This presentation very helpful for the students and scientist who engaged in plant cytogenetics research

1. Cytogenetic stocks in
plant breeding
MANJEET
2017A24D
Ph.D. (Genetics and Plant Breeding)
Email: manjeetsingh125033@gmail.com

2. Introduction
• Wild species are a rich reservoir of useful alien
genes that are not available in the cultivated gene
pool
• Transfer of alien genes into crop plants from wild
and exotic plant genetic resources has invoked
tremendous interest of crop scientists worldwide
• These include resistance to diseases and insect-
pests, tolerance to drought, salinity, temperature
extremities and other abiotic stresses as well as
genes for several quality traits

3. CONT…
• It is very difficult to directly transfer favorable traits
from wild relatives (tertiary gene pool) into cultivated
species (primary gene pool) through conventional
gene recombination due to the absence of pairing and
crossover between chromosomes of species of
primary and tertiary gene pool.
• To enhance the transfer of favorable genes from wild
species into cultivated species, there is need for
development of cytogenetic stocks

4. Cytogenetic stock
• A genotype that carries any kind of
reproducible and maintenable chromosomal
aberration that differentiates it from a normal,
wild type genotype, e.g.
i. Alien addition line
ii. Alien substitution line
iii. Aneuploids
iv. Amphiploids
v. Translocated line

5. Classification of Aneuploids
Types Formula Somatic chromosome
complement
Normal disomic 2n MM NN HH
Monosomic 2n-1 MM NN H-
Double monosomic 2n-1-1 MM N- H-
Nullisomic 2n-2 MM NN --
Double nullisomic 2n-2-2 MM -- --
Trisomic 2n+1 MM NN HHH
Double Trisomic 2n+1+1 MM NNN HHH
Tetrasomic 2n+2 MM NN HHHH
Pentasomic 2n+3 MM NN HHHHH
*For simplicity, only 3 non-homologous chromosomes are depicted here

6. Origin of aneuploids
 Aneuploids originates from aneuploid gametes.
The main modes of origin of aneuploids are
I. During mitosis or meiosis abnormalities, e.g., lagging chromosomes
results in the formation of nuclei with hypoploid chromosome numbers
II. Hypo- and hyper-ploid nuclei may be formed due to
chromosome/chromatid nondisjunction during mitotic or meiotic cell
division, such gametes would, on union with normal (n) gametes, give
rise to aneuploid progeny
III. In the polyploids, especially those with an odd number of chromosome
sets, e.g., triploid, pentaploid etc., univalents are often observed during
meiosis. The anaphase distribution of the univalents is irregular as a
result of which, aneuploid gametes are produced

7. Different types of monosomics and their chromosome
constitution (based on bread wheat; 2n = 6x = 42)
Types of Aneuploid Chromosome constitution Somatic chromosome
number
Primary monosomic 20II + 1I 41
Secondary monosomic
(Mono-isomic)
20II + iI 41
Mono-isodisomic 20II + 1I + iI 42
Monotelosomic 20II+ tI 41
Monotelodisomic 20II + 1I + tI 42
Di-telosomic 20II + tII 42
Di-isosomic 20II + iII 42
Mono-telo-mono-isosomic 20II + tI + iI 42
Tertiary monosomic 19II + 1I + 1I + tertiary 41
*II = Bivalent; I = Univalent; t = telocentric chromosome; i = iso-chromosome

8. A
B
C
D
E
A
B
C
D
E
S
L
A
B
C
D
E
A
B
C
D
E
LL
SS
A
B
A
B
A
B
A
B
C
D
E
C
D
E
C
D
E
C
D
E
A B B A
E D C C D E
S S
L L
LL
SS
Isochromosomes
Telocentrics

9. Source of aneuploids
I. Asynaptic and desynaptic mutants
II. Polyploids and triploids
III. Translocation heterozygotes
IV. Trisomics and monosomics
V. Interspecific and intergeneric crosses
VI. Mutagen treated populations

