situ  hybridization  (FISH).

11

Polyploidy in ornamental  crops-Genome  in  situ hybridization (GISH).

Suggested Text Books

  1. Cytogenetics by P.K. Gupta, Rastogi Publ.

  2. Plant Cytogenetics by Ram J. Singh

  3. Cytology and Cytogenetics. Swanson CP. 1960 . Macmillan & Co.

  4. Plant Biotechnology. BD Singh 2006; Klayani Publishers New Delhi

Session Plan (Theory)

Session 1

Course Overview including course objectives and course outcomes. Architecture of chromosomes.

Lecture Notes 1

https://www.youtube.com/watch?v=i9AbkNGh2u8 (Video: prokaryotic Vs Eukaryotic chromosomes)

https://www.youtube.com/watch?v=63Q3No6shvw (video on Cytogenetics in Plants)

Session 2

Terminology:  Euchromatin  and heterochromatin;  karyotype  and  techniques  for  karyotyping. Banding  patterns  for  identification  of chromosomes.

Lecture Notes 2

https://www.youtube.com/watch?v=Tc4j2E9_dCk  (Video: Karyotype and chromosome banding pattern)

https://www.youtube.com/watch?v=Ml9kiaRSKps (heterochromatin Vs Euchromatin)

Session 3

C value  paradox. DNA  content  (genome  size)  and  adaptability, Split gene concept. Special types  of  chromosomes.

Lecture Notes 3

https://www.embibe.com/study/special-type-of-chromosomes-concept (video on special types of chromsomes)

https://www.youtube.com/watch?v=yzumLbQrmvo (Split gene concept)

Session 4

Theory: Cell Cycle; Mitosis  cell  cycle; significance  of  mitosis.

Practice-3:  Studies  on  the  course of  mitosis  in  wheat, pearl  millet-Studies  on  the  course  of  mitosis  in  onion  and  Aloe  vera.

Practice-3 (practical manual)

https://www.youtube.com/watch?v=5-ur7bWqlDQ (Practice-3)

Lecture Notes 4

https://www.youtube.com/watch?v=RNwJbMovnVQ (video on Mitotic cell cycle)

PDF slides on mitosis

Session 5

Theory: Meiosis  cell  cycle; significance  of  meiosis. Difference between Mitosis and Meiosis.

Practice-4: Studies  on  the  course  of meiosis in cereals, millets and pulses-Studies on the course of meiosis in oilseeds and forage crops.

https://www.youtube.com/watch?v=XOAAIeZXoxU

Lecture Notes 5, Practice-4  (practical manual)

https://www.youtube.com/watch?v=16enC385R0w (Meotic cell cycle)

PDF slides on mitosis Vs meiosis

Session 6

Crossing over mechanisms. Theories of crossing over.

Lecture Notes 6

https://www.biologydiscussion.com/genetics/crossing-over/mechanism-of-crossing-over-4-theories-with-diagram/37129 (Link on theories of crossing over)

https://www.youtube.com/watch?v=g_rcR0FSOJ8 (Mechanism of crossing over)

Session 7

Recombination models  and  cytological  basis  of  crossing  over

Lecture Notes 7

https://www.youtube.com/watch?v=5638I3fGuW4 (Holliday model of recombination)

Session 9

lnversions, Breeding  behaviours  of  translocation heterozygote; Permanent  hybrids  in Oenothera.

Lecture Notes 9

PDF slides on structural aberrations

https://www.youtube.com/watch?v=7_p14l8gPhk (Video on Chromosomal Aberrations | Deletion | Duplication | Inversion | Translocation)

Session 10

Theory: Numerical  chromosomal  aberrations: classification; Euploidy;  Haploidy, Terminology and classification of haploids; Origin,  occurrence and  production  of  haploids.

Practice-7: Induction and  identification  of  haploids; Anther  culture  and  Ovule  culture. Morphological observations on synthesized autopolyploids

Lecture notes 10

https://www.youtube.com/watch?v=qri2F510qsU (Video on numerical chromosomal aberrations)

Session 11

Detection  of  haploids  Phenotypic  effects  of  haploids, Meiosis  and Breeding behaviour of haploids. Use of haploids in plant breeding.

Lecture notes 11

https://www.youtube.com/watch?v=zpoAXBu-22o (video on haploids, doubled haploids and its applications)

Session 12

Polyploidy - autopolyploidy,    Origin    and    types    of    autopolyploids,    Meiotic    behaviour    in autopolyploids - Autotriploids  and  autotetraloids.

Lecture notes 12

https://www.youtube.com/watch?v=-LYPwNLLq8A (autopolyploidy vs allopolyploidy)

Session 13

Allopolypioidy-segmental  allopolyploidy, Genome analysis of  allopolyploids; Evolution of important polyploid crops Wheat, Tobacco, Brassica and Cotton.

Lecture notes 13

allopolyploidy in plant breeding (PDF file on allopolyploidy in plant breeding)

Session 14

Aneuploidy: hyperploids- trisomics  and  tetrasomics;  primary  trisomics  and  secondary trisomics,  Meiotic  behaviour  in  trisomics  and  uses. Balanced  tertiary  trisomics  (BTT) in  hybrid  seed production-Trisomics  in  polyploids;  tetrasomics.

