MENDEL; 150 years on

MAHESH R HAMPANNAVAR
PG14AGR6062
Dept. of Genetics and plant breeding3/7/2016 1MAHESH R HAMPANNAVAR

3/7/2016 2MAHESH R HAMPANNAVAR

MENDEL, 150 years on
MENDEL (1865) to MAHESH (2015)

Content
Mendel experiment
Genetics and genomics of Pea
Characterization of Mendel’s seven genes

1
5
4
3
2
Removed stamens
from purple flowerTransferred sperm-
bearing pollen from
stamens of white
flower to egg-
bearing carpel of
purple flower
Parental
generation
(P)
Pollinated carpel
matured into pod
Carpel
(female)
Stamens
(male)
Planted seeds
from pod
Examined
offspring:
all purple
flowers
First
generation
offspring
(F1)
Selection of material for
experiment
Collection of material

1. Length of stem
2. Color of stem
3. Size of leaves
4. Form of leaves
5. Position of flower
6. Color of flower
7. Size of flower
8. Length of flower
stalk
9. Color of pods
10.Form of pods
11.Size of pods
12.Form of seeds
13.Size of seeds
14.Color of seed coat
15.Color of
albumin(cotyledon)
Pisum sativum
Phaseolus nanus,
Pisum saccharatum

Dominant traits Recessive traits Ratios of dominant
and recessive in F2
generation
No. of plant
observe
Round seed Wrinkle seed 2.98:1 5474:1850
Yellow seed Green seed 3.01:1 6022:2001
Inflated seed Constricted pod 2.95:1 822:299
Green pod Yellow pod 2.82:1 428:152
Purple flower White flowers 3.14:1 705:224
Flower on stem Flower at tip 3.14:1 651:207
Tall stem Dwarf stem 2.84:1 787:277
Average ratio of all
traits
3:1
Ratios of Dominant to Recessive in Mendel’s plants

• ‘‘Versuche’’ : cited 15 times 1865-1899
• Most unfortunately, his scientific records were
apparently burned around the time of his
death (Olby, 1985; Orel, 1996).

G
E
N
E
T I C
S

Pea genetics and genomics
• The standard Pea comprises seven SEVEN
linkage group
• Different types of polymorphism in pea
genomes

• latest consensus map published in pea (2011)
which includes
– Functional markers(214),
– SSR(180),
– RAPD(133)
– Morphological markers(3).

• Completed and annotated genomes of five
model legumes like –
• Comparative genomics is potential tool

• International Pea Genome Sequencing
Consortium
• John Innes pisum collection – JI5 and five
other related are referred as mummy plant
have fascinated character

Seed shape
•Bhattacharyya et al., (1990)
•Controlled by R locus (rugous)
•Linkage group V
•JI15(RR) JI1194(rr)

Compound Round seed Wrinkle seed
Starch (% dwt) 45-49 33-36
Amylose (% total starch) 33 71
Sucrose (% dwt) 5-7 9-12
Lipid (% dwt) 2.4 4.2
Legumin (% protien) 33 36
Composition of mature round and wrinkle seed
•Effect of higher sugar content
•Lack of starch biosynthesis pathway

Fructose-6-P
Glucose-6-P
Glucose-1-P
ADP-glucose
Amylose
Starch
Phosphoglucomutase
Hexose phasphate isomerase
ADP-glucose phosphorylase
Starch synthetase
Starch Branching Enzyme
STARCH BIOSYNTHESIS PATHWAY

SBE1 enzyme lack in rr line
• Antibody raised against SBE1
protien and conduct the
western bloting
• Bond at 114 kDa in RR line

Cloning of cDNA for SBE1 gene
• Screening cDNA library in gt11 of RR line
• pJSBE5 (2.7kb) clone
• Northern blot analysis

• Molecular organization of
the cDNA
Genomic DNA of near
isogenic line ( RR and rr)
digested with EcoRI, EcoRV,
and HindIII and probed with
pJSBE5
4.1kb
3.3 kb

Linkage analysis
of the SBE1 gene
and ‘r’ locus
JI15(RR)  JI1194(rr)
40 F6 plants

Molecular analysis of the insertion in the rr
allele
pJSBE206

 Southern blot
genomic DNA
were
hybridized
with the HincII
fragments
from pJSBE102
and pJSBE206

• compare sequences the
ends of pJSBE102 and
pJSBE206
• Insertion was located in exon
• inserted sequence had 12
inverted repeats at its
termini flanked by 8bp direct
repeats from SBE1 gene
5’……..AGTAGAATTAGGGGTGGCAAAA……………………………….AATTGCCACCCCTAAGTAGAAT………3’
Ips-r