10. Utilization of cytogenetic stocks

11. CONT…
• One of the major application of cytogenetic stocks in plant
breeding is our ability to transfer alien genes to cultivated
species through wide hybridization and chromosomal
manipulation
 These transfers are possible :-
(i) At the level of whole genome for the production of amphiploid (as
done for the production of triticale)
(ii) At the level of individual whole chromosome for the production of
individual alien chromosome addition and substitution lines (when
amphiploid did not give any desirable synthetic new crop)
(iii) At the level of a chromosome segment as done in the production of
terminal and intercalary translocations (to break undesirable part of
alien chromosome)

12. At the level of whole genome

17. Synthetic wheat derived cultivars that have been
released Worldwide
YEARS VARIETY NAME COUNTRY PEDIGREE
2017 Shumai 830 China SHW-L1/Chuannong 16//Pm99915-
1/3/Chuannong 24
2017 Shumai 580 China SHW-L1/Chuanyu 17//Chuanyu
18/3/Chuanmai 107
2017 Talaei Iran Pastor//Site/MO/3/Chen/Ae.
squarrosa (TAUS)//BCN/4/WBLL1
2016 Wane (ETBW 6130) Ethiopia Sokoll/Excalibur
2016 HPBW 01 India T. dicoccon CI9309/Ae. squarrosa
(409)//Mutus/3/2
2016 PBW 677 India Pfau/Milan/5/Chen/Ae.
squarrosa//BCN/3/VEE#7/BOW/4/P
astor
2016 WB2 India T. dicoccon CI9309/Ae. squarrosa
(409)//Mutus/3/2*Mutus
2016 Kenya Hornbill Kenya Pastor//HXL7573/2*BAU/3/Sokoll/
WBLL1
2016 Borlaug 2016 Pakistan Sokoll/3/Pastor//HXL7573/2*BAU
2015 WH 1142 India Chen/Ae. squarrosa
(TAUS)//FCT/3/2*Weaver

18. At the level of individual whole
chromosome
Alien Addition Line
• A line containing an unaltered chromosome
complement of one species (generally a
cultigen) plus a single chromosome
(monosomic alien addition lines, MAALs)
• or a pair of chromosomes (disomic alien
addition lines, DAALs) from an alien species

19. How to produce Alien addition line
• The procedure of addition involves crossing the
higher chromosome number species as pistillate
parent with the lower chromosome number pollen
donor.
• The F1 is generally sterile but by doubling their
number (with colchicine) may result in a fertile
amphiploid.
• Upon the recurrent back crossing of the
amphiploid with the recipient parent, monosomy
results for the donor’s chromosomes

20. Cont…
• After repeated backcrossing in large populations,
one may obtain plants with single monosomes for
all chromosomes of the donor. These are called
single monosomic addition lines.
• Disomic additions are obtained by selfing such
monosomics. These carry an extra pair of
chromosomes
• The purpose of addition is that occasionally the
added chromosome, containing agronomically
useful genes, may get substituted for its
homoeolog and lead to a substitution line

21. Method for obtaining monosomic and disomic
addition lines of wheat (2n = 6x = 42)–rye (2n = 2x =
14).

22. Alien Substitution line
• Alien Substitution takes place when
chromosome(s) of another species replace the
own chromosome(s) of a species
• Alien substitutions may be obtained from alien
addition lines
• However, monosomic lines are used most
commonly

23. Methods of production of substitution line in wheat
using monotelocentric stocks
(20IIR+tI) (21IID)

24. Alien gene(s) introgression at the level of a
chromosome segment
 In transferring alien genes through whole chromosome
substitutions, it has been observed that the transfer of
desirable genes is accompanied with the transfer of
undesirable genes. So, it is desirable to transfer only a
part (segment) of alien chromosome, this can be achieved
by spontaneous or induced translocation.
Interchanges (translocation) can be achieved by
(i.) Irradiation
(ii.) Using homoeologous recombination (as in
wheat)

25. Cont…
1. Irradiation :- produce random interchange between non-
homoeologous chromosome. e.g., segment from Ae.
umbellulata chromosome (6U) carrying resistance to
wheat leaf rust (Lr9), to chromosome arm 6BL of wheat
(Sear, 1972)
2. Homoeologous recombination :- produce interchange
between homoeologous chromosome segments
 Homoeologous Recombination in wheat is facilitated by
i. Removal of 5B chromosome (nulli-5B+tetra5D)
ii. Suppression of 5B effects by the genome of Ae.
speltoides or Ae. mutica
iii. Utilization of recessive mutant of the Ph1 locus on 5B

