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First published online October 9, 2009; 10.1105/tpc.109.069856 The Plant Cell 21:3063-3077 (2009) © 2009 American Society of Plant Biologists OPEN ACCESS ARTICLE
Endogenous, Tissue-Specific Short Interfering RNAs Silence the Chalcone Synthase Gene Family in Glycine max Seed Coats[W],[OA]Department of Crop Sciences, University of Illinois, Urbana, Illinois 61801S 1 Address correspondence to l-vodkin{at}illinois.edu.
Two dominant alleles of the I locus in Glycine max silence nine chalcone synthase (CHS) genes to inhibit function of the flavonoid pathway in the seed coat. We describe here the intricacies of this naturally occurring silencing mechanism based on results from small RNA gel blots and high-throughput sequencing of small RNA populations. The two dominant alleles of the I locus encompass a 27-kb region containing two perfectly repeated and inverted clusters of three chalcone synthase genes (CHS1, CHS3, and CHS4). This structure silences the expression of all CHS genes, including CHS7 and CHS8, located on other chromosomes. The CHS short interfering RNAs (siRNAs) sequenced support a mechanism by which RNAs transcribed from the CHS inverted repeat form aberrant double-stranded RNAs that become substrates for dicer-like ribonuclease. The resulting primary siRNAs become guides that target the mRNAs of the nonlinked, highly expressed CHS7 and CHS8 genes, followed by subsequent amplification of CHS7 and CHS8 secondary siRNAs by RNA-dependent RNA polymerase. Most remarkably, this silencing mechanism occurs only in one tissue, the seed coat, as shown by the lack of CHS siRNAs in cotyledons and vegetative tissues. Thus, production of the trigger double-stranded RNA that initiates the process occurs in a specific tissue and represents an example of naturally occurring inhibition of a metabolic pathway by siRNAs in one tissue while allowing expression of the pathway and synthesis of valuable secondary metabolites in all other organs/tissues of the plant.
Knowledge of the RNA silencing pathway in plants (also known as RNA interference) is now advanced (reviewed in Baulcombe, 2004  ; Matzke and Matzke, 2004; Zamore and Haley, 2005; Chapman and Carrington, 2007; Eamens et al., 2008; Ramachandran and Chen, 2008; Carthew and Sontheimer, 2009), but relatively few examples exist of regulation of a specific plant phenotype by naturally occurring variation in the pathway. The soybean (Glycine max) I (inhibitor) locus, an unusual cluster arrangement of chalcone synthase (CHS) genes that inhibits seed coat pigmentation, is one such example of a silencing locus (Todd and Vodkin, 1996; Tuteja et al., 2004) mediated through posttranscriptional RNA silencing that can be suppressed by a viral silencing suppressor protein (Senda et al., 2004). CHS is the first committed enzyme in the pathway to an extraordinarily diverse set of secondary products, including isoflavones in the seed cotyledons, defense compounds in the leaves, phenolic exudates of the roots, and anthocyanin pigments in the hypocotyls, trichomes, pods, and seed coats of certain genotypes. In this article, we report RNA analysis and high-throughput sequencing of small RNAs to detail that the biogenesis and accumulation of the CHS short interfering RNA (siRNA) silencing signal is limited to the seed coats of dominant I genotypes, thus explaining how the soybean plant can still express CHS transcripts required for the synthesis of secondary products in other tissues with I silencing genotypes.