• These 12 inverted repeats have high homology with
Ac transposon of maize

DNA
RNA
Protien

Stem length
•Lester et al.,
(1997) Martin et al., (1997)
•Stem length is governed by Le
locus linkage group III
Alaska (Le), line 58(le)3/7/2016 30MAHESH R HAMPANNAVAR

• Role of various harmones
• Gibberelic acid
• GA20 GA1
• GA1 is bioactive form
• Dwarfism because reduced endogenes activity
of GA1

• GA20 GA1
• Lack of 3- hydroxylase in dwarf plants
• Mutation in 3- hydroxylase enocode gene
• Dwarf gene in other species like dwarf 1 in
maize, rice dy, Arabidopsis ga4
GA 3- hydroxylation

Isolation of GA4 related gene from Pea plant
• Screening of pea cDNA library (Alaska) with
portion of arabidopsis GA4 cDNA produced
Partial cDNA – pDO3c probe
• pDO3c that shows high homology
• 2ODD , the family of enzyme to which GA 3-
hydroxylase belongs
• This enable the isolation of full length le and
Le genomic sequence.

• Genomic DNA gel blot
analysis with pOD3c
and digest with EcoRI,
EcoRV, HindIII
• RFLP only seen with
HindIII

Restriction mapping of GA4 related gene in pea
• 1.2kb fragment spanning RFLP region is amplified by PCR
and digest with HindIII
•PCR product of LE line remain intact(1.2kb band) where as
le produce 0.2kb and 1 kb band

• Linkage analysis of GA4 related gene with stem
length
•All dwarf plants
1.0- and 0.2-kb
bands only.
• Tall plants 1.2, 1 .O,
and 0.2 kb,
• Some tall plants
1.2-kb band only

Comparision of GA4 gene of arabidopsis with pea GA4
related gene
• A 2.2-kb fragment encompassing the coding region, the TATA
sequence, and some of the flanking sequence of the GA4-
related gene from the Le line
• By comparing the open reading frame of the Le allele with
GenBank sequences, we determined that this sequence is
most highly matched with the GA4 cDNA
• The 1122-bp nucleotide sequence is 61 % identical to the
GA4 cDNA, which translates to an identity of 53% for the
predicted 374-amino acid

Alignment of Deduced Amino Acid Sequences of the Pea (Le) GA 3P-Hydroxylase
Gene and the Arabidopsis GA4 gene

Mutation study in le allele
• Extra two nucleotide bases, AT, in intron
region (1954)  HindIII polymorphism
• G to A transition in exon region (2654)
amino acid changes

• le line code for threonin instead of alanine in
tall plant (229)

Cotyledon color in pea
• Sato et al.,2007
• I locus present on LG1
• Yellow cotyledon(II/Ii) and green cotyledon(ii)

• Mechanism takes place at the time of
senescence
– Unmasking the pre-existing caretinoids compound
– Existing chlorophyll degradation
• In higher plants chlorophyll exist – chl a and
chl b
• Chl a contain PSI and PSII

Chlorophyll
Chlo b Chlo a
Chlorophyllide a
(chide a)
Pheophorbide a
(Pheide a)
Red chlorophyll
reductase
Chlorophyllase
Mg+2
oxygenase
Chlo b reductase

All these SGR genes present in almost all
plants, these are conserved sequence

• Stay Green Mutant
• SGR yellow color and sgr retain greenness
• Nonfunctional SGR
– lls1/acd1/Pao (Maize)
– Nyc1 (Rice)
– Sgr (Rice)
Mutation is independent

Mendel’s Green Cotyledon Mutation Is a Stay-Green
Mutation
chl a and chlo b content
after 12 days of dark
induced

Sequence of SGR gene in pea
Prepare cDNA clones of both isolines
Primers from sequenced gene
• Pao, NYC1,
SGR
degenerate
PCR
• Partial
sequence
of genes
inverse PCR
and 3’-RACE
Full lentgh
sequence

Pao
NYC1
SGR

• Analysis of PsSGR genome sequence
• PCR analysis of 3rd intron
in parents

Mutation study in PsSGR
• Difference inPsSGRJI2775 and PsSGRJI4 sequence:
In sequence of nucleotides In sequence of amino acids (clustal W)
A- T substitution Thr  Ser at amino acid 12 (T12S)
T A substitution Asp  Lys at amino acid 38 (N38K)
6 bp insertion
two-amino acid insertion of Ile and
Leu at 189

