Association of Inorganic Phosphorus with a Molecular Marker Linked to a Novel Low Phytate Association of Inorganic Phosphorus with a Molecular Marker Linked to a Novel Low Phytate MutationMutation

Sarah Burleson, Dr. Katy Martin Rainey, Laura Maupin, Dr. Luciana RossoSarah Burleson, Dr. Katy Martin Rainey, Laura Maupin, Dr. Luciana Rossosburleso@vt.edu, Department of Crop and Soil Environmental Sciences, Virginia Tech, Blacksburg, VAsburleso@vt.edu, Department of Crop and Soil Environmental Sciences, Virginia Tech, Blacksburg, VA

Introduction 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.

ConclusionsConclusions• 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.

ReferencesReferences 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.

ObjectivesObjectives• 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 MethodsMaterials 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.

ResultsResults 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).

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).

Figure 3. Mean Pi Concentrations of Each Marker Allele Class

Table 2. High-yielding lines selected from the pedigree V99-5089 S97-1688, marker class, and mean Pi concentration. Lines highlighted in blue are low phytate based on Pi concentration.

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).

783.10BV07-1979

410.68CV07-6720

1725.37CV07-1978

1796.22CV07-2013

382.49CV07-2044

447.02CV07-2074

383.95CV07-2010

388.14CV07-2040

1732.73CV07-2037

2006.38CV07-2061

358.28CV07-2072

502.63CDV07-1998

861.29CDV07-1997

1147.01CDV07-2020

992.14CDV07-1989

331.62DV07-2051

423.928DV07-2064

381.005DV07-2000

427.99DV07-2017

319.58DV07-2058

325.09DV07-2055

396.88DV07-2033

276.57DV07-2036

334.59DV07-2063

334.97DV07-2065

394.96DV07-2035

326.29DV07-2059

Pi Concentration (μg/g dry weight)Marker ClassSample

Pedigree A B C D AB AC BD CD AD total

(V99-5089 x V97-7158) x (V99-8097 x GP2608) 4     3         2 9

(V99-5089 x V99-8060) x (V99-8097 x GP2608) 2   2             4

V94-2800 X (V95-7456 X V99-5089) 5                 5

V95-7456 x V99-5089     1 3           4

V97-9003 X (V99-5089 X MN 1401) 5   2     1       8

V97-9003 X (V99-5089 X V99-8060) 4   1 1 1         7

V99-5089 x Mn1401 1 6   2   2 1     12

V99-5089 x S97-1688   1 9 12       4   26

V99-6364 X V99-5089     1             1

TOTAL 21 7 15 21 1 3 1 4 2

Figure 1. Colorimetric assay of Pi content

n = 25n = 8

n = 16

n = 22n = 1

n = 3

n = 2

n = 4

n = 1

Figure 5. Mean Stachyose % in Each Marker Allele Class

Figure 4. Mean Sucrose % in Each Marker Allele Class

Table 1. Number of lines of each pedigree in each marker class

Disassociation of the marker from the gene may be exacerbated by poor germination in the low phytate lines of this pedigree. Two pedigrees did not produce 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 these

pedigrees may not have been selected among the top yielding 20%.