Seed genomics for seedling vigour and initial high crop stand

Seed production

SEED TO SEED

Pre-sowing seed management

Seed agronomy, type of sowingSpacing, fertilizer, pesticides, irrigation, weeding

Physiological

maturation

Pre-harvest sanitation

Harvesting

Extraction

Drying

Processing

Drying

Seed treatment

Storage

Selection of seed and land

Season

Seed Genetics

Roguing

Quality seed

• Quality seed is defined as purevariety with a high germinationpercentage, free fromdisease/disease organisms and witha proper moisture content andweight.

• Quality seed insures goodgermination, rapid emergence andvigourous growth. These aspectstranslate to a good field stand.

• Seed quality is the sum of allproperties contributing to seedperformance.

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Structural concept of seed quality

Knowledge about the various seed quality aspects of seeds

greatly contributed to agricultural development in the past

and will continue to play a major role in future

enhancement of crop production. Seed quality is a

multiple concept comprising several components

(Thomson, 1979)

Structural concept of seed quality ( Huda, 2001)6

Seed vigour

Seed vigour is a sum of those properties that determine the activity

and level of performance of seed lots of acceptable germination in a

wide range of environments.

◦ Rate and uniformity of seed germination and seedling growth.

◦ Emergence ability of seeds under unfavourable environmental

conditions.

◦ Performance after storage, particularly the retention of ability to

germinate.

◦ A vigorous seedlot is one that is potentially able to perform well

even under environmental conditions that are not optimal for

species.7

Seed vigour characteristics

Comparison of the characteristics of high and low

vigour seed lots.Characters Vigour level

High Low

Mean germination Fast Slow

Synchrony of germination Good Poor

Mean seedling size Large, uniform Small, variable

Emergence potential Good in most soil

condition

Poor in less than

optimum soil

condition

Storage potential Good poor

Encyclopedia of seeds, pg. 7428

Components of seed vigour

Yield

S:R ratio

Germination

Plant height

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Relationship between vigour, field performance and yield

The germination ability and vigour of a seed lot is directly

related to performance in the field.

Seeds low in vigour generally produces weak seedlings that are

susceptible to environmental stresses.

High level of vigour in seeds can be expected to provide an

early and uniform stands which give the growing seedlings the

competitive advantage against various environmental stresses.

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Provides a very good estimate of potential field performance

and subsequently, the field planting value

The ability of the germinating seed to continue to grow and

survive then determines crop establishment

Low seed vigour contributes to the development of smaller and

uneven seedlings

Which leads to poor plant stand and growth (weaker seedlings)

and uneven time of maturity, resulting in the possibility of yield

loss

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The central dogma of molecular biology was first articulated by Francis Crick in 1958

Explained the of the flow of genetic information

within a biological system

DNA mRNA Protein

Transcription

Translation

It is the link between genotypes and phenotypes

Genome: Total amount of DNA/RNA, genetic information

present in a cell.

Gene: Segment of DNA contains biological information & codes

for RNA/ polypeptide molecule

Most of the eukaryotic genome: non-coding

Humans – only 5% coding DNA, plants have more repetitive

DNA

Genomics is a discipline in genetics that applies recombinant

DNA, DNA sequencing methods, and bioinformatics to

sequence, assemble, and analyze the function and structure

of genomes

1. Structural Genomics: is a worldwide effort aimed at determining the

three-dimensional structures of gene products in an efficient and high-

throughput mode. When the focus is on proteins, this effort may be

called Structural Proteomics

2. Functional Genomics: To understand the function of

information in genome

3. Comparative Genomics: Comparing the genomes of different organisms

TYPES

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Quantitative trait locus

A quantitative trait locus (QTL) is a section of DNA (the locus) that correlates with variation in a phenotype (the quantitative trait).

The QTL typically is linked to, or contains, the genes that control that phenotype.

QTLs are mapped by identifying which molecular markers (such as SNPs or AFLPs) correlate with an observed trait. This is often an early step in identifying and sequencing the actual genes that cause the trait variation.

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Lipid peroxidation plays a major role in seed longevity and

viability.

In rice grains, lipid peroxidation is catalysed by the enzyme

lipoxygenase 3 (LOX3).

Grain from the rice variety DawDam in which the LOX3 gene

was deleted had less stale flavour after grain storage than

normal rice.

The transgenic plants exhibited a marked decrease in LOX

mRNA levels.

LOX3 activity and its ability to produce (9-HPOD) from

linoleic acid were significantly lower in transgenic seeds than in

wild-type seeds

TS-91WT; Hang 1

Huibin Xu et al. (2015)18

The suppression of LOX3 expression in rice endosperm

increased grain storability.

The germination rate of TS-91 (antisense LOX3 transgenic

line) was much higher than the WT (29% higher after artificial

ageing for 21 days, and 40% higher after natural ageing for 12

months).

