Differential RNA expression of Glyma09g36983 and related MYB transcription factor genes in seed color isolines.

<p>RPKMs are shown for seed coats from seed of two seed fresh weight ranges denoted 300–400 and 400–500 mg as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111959#pone-0111959-g004" target="_blank">Figure 4A</a>.</p><p>Superscripts 1 and 2 for the 400–500 mg seed fresh weight represent two independent RNAseq extractions and sequencing determinations.</p><p>Glyma09g36966, Glyma09g37010, and Glyma18g49670 have transcript sequence similarities of 90%, 87%, and 84%, respectively, to Glyma09g36983.</p><p>Glyma09g36983 in Phytozome <i>G. max</i>_189 a1_1.1 was previously named Glyma09g36990 in Phytozome <i>G. max</i>_109 a1_1.0.</p><p>Glyma09g36966 in Phytozome <i>G. max</i>_189 a1_1.1 was previously named Glyma09g36970 in Phytozome <i>G. max</i>_109 a1_1.0.</p><p>Differential RNA expression of Glyma09g36983 and related MYB transcription factor genes in seed color isolines.</p

Differential Expression of Four Genes That Function in the Last Steps of Anthocyanin and Proanthocyanidin Synthesis Between the Black Seeded RM30-<i>R</i>* and Brown Seeded RM38-<i>R*</i> Lines.

<p>Transcript levels are in RPKMs plotted against the same five stages of seed coat development as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111959#pone-0111959-g004" target="_blank">Figure 4</a> for RM30-<i>R</i>* where the <i>R*</i> allele is interrupted by the 13 kb <i>TgmR*</i> insertion (profile in blue) and the RM38-<i>r</i> line where the <i>r</i> allele is not interrupted by <i>TgmR*</i> but has a “C”-nt deletion in Exon2 (red profile). Graphs have different scales. The Glyma models for the indicated genes are as following. (A) ANS3 (Glyma11g03010.1); (B) UFGT2 (Glyma08g07130.1); (C) ANR1 (Glyma08g06630.1) and (D) AOMT (Glyma05g36210.1. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111959#pone-0111959-g007" target="_blank">Figure 7</a> for the pathway abbreviations.</p

The 13 kb CACTA Insertion, Designated <i>TgmR*</i>, and its Subterminal Repeats Within the <i>r^m</i> and <i>R*</i> Alleles.

<p>(A) Schematics of the CACTA transposon (<i>TgmR*</i>) insertion in Intron2 of the <i>R</i> locus encoding a R2R3 MYB transcription factor. The <i>R</i> locus gene, Glyma09g36983, depicted in gray (exons) and blue (introns) has a 13 kb CACTA transposon insertion 110 bp from the beginning of Intron2 in the <i>R</i> locus of both the variegated RM55-<i>r^m</i> and the black-seeded revertant RM30-<i>R</i>* soybean isolines. The ATG duplication is shown at the site of insertion. The locations of the two primers used to amplify the 14 kb fragment containing the transposon insertion are depicted by the two small arrows, FP1 at the 5′ UTR and RPB in Intron2 near the insertion site. The 13 kb <i>TgmR*</i> transposon is a CACTA element based on the terminal ends sequences and the <i>Tnp2</i> transposase it encodes. Both the location of the transposase and the end sequences are marked. Sequences of high homology to this transposase region were found in chromosomes Gm05 and Gm15 and the Glyma models predicted for the Williams 82 genome are indicated. Likewise, the 3′ end of <i>TgmR*</i> has high homology to a sequence in chromosome Gm08 and to <i>Tgm1</i>, another smaller CACTA element of the soybean. Green and blue arrows represent the putative ORF1 (Tnp2 transposase) and ORF2 respectively. (B) Alignment of multiple CACTA transposon subterminal repeated motifs. The consensus sequences of the <i>TgmR*</i> subterminal direct repeats were aligned to other CACTA consensus sequences from other <i>Glycine max</i> transposons (<i>Tgm1, Tgmt*, Tgmw4m</i> and <i>Tgm-Express1</i>) in addition to other subterminal repeats from CACTA transposons in other plants. Portions of the sequence domains for TNPA-like protein binding are boxed. One of these motifs are GC rich (left) and the second AT rich (right). Based on the similarities of these terminal repeat motifs, it is clear that <i>TgmR*</i> is more closely related to <i>Tgm1</i> than to the other three characterized <i>G. max</i> transposons and all of the other CACTA transposon motifs in the lower panel.</p

