Molecular analysis of Pina and Pinb genes of wheat

in Southern European landraces.

Fig-2: Structure of wheat grain (http://www.classofoods.com/page1_1.html, viewed

September 2015).
The major part of wheat composition is endosperm, which is powdered into white flour, to
produce variety of food product for human consumption (shewry, 2002). The main
ingredients of white flour are starchy endosperm with high amount of gluten and starch. The
wheat germ (a part of embryo) is removed during the milling process and outer layer of wheat
baking property of polymer makes wheat grain very useful food substance. As a result the
factors that determine composition and grain quality of wheat are significance in terms of
trade and nutrional value of wheat (Morris, 2002). Wheat quality is an important for its
specific end use and it can be determined by number of physical characteristics such as grain
color, shape, weight, hardness and vitreousness (Gaines et.al, 1996). Moreover, baking,
rheological dough properties, chemical and milling parameters can affect grain quality.
1

1.4 Wheat hardness/texture and its measurement (characterization).

Wheat hardness or texture is most the significant characteristic that determines the
technological utilization and marketing value of grain (Morris, 2002). Moreover, softness and
hardness of the wheat grain is useful for its classification and it is important for the millers,
bakers and breeders. The grain hardness significantly affects baking and milling properties in
the flour industry (Gaines et.al, 1996). For instance, flour density, flour particle size, water
absorption, starch damage and milling yield are physical properties that influenced by grain
For instance, damaged starch granules of hard wheat allow better water absorption and it is
important for the bread making. Moreover, damaged starch granules increase available
carbohydrate to yeast for fermentation activity and increased gas production and bread loaf
volume. The degree of interaction between starch granules and protein matrix are difference
between hard and soft wheat (Csoti et.al, 2005).
Table 1.1: comparison between hard and soft wheat (Adapted from Ramalingam, 2012)
Soft wheat
Useful to makes cakes,
biscuit
Less power require to
breakdown the kernels,

Hard wheat
Bread (common wheat)
Pasta, spaghetti (durum wheat)
More power require to breakdown
the kernels

Intact starch granules, less

available carbohydrate

Broken starch granules, more

carbohydrate available

Flour in powder form

Flour in coarse textured

Hardness values between 1440 ( SKCS)

Low moisture content,
less water absorption
Less connection between
protein matrix and starch
granules

Reference
Morris (2002),
Nadolska-Orczyk
(2009)
Devaux et al.
(1998), Ikeda
et al. (2005),
Jolly et al.
(1993)

Hardness values between 45-90

( SKCS)
More water absorption, Easily
hydrolysed by -Amylase

Beecher et al.
(2002)
Brites et al. (2008)

More connection between protein

matrix and starch granules

Csti et al. (2005),

Simmons et al.
(1973)

Most common methods used to measure hardness of wheat grain are Particle size index (PSI),
Near-infrared reflectance (NIR) spectroscopy, and Single kernel characterization system
(SKCS). Measurement of wheat grain texture using above three methods can quantify the
texture of individual kernels or bulk grain and provide a numerical separation of the
qualitative classes of wheat (Morris, 2002). There are number of factors that affect the wheat
grain hardness, up to 40% of variation due to pest infections, tillage systems and
2

environmental factors such as rainfall, location, temperature etc. and remaining 60% due to
Hardness genes which determined grain texture of wheat (Alfred, 2013).

1.5 Hardness locus and discovery of friabilin.

Hardness (Ha) locus (located on the short arm of chromosomes 5D) is associated with the
molecular basis of grain texture in wheat (Pickeering &Bhave, 2007). The 15 kDa protein
was isolated from water washed starch granules using SDS-PAGE (Sodium dodecyl sulfate
polyacrylamide gel electrophoresis) (Greenwell and Schofield, 1986). Moreover, SDS-PAGE
electrophoresis analysis showed 15 kDs band in soft wheat, no band in durum wheat and faint
band in hard wheat. Greenwell and Schofield (1986) demonstrated that this 15 kDa protein
was present in small amount in hard wheat and lacked in durum wheat while soft wheat
phospholipids and glycolipids. Moreover, the amount of polar lipids were more in soft wheat
compare to hard wheat on the surface of water washed starch granules (Greenblatt e that
friabilin is composed of multiple proteins and some of the proteins show the anti-microbial
activity.

1.6 Molecular basis of grain hardness and discovery of puroindoline.

Blochet et.al. (1993) identified two protein sequences with high homology from wheat flour
using TritonX-114 detergent (for the lipid extraction from the wheat flour). The analyzed
protein sequences showed tryptophan domain and it is called as puroindoline (derived from
the two Greek word, puro related to wheat and indoline related to the indoline ring of
tryptophan (Trp) amino acid). Latter on, Gautier et.al. (1994) determined two types of
complementary DNA (cDNA) clones and its predicted protein sequence were related to
puroindoline sequence and termed as puroindoline a (PINA) and puroindoline b (PIstion
about the role of Gsp1 protein in grain texture.