26. Identification of cytogenetic stocks
1. Morphology of plant
2. Karyotype of the added chromosomes
3. Meiotic chromosome pairing in F1 plants
4. Isozyme analysis
5. Molecular markers

27. Cytological Identification
1. Chromosome morphology
I. Total length
II. Relative length
III. Arm ratio and centromeric index
2. Cytological chromosome markers
I. Heterochromatic and euchromatic distribution
II. Heterochromatic knobs
III. Kinetochore position
IV. Presence of satellite
V. Secondary constriction
VI. Patterns and distribution of chromomeres

28. Cytological techniques for identification of
cytogenetic stocks
1. Chromosome banding techniques
I. G - banding
II. R - banding
III. Q - banding
IV. C - banding
2. Molecular cytogenetic techniques
I. ISH (in-situ hybridization technique)
II. FISH (Fluorescent in-situ hybridization techniques
III. GISH (Genomic in-situ hybridization techniques

29. Utilization of cytogenetic stocks
Gene/allele Location Source Reference
Genes encoding resistance for powdery mildew
Pm21 6VS-6AL Haynaldia villosa Xie et al. (2012)
PmT7A.2 7A T. boeoticum Chhuneja et al. (2012)
PmG25 5BL T. turgidum Alam et al. (2013)
Pm35 5DL Ae. tauschii Miranda et al. (2007)
Pm36 5BL T. dicoccoides Blanco et al. (2008)
Genes encoding resistance for stem rust
Sr44 7D Th. intermedium Liu et al. (2013)
Sr51 3AL Ae. searsii Liu et al. (2011)
Sr 54 2B Ae. speltoides Ghazvini et al. (2013)
Genes encoding resistance for leaf rust
LrAC 5DS Ae. caudate Riar et al. (2012)
Lr58 2BL Ae. triuncialis Kuraparthy et al. (2007)
Genes encoding resistance to stripe rust
Yr53 2BL T. durum Xu et al. (2013)
Yr50 4BL Th. intermedium Liu et al. (2013)
Yr42 6AL (6L.6S) Ae. neglecta Marais et al. (2009)

30. Case study 1

31. Introduction
• Gossypium australe is a diploid wild cotton species (2n = 26,
GG) native to Australia that possesses valuable characteristics
unavailable in the cultivated cotton gene pool, such as delayed
pigment gland morphogenesis in the seed and resistances to
pests and diseases
• However, it is very difficult to directly transfer favorable traits
into cultivated cotton through conventional gene
recombination due to the absence of pairing and crossover
between chromosomes of G. australe and Gossypium hirsutum
(2n = 52, AADD)
• To enhance the transfer of favorable genes from wild species
into cultivated cotton, they developed set of hirsutum–australe
monosomic alien chromosome addition lines

32. Scheme for the development of alien chromosome addition
lines of G. australe in G. hirsutum

33. Identification of complete set of MAALs
Whole plant traits of G. hirsutum acc. TM-1 (A), G. australe (B), and
MAAL-11G (C)
A B
C

34. leaf shapes and fiber traits of MAALs of G. australe individual
chromosomes in G. hirsutum

35. Flower traits are shown for MAALs of G. australe individual
chromosomes in G. hirsutum

36. Genomic in situ hybridization (GISH) of the alien chromosomes
of G. australe in the G. hirsutum background
Mitotic chromosome spread of the (a) 52 chromosomes of G. hirsutum (b) 26 chromosomes of G. australe (c) 52 G. hirsutum
(blue) chromosomes and individual chromosomes 3G (d) 6G (e) 10G of G. australe (Red). (f) Mitotic chromosome spread
showing the 52 G. hirsutum (blue) chromosomes and two 2G, 10G (g) two 10G, 13G (h) three (7G, 8G, 10G) chromosomes of G.
australe (Red).
(i) Meiotic chromosome spread showing 26 bivalents of G. hirsutum (j) 26 bivalents of G. hirsutum plus 1 univalent of
chromosomes 5G (k) 6G (l) 10G from G. australe (white arrowhead)