Methylation Affects Transposition and Splicing of a Large CACTA Transposon from a MYB Transcription Factor Regulating Anthocyanin Synthase Genes in Soybean Seed Coats

<div><p>We determined the molecular basis of three soybean lines that vary in seed coat color at the <i>R</i> locus which is thought to encode a MYB transcription factor. RM55-<i>r^m</i> is homozygous for a mutable allele (<i>r^m</i>) that specifies black and brown striped seeds; RM30-<i>R*</i> is a stable black revertant isoline derived from the mutable line; and RM38-<i>r</i> has brown seed coats due to a recessive <i>r</i> allele shown to translate a truncated MYB protein. Using long range PCR, 454 sequencing of amplicons, and whole genome re-sequencing, we determined that the variegated RM55-<i>r^m</i> line had a 13 kb CACTA subfamily transposon insertion (designated <i>TgmR*</i>) at a position 110 bp from the beginning of Intron2 of the <i>R</i> locus, Glyma09g36983. Although the MYB encoded by <i>R</i> was expressed at only very low levels in older seed coats of the black revertant RM30-<i>R*</i> line, it upregulated expression of anthocyanidin synthase genes (<i>ANS2, ANS3</i>) to promote the synthesis of anthocyanins. Surprisingly, the RM30-<i>R*</i> revertant also carried the 13 kb <i>TgmR*</i> insertion in Intron2. Using RNA-Seq, we showed that intron splicing was accurate, albeit at lower levels, despite the presence of the 13 kb <i>TgmR*</i> element. As determined by whole genome methylation sequencing, we demonstrate that the <i>TgmR*</i> sequence was relatively more methylated in RM30-<i>R*</i> than in the mutable RM55-<i>r^m</i> progenitor line. The stabilized and more methylated RM30-<i>R*</i> revertant line apparently lacks effective binding of a transposae to its subterminal repeats, thus allowing intron splicing to proceed resulting in sufficient MYB protein to stimulate anthocyanin production and thus black seed coats. In this regard, the <i>TgmR*</i> element in soybean resembles McClintock's <i>Spm-</i>suppressible and change-of-state alleles of maize. This comparison explains the opposite effects of the <i>TgmR*</i> element on intron splicing of the MYB gene in which it resides depending on the methylation state of the element.</p></div

Amplification of a Large Insertion in the Glyma09g36983 Gene of RM55-<i>r^m</i> and the Revertant Black RM30-<i>R</i>* Isolines.

<p>(A) Agarose gel displaying a ∼14 kb PCR fragment in the RM55-<i>r^m</i> mutable and RM30<i>-R</i>* revertant alleles of the <i>R</i> locus. A small 929 bp fragment was the only amplification product from the <i>r</i> allele of the RM38-<i>r</i> brown-seeded line and it was the expected size of the DNA fragment comprised between the two oligo DNA primers (FP, forward primer: R6990FP1 and RP, reverse primer: R6990RPB) designed to amplify the 5′end portion of the Glyma09g36983 gene encoding a putative R2R3 MYB transcription factor. The 929 bp fragment was also an amplification product from the DNA of the mutable RM55-<i>r^m</i>. These results predict an insertion of 13 kb in one allele of the mutable RM55-<i>r^m</i> line that is maintained in the revertant RM30-<i>R</i>* black seed isoline. (B) Agarose gel displaying a 1.45 kb PCR fragment of the Glyma09g36983 gene amplified from four other soybean varieties with the standard <i>R</i> allele as well as from two soybean lines with an <i>r</i> allele (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111959#pone-0111959-t001" target="_blank">Table 1</a> for full genotypes). The reverse primer in these instances was R6990RP1, situated 491 bp downstream from R6990RPB used in (A). The size marker “M” is a 1 kb ladder.</p

Bismark bisulfite-seq methylation results for the Tgm<i>R*</i> CACTA transposon insertion and upstream/downstream regions of Glyma09g36983 in three soybean lines: RM30-<i>R*</i> black, RM55-<i>r^m</i> striped and UC44-<i>R</i> black seed coat phenotypes.

<p>Bismark bisulfite-seq methylation results for the Tgm<i>R*</i> CACTA transposon insertion and upstream/downstream regions of Glyma09g36983 in three soybean lines: RM30-<i>R*</i> black, RM55-<i>r^m</i> striped and UC44-<i>R</i> black seed coat phenotypes.</p

Differential Expression of Genes that Function in the Early Steps of the Anthocyanin and Proanthocyanidin Biosynthetic Pathways Between the Black-Seeded RM30-<i>R</i>* and Brown-Seeded RM38-<i>r</i> Lines.