1.7 Biochemical properties of PIN proteins.

PINA and PINB proteins have a conserved region of tryptophan rich domain (TRD) and
cysteine rich backbone (Cys) which makes them unique among the other plant proteins
(Blochet et.al., 1993; Gautier et.al., 1994). There are 5 Trp residues presence in TRD of PINA
(WRWWKWWK) and 3 Trp residues presence in TRD of PINB (WPTKWWK). Both
proteins are 59.5% identical and 67.5% similar. Both proteins consist of 148 amino acids
with a molecular mass around 13 kDa and are highly basic with an isoelectric point of 10.5
and 10.7 for PINA and PINB, respectively (Gautier et.al, 1994). Moreover, both proteins have
N-terminal cleavalble peptide; act as signal peptides (the first 28 th amino acid for PINA &
first 29th amino acid for PINB). Gautier et.al. (1994) suggested that above signal peThe genes
(Pina and Pinb) that encode PINA & PINB proteins are intronless and 447 bp long (Gautier
et.al, 1994). Chantret et.al. (2004) reported that Gsp-1 and Pina and Pinb genes are separated
by 37kbp and 32 kbp respectively. Morris (2002) stated that Pina-D1 and Pinb-D1 genes
(wild type) are found in functional form in soft wheat. However, one of these genes possess
3

mutations or is absent in hard wheat (Giroux and Morris, 1997). Gene Pina and et.al (2005)
and other researchers stated that Ae. Tauschii, a donor of D genome, restored the texture of
soft wheat during the formation of hexaploid wheat.

1.8 Allelic variation in the Pin genes.

The allelic variation in Pin genes is due to mutations and most commonly due to SNP (Single
Nucleotide Polymorphism) or single nucleotide change in the coding sequence that results in
1) frameshift caused by base insertion or deletion (INDELs) 2) substitution of amino acid
with another amino acid 3) an amino acid substitution leading to premature stop codon
(Shewry, 2009).
The wild-type Pin alleles Pina-D1a and Pinb-D1a, both requires for soft wheat phenotype. In
hard wheat, one of the wild forms of PIN proteins deliver partial softening effect either in the
presence other variant form of PIN protein or alone. These types of kernels of wheat have
intermediate level of hardness (reviewed in Bhave and Morris, 2008a). s compare to other
mutations. For instance, allele Pina-D1b is assigned as absent of Pina gene and as a result
absent of PINA protein (Gautier et.al, 1994). Moreover, the frequency of null mutation has
been observed more compare to SNPs or INDELs mutations in Pina gene. Feiz et.al, (2009a)
reported that PINB null has a less effect on grain hardness compare to PINA null mutation.
The mutations in Pina-D1 allele are concise in appendix-1.

1.8.2 Mutations in Pinb-D1 gene.

Giroux and Morris (1997) reported the first mutation Pinb-D1b allele (SNP leading to
Gly46Ser substitution in mature protein) associated with hard texture in common wheat. The
mutation change the tertiary structure of PINB protein at TRD and effect the starch granule
and lipid interaction, resulted into wheat hardness. The frequency of SNP leading to amino
acid substitution, pre-mature stop codon and addition or deletion of amino acid are more
compare to null mutation in Pinb gene. The mutation in Pina-D1 allele is concise in
appendix-2.

1.8.3 Mutations in both Pin genes.

Researchers have found only few mutations in both genes and it is rare event. Some of the
examples are Pina-D1b/Pinb-D1b haplotype (Red Egyptian cv.), Pina-null/Pinb-D1b
(common wheat) and Pina-null/pinb-D1b (Tachun-3) (Reviewed in Bhave and Morris,
2008a; Chang et.al., 2006).

1.9 Restriction Fragment length Polymorphism (RFLP) with Pin alleles.

RFLP technique is a widely used technique to identify specific mutation by using restriction
enzyme. CAPS (Cleaved amplified polymorphic sequence) is an extension of RFLP method
using PCR (Polymerase chain reaction). It can be useful to identify mutations in Pin alleles.
For instance, the most common mutation in Pinb gene is Pinb-D1b, involved 1 SNP that
change amino acid sequence, Gly46Ser in mature protein (Pickering and Bhave, 2007). The
4

nucleotide substitution occurred at position 223 of Pinb gene coding region (Giroux and
Morris, 1997). It can be distinguished from non- Pinb-D1b allele using BsrB1 restriction
enzyme. The restriction digestion of non Pinb-D1b allele gives 2 fragments. However, the
change from GGC to AGC in Pinb-D1b allele, create another restriction site and it gives 3
fragments (Fig-3).