37. Case study 2

38. Introduction
• Yellow rust is one of the most imortant foliar disease
of wheat worldwide. Epidemic of stripe rust can
reduce the wheat yield up to 75%.
• Breeding resistant cultivars offers the most
economical method of disease control
• Dasypyrum breviaristatum harbour novel and
agronomically important genes for resistance against
multifungal disease including yellow rust

39. Material and methods
1. Plant material
(i) D. breviaristatum accession PI 546317
(ii) T. turgidum cv. Jorc-69
(iii) wheat cultivar MY 11
2. FISH analysis
(i) probe pAs1 = for D genome identification
(ii) probe pHvG38 = for B genome identification
(iii) probe pDb12H = for Vb genome identification
3. Molecular marker analysis
PLUG primers were used to distinguish all the crosses and
progenies
4. Disease resistance screening
The genotypes were evaluated against stripe rust strains CRY
30, CRY 32 & 33 during 2011-2013

40. Scheme used for generated the substitution line
T. turgidum
2n = 4x = 28
(AABB)
× D. breviaristatum
2n = 2x = 14
(VbVb)
F1 (ABVb)
Chromosome doubling by colchicine treatment
TDH-2
2n = 6x = 42
(AABBVbVb)
Synthetic
amphiploid
×
MY 11
2n = 6x = 42
(AABBDD)
F1 (AABBDVb)×MY 11BC1
D 11-5 (Disomic substitution line)
(2n = 6x =42 -2D+2Vb)
Continous selfing
BC1F4

41. FISH analysis for chromosome
characterization
(a.) Hybridization with the pDb12H probe indicated a pair of Dasypyrum V b -genome chromosomes in D11-5
(b.) C-banding of D11-5 shows 14 large chromosomes with strong C-bands that represent wheat chromosomes 1B-7B. D.
breviaristatum chromosomes of line D11-5 showed no characteristic C-bands compared with the C-banding pattern of
wheat chromosome.
(c.) probes pAs1(red) detecting D-genome chromosomes and pSc 119.2(green) identifying B-genome chromosome
(d.) probe pHvG38 was also hybridized to the spread chromosomes of D11-5 and confirmed that all B-genome
chromosomes existed in D11-5, while the D. breviaristatum chromosomes have no significant hybridization signal

42. Molecular marker based detection
PCR using PLUG primers (a.) TNAC1182 and (b.) TNAC1142. The arrows indicate the D. breviaristatum-
specific bands, asterisks show the chromosome 2D-specific band which was absent in D11-5. N2AT2D,
N2BT2D and N2DT2B represent the lines nullisomic-2A tetrasomic-2D, nullisomic-2B tetrasomic-2D and
nullisomic-2D tetrasomic-2B of CS, respectively

43. Results
1. Plant morphology
 D11-5 plants were 80-95 cm in height, produced 5–8 spikes per plant, and
had higher tilling ability than the recipient parent MY11 (3-5 spikes)
 D11-5 plants had 24-26 spikelets per spike, closely resembling TDH-2
 The spike length of D11-5 was 12-13 cm, which was larger as compared to
the wheat parents (9-10cm)
 The grain weight and size of D11-5 was clearly higher than those of its
parents
2. Disease response
 D. breviaristatum partial amphiploid TDH-2 was immune to yellow rust
whereas the wheat parent MY11 was highly susceptible
 Similarly, D11-5 was highly resistant to stripe rust
 These results indicated that the stripe rust resistance in D11-5 was inherited
from the TDH-2 parent and originates from D. breviaristatum

44. Results
(A.) The spikes, seed morphology (B.) Leaf response to stripe rust
A
B

45. Case study 3

46. material and methods
 Plant material
The wheat-D. breviaristatum 2Vb deletion lines selected
from M3 generation of D 11-5 with seeds treated with
gamma rays
 FISH :- Synthetic oligonucleotide probes
i. Oligo-pSc119.2
ii. Oligo-pTa535
iii. Oligo-(CAA)7
 Agronomic traits and stripe rust response observation :-
All lines were grown during the 2015-16 and 2016-17 cropping
seasons with 3 replications at the Xindu experimental station,
China . The stripe rust susceptible cultivar Taichung 29 was
used as an inoculum spreader to ensure uniform disease
development throughout the field