<p>Transcript levels are in RPKMs plotted against the same five stages of seed coat development as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111959#pone-0111959-g004" target="_blank">Figure 4</a> for RM30-<i>R</i>* where the <i>R*</i> allele is interrupted by the 13 kb <i>TgmR*</i> insertion (profile in blue) and the RM38-<i>r</i> line where the <i>r</i> allele is not interrupted by <i>TgmR*</i> but has a “C”-nt deletion in Exon2 (red profile). Graphs have different scales. The Glyma models for the indicated genes are as follows: <i>CHS7</i> (Glyma01g43880.1); <i>CHS8</i> (Glyma11g01350.1); <i>CHI1A</i> (Glyma20g38560.1); <i>CHI2</i> (Glyma20g38580.1); <i>F3′H</i> (Glyma06g21920.1); <i>F3′5′H</i> (Glyma13g04210.1); <i>F3H</i> (Glyma02g05450.1); <i>DFR1</i> (Glyma14g07940.1); <i>DFR2</i> (Glyma17g37060.1. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111959#pone-0111959-g007" target="_blank">Figure 7</a> for the pathway abbreviations.</p

Genotypes and phenotypes of soybean cultivars and mutant isolines used in this study.

<p>All cultivars are homozygous for the alleles indicated. The varieties are searchable by the PI number in the USDA Germplasm Resources Information Network (GRIN). NA, not applicable.</p><p>L numbers are isoline number designations. The <i>r^m</i> allele from PI91073 that specifies a variegated phenotype had been repetitively backcrossed into the Clark brown seeded isoline L67-3484 (here designated as RM38 with the <i>r</i> allele) to create the variegated Clark isoline L72-2040 (here designated as RM55 with the <i>r^m</i>) by R.L. Bernard of the USDA. The L72-2040 line demonstrated somatic and germinal instability to yield fully black seed. One of these lines is here designated as RM30 and the gene symbol R* is used to designated this revertants of the <i>r^m</i> line from the standard <i>R</i> allele that specifics black seed coats.</p><p>Genotypes and phenotypes of soybean cultivars and mutant isolines used in this study.</p

Phenotypes of Seeds Coats at Late Developmental Stages for Three Soybean Isolines.

<p>Differential anthocyanin expression in the seed coats of three soybean isolines with variants of the <i>R</i> locus alleles. RM55-<i>r^m</i> with variegated black-brown seed coat; RM30-<i>R</i>* a stable revertant black-seeded isoline derived from RM55-<i>r^m</i>; and RM38-<i>r</i> a brown seeded isoline used as the recurrent parent. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111959#pone-0111959-t001" target="_blank">Table 1</a> for more information. Left, green seed of approximately 300–400 mg fresh weight; middle, dessicating seed of approximately 300–400 mg fresh weight; right, mature dry seed.</p

Distribution of Methylation in the <i>TgmR</i>* Transposon (A) and in the Upstream (B) and Downstream Regions (C) of the <i>R</i> locus in the Black-seeded RM30-<i>R*</i> and the Mutable RM55-<i>r^m</i> Isolines, and of the UC44 line with a Standard <i>R</i> Allele.

<p>All graphs were created by the sequence alignment tool of the Integrative Genomics Viewer (IGV) browser. (A) <i>TgmR*</i> methylation regions. The 13 bp CACTA inverted repeat ends and subterminal repeats are marked IR. Lower levels of methylated sequences (marked by red arrows) are found aligning to the subterminal repeats of the <i>TgmR</i>* element and the 3′-end of the predicted ORF2 transcript in the striped mutable RM55-<i>r^m</i> line. The black-seeded RM30-<i>R*</i> line containing the <i>TgmR*</i> insertion and the black-seeded UC44-<i>R</i>, line which lacks the <i>TgmR*</i> insertion, present nearly identical patterns of methylation at those three differentiated areas of the <i>TgmR</i>* element. (B) Upstream regions and (C) downstream regions of the <i>TgmR*</i> insertion site in the <i>R</i> gene. A few methylation differences appear between the three lines in the upstream and downstream regions, but the most distinct are those for UC44-<i>R</i> (Williams self-black seed) which may represent varietal differences. Gene represented by green arrows are: A (Glyma09g36941); B (Glyma09g36950); C (Glyma09g36966); D (Glyma09g36983); E (Glyma09g37000) and F (Glyma09g37010). The upstream and downstream schematics are not drawn to scale.</p