plant breeding lecture 6-starch-sucrose-fructans-final.pdf

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Breeding for carbohydrates

Luisa Trindade

Bioresource (PBR 31306)

Plant components


Carbohydrates Easily degradable sugars: starch, sucrose, fructans Insoluble sugars: plant cell walls: (hemi) cellulose, pectin

Lipids Membranes Oils, waxes

Proteins and free aminoacids Secondary metabolites

Vitamins Flavonoids / terpenoids

Water and minerals

Prim

ary

meta

bol

ites

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Program for today


Easily fermentable sugars:

● Starch: potato

● Sucrose: sugarbeet and sugarcane

● Fructans: grasses

Plant cell walls

● Maize as a model

Starch


http://www.google.nl/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0CAcQjRw&url=http://www.marutiinternational.in/starch-powder.htm&ei=pcQJVfr5GoqBPZmzgMAP&psig=AFQjCNHcjBZipnO9yAvT4VeUzkDU4bJHRw&ust=1426789844197732

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Starch crops

The main sources of starch are Europe are:

● Cereal crops

● Rice

● Maize

● Wheat

● Potato

...in other part of the world other crops are used: e.g cassava, taro...


The Netherlands is the largest producer world wide

Potato is the major starch crop in The Netherlands

Breeding targets in starch crops

Starch yield

Starch composition

Starch granule size

Starch properties


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Breeding for starch yield

What are the key factors behind starch yield?

1. Starch synthases expression play a (small) role depending on

the crop and growth conditions


Overexpression of glycogen synthases in wheat lead to increase in starch content: • in line 72 only at high

temperature

• In line 79 at both high and low temperature

Reference: Burrell. J Exp.Bot. 54(382): 451-456

In this approach the biosynthesis of starch is stimulated/induced


2. Very recently reported: starch content is inversely correlated

with sugar content in potato:

High sugar content ->Low starch content

in the tuber

● The sugars are probably originary from

starch.

● This approach focuses on reduction of

starch degradation

● What are the factors that influence starch

degradation?


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In approach 1 - target genes in red

● Synthases

In approach 2 – target genes in blue

● Phosphorylation

● Amylases

● Debranching

Reference: Schreiber et al. 2014. 4 (10): 1797-1811

Breeding for starch content in potato


What are potato characteristics:

● Potato is a cross pollinator

● Tetraploid (most varieties)

● Highly heterozygous

● Clonally propagated crop

Tools available

● Genome sequence

● Genetic maps of different diploid

populations and markers for

different traits

● Starch content is correlated with

underwater weight

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Traditionally breeding for starch content targeted:

● High yield of tubers

● High underwater weight

Breeding methods

1. Crosses between diverse genotypes

2. Selection of genotypes with good performance (for both

yield and resistance to Phytophthora)

3. Different rounds of selection and generation of elite lines

4. Clonal propagation of elite genotypes



Current/modern breeding for starch content targeted:

● High yield of tubers

● High underwater weight

Breeding methods: Marker assisted selection

1. Identification of genetic markers (QTL’s) for the above traits

2. Identification of strong alleles

3. Introgression of the right alleles in elite lines

Future breeding: make use of diploid lines

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Starch quality


Starch quality

Starch needs to be derivatized before it can be used for different application


modified starch treatment

(example)

advantage food use non-food

use

pregelatinized heat/moisture cold-water soluble pie fillings, “in-

stant” products,

coatings

oil-drilling,

mining

acid-thinned acid low hot paste

viscosity, high gel

viscosity

gums, jellies textiles,

laundry

starch

oxidized hypochlorite increased clarity,

reduced set-back

gravy, sauces

thickeners,

jellies

paper, tex-

tiles, spray

starch,

adhesives

hydroxyalkyl ethers propylene

oxide

increased clarity and

stability

salad dressings,

pie fillings

paper and

textile sizes

esterified acetic

anhydride

reduced set-back,

increased clarity,

forms films/fibres

“instant” foods,

frozen foods

textiles,

paper,

packaging,

film

monophosphates phosphoric

acid

increased stability

to freeze/thaw

cycles

frozen foods,

infant formulae

paper, tex-

tiles, metal

refining

cross-linked phosphorous

oxychloride

increased stability

to heat, pH, freeze

/thaw cycles

wide range of

canned and

frozen foods

paper, metal

se-

questrants

cationic starches tertiary or

quaternary

amines

increased cold-water

solubility/dispersa-

bility, increased

binding to negatively

charged materials

paper, mining

Derivatization implies:

• Use of chemicals

• Energy

• Reduces the quality of

starch

Improvement of starch in

plant has many

advantages:

• Environmental impact

• Improve quality of

starch

• More economical

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Starch granule composition

Amylose: 20-30% Mw. 5×105 to 106

Amylopectin: 70-80% Mw. 107 to 108

Proteins

Lipids

Phosphate

“Contaminating” substances

Possibly starch modifications

Amylose and amylopectin ratio

Size of the granules

Length of the amylopectin chains

Phosphate content

Protein and lipids content

Expression of heterologous proteins:

● New polymers

● New linkages

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Potato amylose –free starch


Bakery creams ( 20% expensive alginates) Fruit filling (shiny texture)

Powdered soups, sauces & processed meat products (salt stability)

Noodles (hydration at lower temperatures)

Snacks ( crispness)

What happens when you remove amylose?

Interesting breeding target!!

Breeding for amylose-free potato

How do you identify amylose free lines?

● Based on phenotype

● Based on genotype

● Search for lines where

GBSS has been mutated

● Induce mutations in GBSS

(Tilling)


Wild type Amylose-free

Iodine staining

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Breeding for amylose-free potato

Breeding strategy: mutation breeding (non-GM)

● As potato is a highly heterozygous and tetraploid crop it is

difficult to combine all the desired traits in one genotype

● One tool is to do the first breeding steps in diploid (or even

monoploids) to generate a line homozygous for non-functional

GBSS

● Introduce the non-functional GBSS in an elite line (this can

take many years!)

● A GM approach is much faster and effective in this case

Amflora


Attempts to breed for high-amylose

starch in potato


Advantages of high amylose starches:

● Form strong gels

● Applications in jelly-gum

candies and coating of

deep-fried foods

No 100% amylose potato could be achieved

● Probably lethal

Large impact on tuber yield

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Starch phosphate content

Where is P in starch?

● Amylopectin: C3 and C6 positions

What is the effect of P on starch prop.?

● Higher phosphate content

● High swelling power

● Increased solubility

● Resistance starch – effective in lowering glycemic index

● Low phosphate content

● Higher digestibility

● Low viscosity


Breeding for phosphate content

The genes involved in phosphorylation of starch are known: GWD (glucan-water dikinase), PWD (phophoglucan water dikinase)

Phosphatases are known: sex4 (starch excess)

Phosphate content is not only important for starch properties but for survival of plant:

● Paradox:

● P is needed for starch for degradation in the plant

● P is reducing digestibility of starch in humans


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Breeding for phosphate content

Breeding approach: Reverse genetics (candidate gene)

Breeding strategy:

1. Alleles of GWD, PWD and SEX4 have been identified in a wide

collection of potato genotypes

2. These same genotypes have been characterized for P content

3. Alleles correlating with high or low P content have been

identified

4. Epistatic effects of starch phosphorylation are being analysed

(specially effect of reduced P on yield)

5. Positive and strong alleles will be introgressed in potato elite

lines


Tailoring of potato starch (GM)


E. coli

Branching

enzyme (GlgB)

Streptococcus downei

Mutansucrase gtfi

Human - Laforin

CBM25

A. thaliana

Water dikinase

Bacterial Glucansucrases

L. mesenteroides

Dextransucrase

dsrs

L. mesenteroides

Alternansucrase asr

Expression of different heterologous proteins lead to changes in starch granule morphology and properties of starch