Variation in germinability after artificial ageing for

0–30 days.

Huibin Xu et al. (2015)19

Variation in germinability after natural ageing

Huibin Xu et al. (2015)20

Scanning electron microscope images of starch granules in antisense LOX3

transgenic plants and wild-type (WT) plants. (a) WT seeds without ageing.

(b) WT seeds aged for 30 days. (c) Antisense LOX3 transgenic seeds without

ageing. (d) Antisense LOX3 transgenic seeds aged for 30 days. Bar = 5 lm.

Increasing longevity of seeds with genetic

engineering

• Researchers of the IBMCP (Institute for Plant Molecular and Cell

Biology) traced half a million seeds, related to one hundred

thousand lines of Arabidopsis mutated by T-DNA insertion, using

the natural system of Agrobacterium tumefaciens.

• The key is the over expression of the ATHB25 gene. This gene

encodes a protein that regulates gene expression, producing a new

mutant that gives the seed new properties.

• Researchers have proven that this mutant has more gibberellin -the

hormone that promotes plant growth-, which means the seed coat is

reinforced as well.

Eduardo Bueso et al. (2013)

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"The seed coat is responsible for preventing oxygen from

entering the seed; the increase in gibberellin strengthens it and

this leads to a more durable and longer lasting seed," (Eduardo

Bueso, researcher at the IBMCP ).

Researchers compared the longevity of genetically modified

Arabidopsis seeds and seeds which were not modified. In order to

do this, they preserved them for thirty months under specific

conditions of room temperature and humidity. After thirty

months, only 20% of the control plants germinated again,

whereas almost the all of the modified plants (90%) began the

germination process again.

The increase of the lifespan of seeds would mean a reduction in

their purchase price."

Eduardo Bueso et al. (2013)

The sweet corn inbred line P39 and the field corn inbred line

EP44 were used as plant material.

Bulks of living and dead seeds after 20 and 22 years of

storage were compared by using simple sequence repeats

(SSRs).

Differences between dead and living seeds could be explained

by residual variability, spontaneous mutation, or ageing.

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Revilla et al. (2009)

Variability was larger for chromosome 7 than for other

chromosomes, suggesting some relationships between position

in the genome and viability in aged seed.

Polymorphic SSRs between living and dead seeds were found

in six known genes, including pathogenesis-related protein 2,

superoxide dismutase 4, catalase 3, opaque endosperm 2, and

metallothionein1 are related to germination, along with golden

plant 2.

Revilla et al. (2009)

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Phospholipase Da1 is the most abundant form of PLD in A.

thaliana

Phospholipase D (PLD), which cleaves phospholipids to

generate phosphatidic acid (PA), has been proposed to

catalyze an early step in the process of membrane degradation

and seed deterioration.

The PLDa1 knockout (KO) mutant, designated plda1-1, had

no detectable PLDa1 protein.

Shivakumar et al. (2007)

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Immunoblotting of PLDa1 protein (upper

panel) and PLDa1 activity (lower panel) in seeds

of WT, plda1-1 (KO) and plda1-AS (AS).

The western blot (sometimes called the protein immunoblot) is

used to detect specific proteins in a sample of tissue

homogenate or extract.Shivakumar et al. (2007)

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Germination rate of WT and PLD mutant seeds without (C = control seeds)

and with aging (AA = accelerated aging) treatment. Accelerated aging was

performed by placing the seeds at 43C in a tightly closed box with 100%

relative humidity for 48 h.

Shivakumar et al. (2007)27

• Maize seeds homozygous for luteus2 and luteus4 genes lose

viability more quickly than seeds homozygous for any of the

remaining six luteus gene

(Weiss &wentz, 1937).

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Genetic dissection of seed vigour under artificial ageing conditions using

two joined maize recombinant inbred line populations

Yu82 9 X Shen137 and Yu537A 9 X Shen137 crosses were

evaluated for the mean germination time (MGT) under

artificial ageing.

Twenty-two key candidate genes associated with four seed

vigour-related traits mapped to 14 QTLs .

GRMZM2G163749,

GRMZM2G122172/GRMZM2G554885/

GRMZM2G122871 and GRMZM2G150367 genes mapped

within the QTL5–4, QTL6 and QTL8 regions.

Lixia Ku et al. (2014)29

DOG1 is a key regulator of seed dormancy in Arabidopsis

• HISTONE MONOUBIQUITINATION

1 (HUB1) , REDUCED DORMANCY

2(RDO2) and SUPPRESSOR OF ABI3-5,

DOG1 are responsible for dormancy.

• Mutations in most of these genes cause

reduced dormancy, resulting in a higher

germination of freshly harvested seeds.