OsSGR cDNA
Insertion of 6-bp at the site
corresponding PsSGRJI2775
Insert the constract into sgr
mutant
Restore the stay green
mutation

DNA
RNA
Protein

FLOWER COLOR
•Hellen et. al in 2010
•Flower color governed by A locus
•Linkage group II

• Phenyl alanine----> anthocyanin----> Purple
flower
• MYB or bHLH transcription factor or WD40
protien
• Disruption of these above regulation gene
lead to white flower production

Synteny analysis between
Medicago truncatula and pea
plant A locus

BAC
clones
purple
flowered
accession
PI269818
the white
flowered
cultivar
Came´or
BAC112D23 BAC452H2
Two BACs, each over 150 kb, were sequenced and used to
define a gene model for the A locus in pea and to identify
the nature of the mutation in the white flowered variety.
Degenerate PCR by
mtbHLH gene

 Seven exon and six introns
 92% sequence identical to the between two clones
 Sixteen SNP found in open reading frame of bHLH
gene of cameor (white flower)
 One of these silent mutations is a single base
change, from G to A, in the splice donor site of intron
6

Role of GT splice donar site
• GT region present in all introns
• Help to removal of intron region in during
mRNA processing
• Mis-spling

GT-------5775bp------------AG

Organisation bHLH gene in colored flower clone
DNA
mRNA
Protien
Flower phenotype

ATAAATGGGT---------------AG

Organisation bHLH gene in white flower clone
DNA
mRNA
Protien
Flower phenotype
+8 nt

Molecular characterisation of a mutation
in white flowered pea cultivars

•Flowers from the twelve PI lines, JI 2822 and Came´or
used in this analysis.
• RT-PCR products spanning the exon6-exon7 junction.
•79 bp amplify in color flower campare to white flower
(87bp)

DNA
RNA
Protien

Uncharacterized genes
• Position of flower (Fa or Fas)
• Pod color (Gp)
• Pod form (P or V)
Why ?

POSITION OF FLOWERS
• Axil or terminal
• Several genes regulate
the position of flowers-
– det, Fa, Fas
• det locus muntant is
not correlate with mendel
explaination

• Fa and Fas locus on linkage group IV and III
respectivly
• Relation between Fas and Le
• This fascinated type phenotype also seen in
Arabidopsis.
• Failure CLV signalling path way

POD COLOR
• Green color (Gp) > Yellow color (gp) (LG-V)
• Is not contrasting of I locus
• In yellow pods plastids are restricted to single
layer and lack of grana (5%)

• Is it possible to get yellow cotyleden in green
pods?
Gp locus on linkage group on V but I on
linkage group I so mendel 2nd law apply

POD FORM
• Inflated > constricted Pods
• Sclerenchyma layer plays role
• Suger pods
• p or v mutants possess the same phenotype
– Pv – small patch of sclerenchyma
– pV – sclerenchyma along each side
– pv – lack of sclerenchyma
Which mutant mendel
study ?

Mendel found the linkage
•Why did mendel use less
no. sample in
independent assortment
of stem length and pod
form
•he used genotype
mutant in V locous
•V and Le are 15 cM apart
on linkage group III
Mendel not found the
linkage
•If he studied P locous
mutant
•P locus on linkage group
VI
•then no Question of
linkage
Linkage ?

Trait
Dominant
phenotype
Recessive
phenotype
LG Gene function
Molecular
nature of
mutation
Reference
Seed
shape(R)
Round Wrinkle V
Starch branching
enzyme 1
0.8-kb insertion
Bhattacharyya et
al. (1990)
Stem
length(L)
Tall Dwarf III GA3-oxidase1
G-to-A
substitution
Martin et al.
(1997)
Cotyledon
color(I)
Yellow Green I Staygreen gene 6-bp insertion Sato et al., (2007)
Flower
color(A)
Purple White II
bHLH transcription
factor
G-to-A splice site
Hellens et al.,
(2010)
Pod
color(GP/?)
Green Yellow V
Chloroplast
structure
Unknown
Reid and Ross.,
(2011)
Pod
form(V/?)
Inflated Constricted III
Scleronchyma
formation
Unknown
Reid and Ross.,
(2011)
Flower
position(FA/?
)
Axil Terminal IV Meristem function Unknown
Reid and Ross.,
(2011)3/7/2016 71MAHESH R HAMPANNAVAR



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MENDEL TO MAHESH
mahi5295@gmail.com

MENDEL; 150 years on

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