Lecture notes 14

https://www.youtube.com/watch?v=hI5megITBm0 (you tube on BTT)

https://www.youtube.com/watch?v=p05JqlWAv20 (Video on trisomics in crop improvement)

Session 15

Aneuploidy-hypoploidy; Monosomics  and nullisomics; Method   of   production   of   monosomics; Meiotic   behaviours   of   monosomics.

Lecture notes 15

https://www.youtube.com/watch?v=w8it4s9dm0M (You tube on Meotic & Breeding bahaviour in Monosomics)

https://www.youtube.com/watch?v=DMO1OTFb0sU ((You tube on method of production of Monosomics)

monosomics (PDF slides)

Session 16

Monosomics  in maize;  Production  of  nullisomics; Meiotic  behaviour  of  nullisomics,  nullisomic analysis,   use   of   nullisomics   in   locating   genes   on   chromosomes. Aliengene   transfer   through chromosome manipulations-transfer of genome to 4x and 6x wheat; transfer of genome in the genus Arachis.

Lecture notes 16

Aneuploids (PDF)

Session 17

Theory: Transfer  of  individual  whole  chromosome -alien  addition  lines-alien  substitution  lines. Apomixis,  Chromosome  painting.

Practice-9: Cytogenetic   analysis of   interspecific   and   intergeneric   crosses-Maintenance   of Polyploidy in ornamental  crops-Fluorescence  in  situ  hybridization  (FISH).

Practice-7 (practical manual)

PDF slides on FISH procedure

Lecture notes 17

https://www.youtube.com/watch?v=4eIs8k0CwIk (you tube on alien addition line)

PDF slidfes on alienadditionline  

PDF slides on aliensubstitutionlines

Session 18

Chromosome walking; Chromosome  jumping and  Artificial  chromosome  construction  and  its  uses.Reversion  of  autopolyploids to diploids; Genome mapping in polyploids.

Lecture notes 18

https://www.youtube.com/watch?v=jSMNXtppNe4. (you tube on chromsome walking)

chromosome walking & jumping (PDF Slides)

Session 19

Fertilization  barriers  in  crop  plants  at  pre-and  post -fertilization  levels; In  vitro techniques  to overcome  the  fertilization  barriers. Chromosome  manipulations  in  wide  hybridization.

Lecture notes 19

Chromosome manipulation in wide hybridiz(PDF Slides)

barriers in widecrosshybridization (PDF slides)

Session 20

Production and use of haploids, dihaploids and doubled haploids in genetics and breeding.

Lecture notes 20

https://www.frontiersin.org/articles/10.3389/fpls.2015.00384/full (A PDF on Doubled haploid production)

https://www.youtube.com/watch?v=ND0QNuDNaRg (Video on Doubled haploid production)

Session 21

Practice-1: Learning  the  cytogenetics  laboratory,  various  chemicals    to  be  used  for  fixation,  dehydration, embedding, staining, cleaning etc.

Practice-1 (Record Manual)

Session 22

Practice-2: Microscopy: various types of microscopes. Observing sections of  specimen  using Electron microscope; Preparing specimen  for observation-Fixative preparation  and fixing  specimen  for  light  microscopy  studies  in  cereals

Practice-2 (practical manual)

https://www.youtube.com/watch?v=CzQAtSXaKVs (Types of microscopes)

Session 23

Practice-5: Using micrometers and studying the pollengrain size in various crops:   Various methods of staining and  preparation  of    temporary  and  permanent  slides. Pollen  germination in vivo and in  vitro.

Practice-5 (practical manual)

https://www.mccrone.com/mm/permanent-slide-preparation/

Session 24

Practice-6: Microtomy  and  steps  in  microtomy;  Agents  employed for  the  induction  of  various  ploidy  levels; Solution preparation  and  application  at  seed,  seedling level-Identification  of  polyploids  in  different crops.

Practice-6 (practical manual)

https://www.youtube.com/watch?v=MWHxq_Xly-A (Video on Microtome)

Session 25

Practice-8: Observations on C-mitosis, learning on the dynamics of spindle  fibre  assembly. Morphological observations  on  alloployploids-Morphological  observations on   an euploids

Session 26

Practice-10: Cytogenetic   analysis of   interspecific   and   intergeneric   crosses-Maintenance   of Polyploidy in ornamental  crops-Genome  in  situ hybridization  (GISH).

Practice-8 (practical manual)

https://www.intechopen.com/books/plant-breeding-from-laboratories-to-fields/genomic-in-situ-hybridization-in-triticeae-a-methodological-approach (PDF on Methodology of GISH)

Case Studies

Case Studies

Our Main Teachers

Dr. K. Krishnam Raju          Educational Qualifications: B.Sc (Ag), M.Sc Ag (GPB), Ph.D (BARC), PDF (USA); Qualified UGC-JRF (NET);    Awards: DST young scientist Award (2012); Extramural Research Grants: Two;      Experience: 13 years RESEARCH PUBLICATION:  Raju, K. K., S. Gautam, and A. Sharma (2006) Molecules involved in the modulation of rapid cell death in […]