• The dog1 and rdo5 mutants show a complete

loss of dormancy upon mutation.Mutants with reduced seed dormancy.

Germination percentages of freshly harvested

seeds are shown for the wild-type Columbia

accession and the mutants dog1, rdo5, hub1,

and rdo2.

Graeber et al.(2012)30

Genetic mapping within the wheat D genome reveals QTL for

germination, seed vigour and longevity, and early seedling growth

• In wheat D the QTL analysis for the traits related to

germination, seed vigour and longevity, 17 QTL were

identified.

• These loci were distributed on chromosomes 1D (9 QTL),

5D (5), 7D (4), 2D (1) and 4D (1). QTL were detected for

19 of the 38 studied traits, among which nine were for

germination-related traits, two were for seed vigour-

related traits, five were for seed longevity-related traits,

and three were for seedling growth traits.

• QTL responsible for the germination capacity following

AA were located on chromosomes 5D and 1D.

(Landjeva et al., 2009). 31

Quantitative Trait Locus Analysis of Seed

Germination and Seedling Vigour in Brassica rapa

• Doubled haploid population from a cross of a yellow-seeded oil-type yellow sarson and a black-seededvegetable-type pak choi were chosen .

• 26 QTL regions across all 10 linkage groups for traitsrelated to seed weight, seed germination and seedlingvigour under non-stress and salt stress conditionsillustrating the polygenic nature of these traits wereidentified.

• QTLs for multiple traits co-localized, eight hotspots forquantitative trait loci (QTL) of seed weight, seedgermination, and root and shoot lengths.

• A QTL hotspot for seed germination on A02 mapped atthe B. rapa Flowering Locus C (BrFLC2).

32(Basnet, R.K et al., 2015).

• Another hotspot on A05 with salt stress specificQTLs co-located with the B. rapa Fatty aciddesaturase 2 (BrFAD2) locus

• For the 24 seedling vigour traits measured in 2010and 2011 seed batches, 10 QTL regions with 69QTLs were identified.

• The explained variances ranged from 7.1 to24.3%. For the 2010 seed batch, 22 QTLs for 13traits were identified on different linkage groupswith at least one QTL per trait and an additional24 putative QTLs for 13 different traits.

• For the 2011 seed batch, 47 QTLs were identifiedfor 20 traits with at least one QTL per trait and allthose traits also had putative QTLs.

33(Basnet, R.K et al., 2015).

Quantitative genetic analysis of seed vigour and pre-emergence seedling growth traits in Brassica oleracea

• Traits associated with seed vigour and pre-

emergence seedling growth in a segregating

population of 105 doubled haploid

Brassica oleracea lines were studied.

• Quantitative trait loci analyses revealed

significant loci on linkage groups O1, O3, O6,

O7 and O9

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Quantitative trait loci analysis for rice

seed vigour during the germination stage

• RIL population derived from a cross between japonica

Daguandao and indica IR28.

• The results showed that indica rice presented stronger seed

vigour during the germination stage than japonica rice.

• A total of ten QTLs, and at least five novel alleles, were

detected to control rice seed vigour, and the amount of

variation (R2) explained by an individual QTL ranged from

7.5% to 68.5%, with three major QTLs with R2>20%.

• Most of the QTLs detected here are likely to coincide with

QTLs for seed weight, seed size, or seed dormancy,

35(Wang et al., 2010).

• Three QTLs controlling germination rate wereidentified on chromosomes 1, 2, and 11, respectively.

• Among them, qGR-1 might be one major QTL(R2=68.5%), the other two of the minor QTLs (qGR-2and qGR-11).

• Four QTLs associated with germination percentagewere found on chromosomes 4, 6, 8, and 11,respectively and one major QTL qGP-6, Three QTLs(qGI-1, qGI-7, and qGI-11) were responsible forgermination index located on chromosomes 1, 7, and11, respectively

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QTLs for Seed Vigour-Related Traits Identified in Maize

Seeds Germinated under Artificial Aging Conditions

• Twenty-three candidate genes for association with

seed vigour traits coincided with 13 mQTLs.

• The candidate genes had functions in the

glycolytic pathway and in protein metabolism.

• Candidate genes included a calcium-dependent

protein kinase gene (302810918) involved in

signal transduction that mapped in the mQTL3-2

interval associated with germination energy (GE)

and germination percentage (GP).

37(Zanping, H et al., 2014)

• hsp20/alpha crystallin family protein gene

(At5g51440) that mapped in the mQTL3-4

interval associated with GE and GP.

• A cucumisin-like Ser protease gene

(At5g67360) mapped in the mQTL5-2 interval

associated with GP.

• The chromosome regions for mQTL2,

mQTL3-2, mQTL3-4, and mQTL5-2 may be

hot spots for QTLs related to seed vigour traits

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Thank you

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