<p style="text-align: justify;"><b>The Introduced DNA</b></p>

<p style="text-align: justify;">Molecular characterization of 305423 soybean to evaluate insert copy number, insert integrity and presence of plasmid backbone sequences was conducted by Southern blot analysis in combination with sequencing of the inserted DNA in 305423 soybean.</p>

<p style="text-align: justify;">Extensive Southern analyses demonstrate that multiple intact and truncated copies of fragment PHP19340A have been inserted into 305423 soybean that contain, in total, eight copies of the <i>KTi3</i> promoter, seven copies of the <i>gm-fad2-1</i> gene fragment and five copies of the <i>KTi3</i> terminator. A single intact copy of fragment PHP17752A has been inserted into 305423 soybean. In addition, Southern blot analyses confirm the absence of all functional elements from the plasmid backbone in 305423 soybean, despite the presence of a small nonfunctional fragment of the plasmid backbone DNA.</p>

<p style="text-align: justify;">Sequence characterization of the inserted DNA in 305423 soybean confirms that 305423 soybean contains four insertions that comprise:</p>

<ul>
	<li style="text-align: justify;"><b>Insertion 1:</b> one truncated PHP19340A fragment with a truncated <i>KTi3</i> terminator and intact <i>gm-fad2-1</i> gene fragment and intact <i>KTi3</i> promoter, one intact PHP19340A fragment, one intact PHP17752A fragment, one truncated PHP19340A fragment with an intact <i>KTi3</i> promoter and a truncated <i>gm-fad2-1</i> gene fragment, and one truncated PHP19340A fragment with a truncated <i>KTi3</i> promoter and truncated <i>gm-fad2-1</i> gene fragment.</li>
	<li style="text-align: justify;"><b>Insertion 2:</b> one truncated PHP19340A fragment with a truncated <i>KTi3</i> promoter and with intact <i>gm-fad2-1</i> gene fragment and intact <i>KTi3</i> terminator.</li>
	<li style="text-align: justify;"><b>Insertion 3:</b> one truncated copy of the <i>KTi3</i> promoter with a nonfunctional 495 bp fragment of the plasmid backbone; and</li>
	<li style="text-align: justify;"><b>Insertion 4:</b> two truncated PHP19340A fragments in an inverted repeat configuration, both with a truncated <i>KTi3</i> promoter and intact <i>gmfad2-1</i> gene fragment and <i>KTi3</i> terminator.</li>
</ul>

<p style="text-align: justify;">The genetic material that is inserted in 305423 soybean is genetically linked and segregates following a typical pattern of Mendelian inheritance expected for a single, genetically-linked insertion locus.</p>

<p style="text-align: justify;"><b>Expressed Material</b></p>

<p style="text-align: justify;">The 305423 soybean has been obtained by introducing the <i>gm-fad2-1</i> gene fragment and the gm-hra gene into the soybean genome. The <i>gm-fad2-1</i> gene fragment is part of the coding region of the soybean omega-6 desaturase gene 1 (<i>FAD2-1</i>) and does not code for a functional protein. Transcription of the <i>gm-fad2-1</i> gene fragment in 305423 soybean seeds acts to suppress transcription of endogenous omega-6 desaturase, resulting in the high oleic phenotype.</p>

<p style="text-align: justify;">The <i>gm-hra</i> gene encodes the GM-HRA protein, an optimized version of the soybean acetolactate synthase (ALS). Expression of the GM-HRA protein in 305423 soybean, used as a selectable marker, confers tolerance to ALS-inhibiting herbicides.</p>

<p style="text-align: justify;">Northern analyses demonstrate that transcription of the endogenous <i>FAD2-1</i> gene is effectively silenced in 305423 soybean seed as intended by the genetic modification.</p>

<p style="text-align: justify;">The GM-HRA protein levels in 305423 soybean were measured in replicated samples of leaf, root, forage and grain tissues using a quantitative enzyme-linked immunosorbent assay (ELISA). Pioneer reports that the mean GM-HRA protein concentrations in 305423 soybean leaf, root, forage, and grain were 4.0, 0.18, 5.7, and 2.5 nanograms per milligram (ng/mg) tissue dry weight respectively. Pioneer concluded that the above results confirm that the expression of the GM-HRA protein in 305423 soybean is constitutive.</p>