In soybean, two dominant forms (I and ii) of the I locus inhibit pigmentation of the seed coat in a spatial manner resulting in a colorless seed or light yellow on the entire seed coat (I allele) or yellow seed coat with pigmented hilum where the seed coat attaches to the pod (ii allele). By contrast, the homozygous recessive i allele allows for pigment production and accumulation over the entire epidermal layer of the seed coat. Most cultivated soybean varieties have been selected for a yellow, nonpigmented seed coat (homozygous I or ii alleles) to mitigate the undesirable effects of the black or brown anthocyanin pigments on protein and oil extractions during processing of soybean products (Palmer et al., 2004
The I locus was initially identified as a region of duplicated and inverted CHS genes (CHS1, CHS3, and CHS4) (Todd and Vodkin, 1996
The six contiguous CHS1-3-4 genes in the inverted repeat clusters lead to spontaneous deletions and truncations of CHS genes manifested as mutations of the I locus. Spontaneous mutations of the dominant, silencing I or ii alleles to the recessive i alleles involve deletion of CHS promoter sequences from CHS4 or CHS1, paradoxically resulting in increased CHS7/CHS8 transcripts and pigmented soybean seed coats (Todd and Vodkin, 1996
The involvement of gene silencing characterized by the production of the 20- to 30-nucleotide small RNAs in the regulation of plant development is now a well-established occurrence (Carrington and Ambros, 2003 Here, we present results from both small RNA gel blots and deep sequencing of small RNA populations from several genotypes of soybean and demonstrate that the CHS siRNAs accumulated only in the yellow seed coats having either the dominant I or ii alleles and not in the pigmented seed coats with homozygous recessive i genotypes. However, the diagnostic CHS siRNAs did not accumulate in the cotyledons of genotypes with the dominant I or ii alleles, thus demonstrating the novelty of an endogenous inverted repeat driving RNA silencing in trans of nonlinked CHS family members in a tissue-specific manner. This system demonstrates a naturally occurring feature of small RNA biogenesis and accumulation not well defined in other endogenous silencing examples.
Since CHS is the first committed enzyme of the flavonoid pathway, the endogenous tissue-specific silencing phenomenon of the I locus leads to selective downregulation of the flavonoid pathway and pigment inhibition only in the seed coats of silencing genotypes, whereas the cotyledons continue to accumulate high levels of isoflavones, other products of the flavonoid pathway that are characteristic of soybean seed (Dhaubhadel et al., 2007
CHS-Derived siRNAs Found in the Seed Coats of both I and ii Dominant Allele Genotypes The classically defined I locus (inhibitor) is characterized by its four alleles: I, ii, ik, and i (in order of dominant to recessive forms) that affect the production and accumulation of anthocyanins and proanthocyanidins in a spatial manner in the soybean seed coat (Todd and Vodkin, 1993 ; Wang et al., 1994). The dominant I allele inhibits pigmentation over the entire seed coat, resulting in a light or yellow color on mature harvested seeds, whereas the ii and ik alleles restrict pigmentation to the hilum and saddle shaped regions, respectively. The homozygous recessive i allele allows for pigment production and accumulation in the epidermal layer of the seed coat, thus imparting a buff, brown, or black coloration depending upon other anthocyanin pathway alleles present (Palmer et al., 2004).
We investigated the presence of CHS-related siRNA species in seed coats of the nonpigmented (Richland, I), and hilum-pigmented isoline (Williams, ii) along with their corresponding mutant allele lines (T157, i and Williams 55, i) (Table 1
) using RNA gel blotting. The siRNAs were visualized via RNA gel blots probed with an antisense, in vitro–transcribed CHS7 probe. CHS7 was chosen as the probe since the nearly identical CHS7 and CHS8 genes are downregulated by the silencing I locus (Senda et al., 2004
Thus, both the dominant I allele and the dominant pattern form of the I locus, the ii allele typical of the cultivar Williams, result in silencing mediated by CHS siRNA production. The two lines used in our study, Richland (I) and Williams (ii), are the sources of the I and ii alleles in many modern cultivated varieties. Williams is also the cultivar that has recently been sequenced by the Joint Genome Institute (http://www.phytozome.net/soybean).
CHS siRNAs Are Absent in the Cotyledons of Seeds with the Dominant I Genotype
Highly Tissue-Specific Accumulation of CHS siRNA Conferred by the Dominant I and ii Alleles Our analysis of CHS-siRNAs was expanded to other tissues representing the vegetative parts of the plant. LMW RNA fractions from seed coats, cotyledons, roots, and leaves of the two isogenic pairs (Richland and T157 representing an I  i mutation and Williams and Williams 55 representing an ii i mutation) were separated on polyacrylamide gels and the RNA gel blots hybridized to the CHS7 antisense probe as described before.
