Transcript Slide 1 - Confex

Association of Inorganic Phosphorus with a Molecular Marker Linked to a Novel Low Phytate
Mutation
Sarah Burleson, Dr. Katy Martin Rainey, Laura Maupin, Dr. Luciana Rosso
[email protected], Department of Crop and Soil Environmental Sciences, Virginia Tech, Blacksburg, VA
Introduction
Phytate, the main phosphorus storage compound for seeds, is the basis
of several agricultural and environmental problems. Being indigestible
to nonruminant animals, the compound robs phosphorus from their diet
while also chelating important micronutrients (Erdman, 1979). This
poses a problem not only for the animal but also for the producer, as
they must supplement the diet with phytase, adding cost.
Environmentally, phytate causes the animals’ manure to be phosphorus
rich and therefore, when the manure comes into contact with
waterways, eutrophication and other detriments occur.
Soybean line V99-5089, with a naturally occurring gene for low
phytate (LP), was discovered at Virginia Tech in the soybean breeding
and genetics program. One of the benefits of this line is that the LP trait
is controlled by only one gene, making it easier for breeders to work
with, unlike some previously discovered sources which are controlled by
two or more genes (Feng et al., 2007). Also, this gene conditions high
sucrose, low stachyose and low raffinose content, additional important
traits for animal nutrition. One downfall of this gene, however, is low
phytate plants have been known to have poor germination, creating an
obstacle to data collection. Another group at Virginia Tech identified a
molecular marker linked to the trait. To facilitate marker-assisted
breeding, we investigated whether the marker and the LP and modified
sugar traits were associated in diverse pedigrees.
Objectives
• Test the efficacy of VT V99-5089 LP marker in different genetic
backgrounds
• Correlate VT LP marker and phytate content in V99-5089-derived
germplasm
• Correlate VT LP marker and sugar content in V99-5089-derived germplasm
Materials and Methods
Agronomic data were collected in 2008 from three yield tests of
potentially low-phytate lines planted at VAES Eastern Virginia
Agricultural Research and Extension Center in Warsaw, VA. Yield test
consisted of 350 lines from 18 pedigrees in maturity groups IVE through
V. The top yielding 20% from each test were selected using adjusted
estimates of yield from analysis of a modified-augmented design. This
selection resulted in 88 lines from 15 different pedigrees.
We collected the following data on the top yielding 20% of lines:
Three replications of the inorganic P (Pi) colorimetric assay (Figure 1) to
estimate phytate content.
Phytate content is inversely related to inorganic P content (Scaboo et al.,
2009) as seen in Figure 2. Pi concentration is presented as μg/g dry
weight. One replication of sugar extraction and HPLC analysis of sugar
content (Cicek et al., 2006). Sucrose and stachyose are presented as
percent of overall seed composition.
Leaf tissue samples from each line in the three yield tests were
collected during the season, and the top-yielding 20% were genotyped
for the VT LP marker (SATT 453) on an ABI 3100 genetic analyzer.
Results
The mean Pi content of samples was 564.3μg/g dry weight±414.9, with a minimum of
263.2 and maximum of 2006.4. Mean sucrose content was 5.6% on a dry weight basis ±1.5,
with a minimum of 3.3 and maximum of 9.9. Stachyose content had a mean of 3.2% on a
dry weight basis ± 1.2, with a minimum of 0.02 and maximum of 4.7. Figure X shows the
relationship between low phytate and high sucrose in V99-5089-derived material (Pi content
is inversely correlated with phytate, and Pi increases with increasing sucrose content).
Figure 4. Mean Sucrose % in Each Marker Allele Class
Figure 3. Mean Pi Concentrations of Each Marker Allele Class
Among nine pedigrees, four allele classes of the Satt 453 marker were assigned based on
grouping alleles of different fragment lengths. When including allele combinations this
produced nine different allele classes. The C class marker allele was most often associated
with the high PI and sucrose, low stachyose phenotype (Figures 3, 4 and 5). However, not all
lines with the C allele had the desired phenotype. This can be explained if the Satt 453 is
not closely linked to the V99-5089 low phytate gene. For example, lines with the V99-5089
 S97-1688 pedigree produced the most individuals with the C allele (Table 1), only half of
those actually have Pi concentrations associated with the LP phenotype (Table 2).