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Sugarbeet

Crop characteristics:

● Cross pollinator

● Most commonly diploid and tetraploid

● Originally self-incompatible

● Root has high sucrose (sugar) content


Sugar content in sugar beet

History:

● Modern sugarbeets are originated from selections made in the

middle of the 18th century from fodderbeets grown in then

German Silesia

● Breeding of sugarbeet was intensified during the blockade of

shipments of cane sugar to Europe by the British during the

Napoleonic wars

● The sugar extraction process developed in the 19th century has

created a market for sugarbeet and thus breeding of this crop

enforced


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Breeding sugarbeet

Breeding of the first varieties of sugar beet ware done by mass selection

These varieties were followed by better ones produced by anisoploid (triploid) synthetic varieties resulting from crossing diploid and tetraploid lines

Diploid male sterile x tetraploid pollinators

Current sugarbeet varieties are diploid hybrids

1. Making diploid inbred parents containing the required traits

(sugar content, resistance to pathogens, and sugar quality)

2. making hybrid seeds


Synthetic: refers to varieties that result from crossing of multiple (>2) parents

Breeding sugarbeet

Breeding targets:

● Total root yield

● Sucrose yield

● More recently breeding

for sugar quality has

become a target.

Current yield in The Netherlands:

● Average root yield ~ 60 tons/ha

● Average sugar yield ~ 10 tons/ha (16%)


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Sugarcane

Perennial grass

Comprises 6 species but Saccharum

officinarum is the major contributor of

high sucrose in modern varieties

Genetics: 2n=80 chromosomes

Contains a lot of duplications and

unpaired chromosomes

Breeding is a great challenge!!

Breeding targets:

● Sugar yield!

● Ratooning performance, resistance to pathogens, low fibre, cane yield.

Bioresource (PBR 31306) Further reading: Jackson. 2005. 92: 277290

Breeding sugarcane

Breeding of sugarcane happen in three phases:

1. Crossing and selecting among S. officinarum clones: these

varieties had high sugar content and low fibre content, but

they were susceptible to diseases and lacked vigour

2. Development of interspecific hybrids between S. officinarum

and other species (mostly S. spontaneum): these were

vigorous and with good ratooning performance but they had

lower sucrose levels. By backcrossing the hybrids with S.

officinarum rubust high-sugar-high-yielding varieties were

generated: “wonder cane” (ancestry of all modern varieties)

3. Exploitation of interspecific hybrids from phase 2 by recurrent

selection


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Breeding sugarcane

Most modern varieties are made from a small number of original progenitors

Narrow genetic basis of sugarcane breeding programs

Limited possibilities for introgression or improvement of new traits


Breeding sugarcane


Sugar yield/ha has increased

But...that was not to increase in sugar content but due to improvements in total biomass yield

Future:

● Increase genetic basis in

breeding programs

● Developing genetic markers

for different positive and

negative traits

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Fructans in plants

Fructans are the major form of storage carbohydrates in grasses:

● Are important to sustain regrowth of the plant after defoliation

● Nutritive value of feed

● When extracted can be used as sweeteners as humans are not

able to degrade fructans


Breeding fructans in wheat

Genetic markers (QTL’s) for fructan yield in wheat grain have been

(recently) identified

Breeding strategy: Marker

Assisted Selection (MAS)

Identify the genes underneath

these QTL’s would create tools

for breeding in other crops


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Summary

Starch, sucrose and fructans are the main storage forms of “easily

degradable” sugars and they are specific to each crop.

Each crops has different characteristics and different

genetic/breeding tools are available – this determines breeding

strategy

Breeding for sugar/starch content is often indirect. The real target is

crop yield

Starch biosynthetic pathway is well characterized and starch quality

has been mainly tackled using candidate gene approaches both GM

and non-GM.

Breeding of polyploids (potato and sugarcane) is much more difficult

than diploids species

One (future) line of potato breeding is through diploids


plant breeding lecture 6-starch-sucrose-fructans-final.pdf

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