Figure 3
clearly shows that sense CHS siRNAs accumulated in the seed coats of both the nonpigmented Richland (I) and hilum-only pigmented Williams (ii) cultivars. As shown before in Figure 1, no detectable hybridization to the CHS probe was observed in seed coats of their respective pigmented isolines, T157 (i) and Williams 55 (i). Intriguingly, no trace of CHS siRNAs was detected in the cotyledons, leaves, or roots of the yellow seeded cultivars Richland (I) and Williams (ii). These results were in accordance with our previously published findings of exclusive tissue-specific effect on reduction of CHS mRNA transcript levels only in the seed coats of the yellow seeded cultivars and not in the cotyledons and vegetative tissues of the yellow seeded cultivars. Together, these data (Figures 1 to 3
High-Throughput Sequencing of Small RNA Populations from Seed Coats and Cotyledons To ascertain and characterize the identity of the CHS siRNAs detected on gel blots, multiple small RNA libraries were generated and sequenced deeply by the Illumina high-throughput sequence by synthesis technology. Four small RNA libraries were sequenced (Table 1): seed coats from Richland (I, yellow seed coats), seed coats and cotyledons from Williams (ii, yellow seed coats with black hilum), and seed coats from mutant line Williams 55 (i, pigmented seed coats). A total of almost 15 million sequence reads (14,904,022) were obtained from the four libraries. As shown in Table 1, three libraries were sequenced at the same time to approximately three million reads. The fourth library from the pigmented genotype of the Williams 55 line carrying the i mutation produced twice the number of reads (six million) as it was sequenced at a later date when the yield from the Illumina flow cells had increased. The raw sequence reads were processed computationally to remove adapter sequences and from this pool of processed sequences, unique signatures representing at least five reads within each library were identified and selected for further analyses. The number of unique signatures ranged from 28,000 to 92,000 per library (Table 1). Normalization of the total counts of individual signatures was made based on three million raw reads. BLASTn searches to the Sanger miRNA database (http://microrna.sanger.ac.uk/) found relatively few, <1000, with matches to currently known and curated miRNAs, indicating that many represent siRNAs or previously unknown miRNAs. In this report, we focus on the CHS siRNAs found in the different tissues and genotypes that are active physiologically to effect a change in plant pigment phenotype.
Multiple CHS siRNAs Accumulate in the Seed Coats but Not the Cotyledons of the ii Yellow Seeded Genotype
Figure 4 illustrates the striking differences observed when both distribution and total counts of CHS siRNAs from the seed coat and cotyledon libraries of the hilum-pigmented yellow seeded Williams (ii) cultivar were compared. Very high counts of  25,000 CHS siRNAs that align to the individual CHS genes were observed for the highly similar CHS1-3-4 genes found in the inverted repeat CHS clusters of the silencing ii allele. However, only eight or fewer occurrences of CHS siRNAs were found in the cotyledon sequences, thereby providing unequivocal evidence that CHS siRNAs were found uniquely in the yellow seed coats in a tissue-specific manner.
Tissue-Specific CHS siRNAs That Silence CHS7 and CHS8 in the Dominant ii Genotype
The CHS multigene family has been divided into two subgroups on the basis of the degree of nucleotide identity in the open reading frames (Tuteja et al., 2004 Because the size of the target sequence influences the e value obtained from the BLAST algorithm and the BACs vary widely in size from 61,000 to >146,000 bases, we performed the BLAST analysis of each small RNA population to each of the nine individual CHS genes to attain an accurate, comparative number for CHS siRNAs aligning to the individual CHS genes.
All CHS genes contain one intron at the same position, and excluding their introns, the CHS genes are nearly identical in size at 1167 bases (CHS1-6 and CHS9) or 1170 bases (CHS7 and CHS8) from the ATG to the stop codon. Supplemental Data Set 2 and Supplemental Tables 2 and 3 online present the results. The number of unique signatures with 100% identity to each CHS gene in a pairwise comparison (see Supplemental Table 2 online) indicates that while CHS4 has 82% nucleotide similarity to both CHS7 or CHS8, only Specifically, Figure 5A illustrates the alignment of CHS siRNAs from the Williams (ii) seed coat with 100% identity to CHS7. Overall, the CHS siRNAs aligned through almost the entire length of the CHS gene exons and not at all to the introns. In contrast with the large number of CHS siRNA sequences that aligned with exon 2, only a few sequences aligned with exon 1. The majority of CHS siRNAs aligned with exon 2 to form a bell-shaped curve against both the sense and antisense strands. Figure 5 shows only the alignment results of the CHS siRNAs with more than 50 occurrences. As shown in Table 2 , the majority (976) of the total (1118) unique signatures had very few occurrences (5 to 50), while the remaining 13% (141) were represented many times (50 to 1000). Only 38 CHS siRNA unique species, including only three siRNAs with more than 50 counts, aligned with exon 1 of CHS7. None aligned with the intron, although some did appear to span the border, indicating that they arose from processed transcripts.