47. FISH of wheat- D. breviaristatum deletion lines
(a) D2230, (b) D2181, (c) D2326 and (d) D2237. Arrows indicate the D.
breviaristatum chromosomes.
a b
c
d

48. Result
(A) Spike length and (B) stripe rust response of 2Vb lines. Lines D11-5 (2Vb ), D2230
(deletion 2Vb -1), and D2326 (deletion 2Vb -3) are highly resistant to stripe rust, while D2181
(deletion 2Vb -2) and D2237 (deletion 2Vb -4) are highly susceptible to stripe rust

49. Result
• Lines D2230, D2181, D2326, D2237 and D11-5 were challenged
with CYR 32,CYR 33, and CYR 34 races of stripe rust in the
field
• Lines D11-5, D2230, and D2326 with 2Vb , 2Vb-1, and 2Vb-3
deletion chromosomes, respectively, were highly resistant
• Lines D2181 and D2237 with deletion chromosomes 2Vb-2 and
2Vb-4, respectively, were highly susceptible
• This indicates that the D. breviaristatum 2Vb L chromosome arm
carries a gene(s) for stripe rust resistance on the L3 to L4
segment (about FL 0.40–1.00)
• The gene(s) influencing spike length on chromosome 2Vb
were located on the short arm (2Vb S)

50. Case study 4

51. Material and methods
• B. juncea-B. fruticulosa introgression set was developed by first
hybridizing wild crucifer B. fruticulosa (2n =16) with B. rapa (2n = 20).
The synthetic amphiploid (FFAA; 2n = 36)
• This synthetic amphiploid was subsequently backcrossed twice with B.
juncea (RLC-1) and advanced by following single seed descent method to
develop a BC2F5 populations.
• Among the 206 BC2F5 ILs, a set of 93 ILs (BC2F6) were selected on the
basis of high pollen grain fertility, euploid chromosome number, normal
meiosis and disease response and these lines were evaluated for their
resistance response against S. sclerotiorum isolate PAU-4
• High humidity was maintained by using adequately placed foggers

52. Resistance expression of introgression lines against
Sclerotinia sclerotiorum
(A) Highly susceptible (stem lesion length >10.0 cm)
(B) Susceptible (stem lesion length ranging from 7.5 to 10.0 cm)
(C) Moderately resistant (stem lesion length ranging from 5.0 to <7.5 cm)
(D) Resistant (stem lesion length ranging from 2.5 to <5.0 cm)
(E) & (F) Hypersensitive resistance response (stem lesion lengths <2.5 cm)
Introgression lines were grouped into five resistance categories

53. Genomic in situ hybridization on mitotic spreads of
introgression lines (ILs) of B. juncea (AABB)
 A genome is painted in blue while
B genome is painted in red (AABB)
 B. fruticulosa introgressions are
shown in green colour
a) B. juncea with no introgression
b) IL with two fruticulosa segment
substitutions in the A-genome
c) IL with three fruticulosa segment
substitutions in the B-genome
d) IL with three fruticulosa segment
substitutions in the B-genome and
one in A-genome
e) IL with five fruticulosa segment
substitutions in the B-genome
f) IL with six fruticulosa segment
substitutions in the A-genome

54. Results and conclusion
 Among the 93 Introgression lines evaluated, 13 genotypes
showed a highly resistant response with a mean lesion length
less than 2.5 cm
 Dunnet test established them to be significantly superior than
the susceptible recipient (B. juncea L. cv. RLC 1)
 This study indicates that many small segments of B.
fruticulosa chromosomes together contributes to resistance
against sclerotinia stem rot in the background genome of B.
juncea
 This study opens the way for novel engineering of new B.
juncea varieties that express resistance for better management
of this worldwide devastating pathogen of rapeseed-mustard
crops

55. Conclusion
 Cytogenetics has its own niche and complements in
molecular genetics analysis
 Considerable progress has been made in alien gene transfer
into crops with help from cytogenetic techniques in almost
every step
 The role of cytogenetics in identifying new disease
resistance sources, developing resistant germplasm and
breeding for durable resistance to different diseases in
cereals, especially in wheat, cannot be replaced by any other
technique
 Genetic engineering offers opportunities for the future, the
problems of gene identification, gene cloning, and social
acceptance of engineered derivatives are still to be solved