Disassociation of the marker from the
gene may be exacerbated by poor
germination in the low phytate lines of
n=2
n = 25
n=1
n=1
n=8
n = 22
this pedigree.
n=3
Two pedigrees did not produce
n=4
n = 16
expected LP lines with the C class marker
allele or appropriate phenotypes. Because
the LP trait is associated with poor seed
germination, it is possible that the LP lines
in these two pedigrees did not germinate
well or at all. Additionally, the LP lines in
Figure 5. Mean Stachyose % in Each Marker Allele Class
these
A
B
C
D
AB AC BD CD AD total
Pedigree
pedigrees
(V99-5089 x V97-7158) x (V99-8097 x GP2608)
4
3
2
9
may not
(V99-5089 x V99-8060) x (V99-8097 x GP2608)
2
2
4
Table 1.
have been
Number of lines
V94-2800 X (V95-7456 X V99-5089)
5
5
selected
of each
V95-7456 x V99-5089
1
3
4
pedigree in
among the
V97-9003 X (V99-5089 X MN 1401)
5
2
1
8
each marker
top
V97-9003 X (V99-5089 X V99-8060)
4
1
1
1
7
class
V99-5089 x Mn1401
1
6
2
2
1
12
yielding
V99-5089 x S97-1688
1
9
12
4
26
20%.
1
V99-6364 X V99-5089
TOTAL
21
7
15
1
21
1
3
1
4
2
Figure 1. Colorimetric assay of Pi content
Figure 2. Relationship between mean Pi
concentration and sucrose % content per one run
of each of the individual LP lines selected from
(the inverse relationship between phytate and
sucrose).
Sample
Marker Class
Pi Concentration
(μg/g dry weight)
V07-2059
D
326.29
V07-2035
D
394.96
V07-2065
D
334.97
V07-2063
D
334.59
V07-2036
D
276.57
V07-2033
D
396.88
V07-2055
D
325.09
V07-2058
D
319.58
V07-2017
D
427.99
V07-2000
D
381.005
V07-2064
D
423.928
V07-2051
D
331.62
V07-1989
CD
992.14
V07-2020
CD
1147.01
V07-1997
CD
861.29
V07-1998
CD
502.63
V07-2072
C
358.28
V07-2061
C
2006.38
V07-2037
C
1732.73
V07-2040
C
388.14
V07-2010
C
383.95
V07-2074
C
447.02
V07-2044
C
382.49
V07-2013
C
1796.22
V07-1978
C
1725.37
V07-6720
C
410.68
V07-1979
B
783.10
Table 2. High-yielding lines
selected from the pedigree V995089  S97-1688, marker class,
and mean Pi concentration.
Lines highlighted in blue are low
phytate based on Pi
concentration.
Conclusions
• An allele of Satt 453 (marker class C) was discovered that was
associated with the low phytate and stachyose, high sucrose
phenotype in 9 diverse pedigrees, and can be used for marker-assisted
selection.
• However, because the Satt 453 marker produces many potential
alleles and is not tightly linked with the V99-5089 LP, the marker is not
breeder-friendly.
• Two pedigrees did not produce LP lines, which could be due to poor
germination of LP seeds or association of the LP traits with poor yields.
• The Pi assay is faster and easier for identification of LP lines than the
marker or sugar HPLC.
References
Cicek MS, Chen P, Maroof MAS, Buss GR. (2006). Interrelationships among Agronomic and
Seed Quality Traits in an Interspecific Soybean Recombinant Inbred Population. Crop
Science 46: 1253-1259.
Erdman, JW Jr. (1979). Oilseed phytates: nutritional implications. Journal of American Oil
Chemists Society 56: 736.
Feng JY, Hai JZ, Xue LR, Shen LZ, Xu, JF, Qing YS. (2007). Generation and characterization of
two novel low phytate mutations in soybean (Glycine max L. Merr.) Theoretical and
Applied Genetics 115:945-957.
Meis SJ, Fehr WR, Schnebly SR. (2003). Seed Source effect on field emergence of soybean
lines with reduced phytate and raffinose saccharides. Crop Science 43:1336-1339.
Raboy V, Gerbasi PF, Young, KA, Stoneberg SD, Pickett SG, Bauman AT, Murthy PPN,
Sheridan WF, Ertl DS. (2000). Origin and Seed Phenotype of Maize low phytic acid 1-1 and
low phytic acid 2-11. Plant Physiology 124:355–368.
Scaboo et al. 2009. Confirmation of Molecular. Markers and Agronomic Traits Associated
with Seed Phytate Content in Two Soybean RIL Populations. Crop Science 49:426-432.