Since the frequency of each small RNA signature in the library generally reflects its relative abundance in the sample, the sequence repeats provide a quantitative expression measurement. Strikingly, of the 1118 unique siRNA signatures with perfect matches to CHS7 gene sequence, only 149 (13%) match perfectly to CHS4 gene sequence (see Supplemental Table 2 online). This finding illustrates that many of the siRNAs matching 100% to CHS7 originated from CHS7 (or the similar CHS8) transcripts after intron splicing, most likely as a result of amplification by RNA-dependent RNA polymerase (RdRP), dicer-like (DCL), and argonaute (AGO)-like effector complex that synthesize and cleave aberrant double-stranded RNA (dsRNA) into phased 21- to 22-nucleotide secondary siRNAs. CHS8 shows a very similar alignment of the CHS siRNAs (Table 2), as expected from the high sequence similarity between CHS7 and CHS8 (97% similar). The siRNAs that aligned uniquely to CHS1, CHS3, and CHS4 are evidence that they originated from transcripts of the inverted repeat on chromosome Gm8 where those CHS genes reside. We propose that some of these siRNA signatures with perfect matches to genes in the CHS1-3-4 and CHS4-3-1 clusters represent the primary siRNA guides that trigger the silencing of all CHS genes. The CHS7 and CHS8 sequence region that aligned with the largest number of siRNA signatures with very high counts must be the region most targeted by the primary siRNA-guided RNA-induced silencing complexes (RISC) (Figure 5A, framed region). This portion of the sequence comprises the central region 748 bp of exon 2 (975 to 1281 bp relative to the initiation codon). Likewise, the alignments of the Williams (ii) seed coat library CHS siRNAs to CHS1, CHS3, and CHS4 sequences of the inverted repeat also produced a similar alignment pattern (as shown in Figure 5B for the alignment of CHS siRNA with 100% identity to CHS4). This suggests that once silencing of all CHS genes is triggered by the CHS1-3-4 primary siRNA guides, a multitude of CHS siRNAs originating from any of the expressed CHS genes become guides to advance the targeting and posttranscriptional suppression of the entire CHS gene family.
The 21-Nucleotide siRNAs Are the Predominant Size Class of siRNAs with 100% Match to Individual CHS Genes
The Dominant I Allele Also Produces Complex, Heterogeneous CHS siRNAs Sequencing of the small RNA population from the immature seed coats of Richland (I, yellow) at the same stage of development as those of the Williams cultivar (ii, yellow with pigmented hilum) also yielded close to three million raw sequence reads and >30,000 unique small RNAs. Large numbers of CHS siRNAs were found, agreeing with the blot data of Figures 1 to 3  . The total number of CHS siRNAs found in the Richland population that map to each CHS gene is generally similar to that found for Williams (summarized in Table 3
from Supplemental Data Set 2 online). The CHS siRNAs from Richland also represented both strands and primarily mapped to exon 2. Some of the most abundant CHS siRNAs were the same tags as in Williams. For example, Table 4
shows some of the more abundant CHS siRNAs and the counts found in each library. Thus, both the dominant alleles (I and ii) are effective in silencing the targeted CHS genes through production of a heterogeneous siRNA population that largely maps to both strands in the middle of exon 2 of the individual CHS gene family members.