Non Pinb-D1b
130bp fragment

317bp fragment

Pinb-D1b (SNP at position 223 of coding sequence G to A.

130bp fragment

94bp fragment

223bp fragment

Fig-3: shows BsrBI restriction site in Pinb-D1b and non- Pinb-D1b alleles.
Similarway, Different Restriction enzyme can be useful to identify specific mutation in Pinb
gene. For example, PvuII, MnlI/StyI, BstNI and HphI can identify Pinb-D1c, Pinb-D1d, PinbD1e and Pinb-D1f, respectively (Pickering and Bhave, 2007). The mutated allele, which
cannot be determined by RFLP analysis then it, can be determined by DNA sequencing
method (Especially for the Pina gene).

1.10 Diversity of Pina & Pinb allele in Australian wheat germplasm.

Researchers have identified limited variability of Pin alleles in Australian wheat germplasm;
mainly it shows only Pina-D1b and Pinb-D1b mutation (Pickering and Bhave, 2007). The
limited variability does not provide range of food procession qualities in Australian wheat
market. Therefore, it is essential to introduced new genetic resources or wheat line, which
provides range of wheat genotypes for domestic market use and competitive in international
5

trade. Furthermore, analysis of diverse wheat land races offers better unden of pin allele can
be identify and useful to current knowledge of Pin allele.

1.11 Summary of literature review and research question.

The Pin genes relationship with grain composition have prompted the quest for new Pin
alleles which could be significant to genetic improvement of wheat and prica (Morris et.al,
2001), Australia (Pickering and Bhave, 2007) and Northern European countries- Sweden,
Denmark, Norway and Germany (Lillemo and Morris, 2002) and at a smaller scale in some
other countries. It will be valuable to consider the Pin allele in Southern European countries
wheat seeds, to check whether other new varieties Pin allele could be recognized which will
add to the present information of Pin alleles.

1.12 Aim of the project.

It includes molecular analysis of Pina and Pinb genes in hexaploid wheat, mainly southern
European landraces (3 from France, 3 from Portugal and 2 from Croatia); in search of new
Pin allele using CAPS technique or DNA sequencing method.

2. Experimental Design (Methods)

2.1 Isolation and quantification of genomic DNA from wheat seed.
The isolation of Genomic DNA from wheat seed includes treatment of crushed wheat flour
with the extraction buffer (1 M Tris-HCl, 0.5 M EDTA & 10% SDS), followed by treatment
with 6 M ammonium acetate and DNA precipitation using isopropanol. (Adapted from Chao
and Somers protocol & Mohammad et.al, 2012). Quantification of DNA was carried out
using Nanodrop 2000 spectrophometer at 260 nm.

2.2 Isolation of Pina and Pinb gene using PCR amplification.

The Pina and Pinb were amplified using specific forward and reverse primer for individual
gene. The PCR was carried out at 94C for initial denaturation, followed by 35 cycles of
denaturation at 94C for 45 s, annealing of both primers at 54C for 45 s and extension at
72C for 45 s. The PCR products were separated by gel electrophoresis (1.7% agarose gel
with Gelred) and visualized under UV light (Pickering and Bhave, 2007).

2.3 Restriction enzyme digestion of PCR amplified product for

identification of specific mutation (CAPS).
Restriction digestion of specific PCR product involved 10 l PCR products with 1 l of
Restriction enzyme and 1.5 l of Enzyme buffer and make up final volume up to 15 l using
sterile water and incubate the tubes at specific temperature for 1 hour. It is followed by
separation of digested product using 1.7% agarose gel electrophoresis (Pickering and Bhave,
2007).
6

2.4 DNA sequencing of PCR amplified product for identification of specific

mutation.
Some of the mutation cannot identify by CAPS technique that can be identifying using DNA
sequencing method.

References.

Appendices
Appendix -1: Pina-D1 alleles with point mutation in mature protein (PINA). (Adapted from
Bhave and Morris (2008a).
Description
Pina-D1
allele
Pina-D1 (D1a)
Wild type
Pina-D1b
Gene deletion (Null)
Pina-D1k
Multiple deletion (Double null)
SNPs leading to Synonymous mutation
Pina-D1c
1 SNP, (Arg58Gln)
Pina-D1d
2 SNPs, (Arg58Gln and 1
synonymous mutation)
Pina-D1e
2 SNPs, (Arg58Gln and 1
synonymous mutation)
Pina-D1f
3 SNPs, (Arg58Gln and 2
synonymous mutation)
Pina-D1g
1 SNPs, (1synonymous
mutation)
Pina-D1h
2 SNPs, (Arg58Gln and
1 synonymous
Pina-D1i
2 SNPs, (Arg58Gln and
Arg21Ser)
Pina-D1j
3 SNPs, (Arg58Gln, Pro108Arg
and one synonymous)
Pina-D1m
1 SNP, (Pro35Ser)
Pina-D1o
2 SNPs, (Arg58Gln and one
synonymous mutation)
Pina-D1q
2SNPs,(Asn111Lys and
Ile112Leu)
SNP resulting in premature stop codon
Pina-D1n
1 SNP, (Trp43stopcodon)
Point mutations due to INDELs
Pina-D1l
1base deletion,
(frameshift Gln61Lys)
Pina-D1p
1 SNP in signal peptide
and 1 base deletion
(Frame shift at Cys 110
Ala)

Wheat

Reference

T. aestivum
T. aestivum
T. aestivum (Gaiyuerui)

Gautier et.al. (1994)

Giroux & Morris
(1997)

Ae. tauschii
Ae. tauschii

Massa et al. (2004)

Massa et al. (2004)

Ae. tauschii

Massa et al. (2004)

T. aestivum

Massa et al. (2004)

Ae. tauschii

Massa et al. (2004)

Gedye et al. (2004)

Synthetic wheat
Synthetic wheat

Gedye et al. (2004)

Synthetic wheat

Gedye et al. (2004)

T. aestivum
Ae. taushcii
T. aestivum

Chen et al. (2006)

Huo et al.
(unpublished)
Chang et al. (2006)

T. aestivum

Chen et al. (2006)

T. aestivum

Gazza et al. (2005)

T. aestivum

Chang et al. (2006)

Appendix-2: Pinb-D1 alleles with point mutations in mature protein (PINB) (Adapted from
Bhave and Morris (2008a).
Description
Wheat
Reference
Pinb-D1 allele
Wildtype
T. aestivum
Gautier et.al. (1994)
Pinb-D1a
SNPs leading to amino acid substitutions or synonymous mutations (1-2 SNPs)
Pinb-D1b
T. aestivum
1 SNP (Gly46Ser)
and
Morris,
Girou
x
Pinb-D1c
T. aestivum (European)
1 SNP (Leu60Pro)
Lillemo and Morris,
(2000)
Pinb-D1d
1 SNP (Trp44Arg)
Lillemo and Morris,
(2000)
Pinb-D1l
T. aestivum
1 SNP (Lys45Glu)
Pan et al. (2004)
Pinb-D1q
T. aestivum
1 SNP (Trp44Leu)
Chen et al. (2005)
Pinb-D1t
T. aestivum
1 SNP (Gly47Arg)
Chen et al. (2006)
Pinb-D1v
2 SNPs (Ala8Thr and Leu9Ile in T. aestivum
Chang et al. (2006)
the signal peptide)
Pinb-D1w
T. aestivum
1 SNP (Ser115Ile)
Chang et al. (2006)
SNPs leading to amino acid substitutions or synonymous mutations (more than 2, multiple SNPs)
Pinb-D1h
29 SNPs, (12 a.a.s)
Ae. tauschii
Massa et al. (2004)
Pinb-D1i
30 SNPs, (14 a.a.s)
Ae. tauschii
Massa et al. (2004)
Pinb-D1j
19 SNPs, (9 a.a.s)
Ae. tauschii
Massa et al. (2004)
Pinb-D1k
Ae. tauschii
31 SNPs, (14 a.a.s)
Lillemo et al. (2002)
Pinb-D1m
28 SNPs, (14 a.a.s)
Synthetic wheat
Gedye et al. (2004)
Pinb-D1n
29 SNPs, (14 a.a.s)
Synthetic wheat
Gedye et al. (2004)
Pinb-D1o
28 SNPs, (14 a.a.s)
Synthetic wheat
Gedye et al. (2004)
SNP resulting in premature stop codon
Pinb-D1e
T. aestivum (American) Morris et al. (2001)
1 SNP (Trp39stop codon)
Pinb-D1f
1 SNP (Trp44stop codon)
Morris et al. (2001)
Pinb-D1g
1 SNP (Trp56stop codon)
Morris et al. (2001)
Pinb-D1ab
1 SNP (Gln99stop codon
T. aestivum
Tanaka et al. (2007)
Pinb-D1ac
Wang et al. (2008)
2 SNPs (Cys57Tyr and Gln99 T. aestivum
stop codon)
Point mutations due to INDELs
Pinb-D1p
Xia et al. (2005)
1 base deletion (frameshift at T. aestivum
Lys42Asn)
Pinb-D1r
Ram et al. (2005)
1 base deletion, (frameshift at T. aestivum, (Indian)
Glu14)
Pinb-D1s
Ram et al. (2005)
1 base deletion and 1 SNP,
(frameshift at Glu14)
Pinb-D1u
T. aestivum
Chen et al. (2007)
1 base deletion
(frameshift at Glu14)
Pinb-D1aa

1 SNP, 1 base
(frameshift at Lys42)

deletion T. aestivum

Li et al. (2008)

Here; a.a.s Amino acid substitution.