The ii i Mutation Abolishes CHS siRNAs from the Small RNA Population and Restores Pigmented Seed CoatsStructurally, the recessive i locus mutation in Williams 55 line is represented by a deletion that includes the CHS cluster B and extends into the promoter of CHS4 of cluster A (as illustrated in Figure 7 ). Examination of the number and distribution of CHS siRNAs in the seed coats of the two ii and i isogenic lines revealed the presence of a considerably higher number of CHS siRNA reads in the hilum-pigmented yellow seeded cultivar (Williams, ii) relative to the black seed coat of Williams 55 (i) (Table 3). In a Williams 55 seed coat library with >90,000 unique signatures and processed reads, only 16 different signatures totaling around 108 molecules per three million raw sequence reads mapped to any of the individual CHS gene family members. These few constitute only 0.03% of the number of CHS siRNAs found in the Williams (ii, yellow with pigmented hilum) and 0.06% of the CHS siRNAs found in Richland (I, yellow). The presence of large numbers of CHS-specific siRNAs in the seed coats of the dominant I and ii cultivars and not in the recessive i genotype is clear evidence of a suppressive effect of the inverted repeat I locus in soybean, which is mediated by a siRNA silencing pathway. Coupled with the small RNA gel blots of Figures 1 to 3 , these results confirm that the naturally occurring deletions in the CHS genes at the I locus that accompany ii i and I i mutations (Todd and Vodkin, 1996; Tuteja et al., 2004) serve to abolish the production of the small RNAs from the dominant forms of the I locus.
An Endogenous, Inverted Repeat, CHS-Coding Region Generates Heterogeneous CHS siRNAs Sequencing small RNAs to a depth of three million reads from the seed coats of two silencing alleles of the I locus (I, yellow seed coats, and ii, yellow seed coats with pigmented hilum) provided a wealth of data from which to determine the CHS-specific forms produced by these alleles. Based on alignments of these sequence signatures to the different CHS genomic sequences (Figure 4) and on data from RNA gel blots (Figures 1 to 3 ), we conclude that CHS small RNAs were generated and accumulated exclusively in seed coats of yellow seed soybeans with the dominant allele genotypes I and ii and not in cotyledons. The CHS siRNAs found in the seed coats ranged in size from 19 to 24 nucleotides, although the small 21-nucleotide species was the most abundant size class. These sequences aligned to both the sense and antisense strands of exons 1 and 2 of all the CHS gene sequences (CHS1 to CHS9). For all CHS genes, the numbers of signatures aligning to the sense strand were somewhat higher than to the antisense strand (Table 2). Many more siRNAs aligned to exon 2 than to exon 1. In exon 2, the larger numbers of unique signatures as well as the larger number of counts per signature were concentrated to the central sequence portion (Figure 5). Because there is sequence variation among the nine CHS genes, we were able to identify CHS siRNA signatures with 100% sequence identity to individual genes, thereby proving that the CHS siRNAs originated from transcripts of more than one CHS family member.
The silencing ii allele contains six genes CHS1-3-4 and CHS4-3-1 as two perfectly repeated and inverted 10.91-kb clusters separated by a 5.87-kb intervening region as sequenced in two individual BACs (77G7-a and 104J7) (Clough et al., 2004
It is not difficult to envision that long transcripts initiated from one promoter within the two clusters of the ii allele and that span very similar CHS genes in inverse orientation could fold and create aberrant dsRNAs that are then subjected to dicer-like enzyme complexes and resulting in 20- to 22-nucleotide primary CHS siRNAs. In Arabidopsis and other systems, different DCL ribonucleases orthologs (1 to 4) process the dsRNA into different siRNA size classes depending on their catalytic properties (Hamilton et al., 2002
Amplification of the Silencing Signal through the Action of an RNA-Dependent RNA Polymerase Is Deduced from the Specific CHS siRNA Sequences in the Seed Coat In summary, the pathway for CHS siRNA generation and accumulation must account for (1) the large number and distribution of unique siRNA signatures detected; (2) the range of siRNA sizes (20 to 22 nucleotides) with the small 21-nucleotide species being the most abundant, particularly for CHS7 and CHS8; (3) the lack of siRNAs derived from intron sequences; (4) the existence of siRNA with 100% sequence identity to genes CHS7 or CHS8 and lower similarity to genes CHS1, CHS3, and CHS4 and vice versa; (5) silencing of all nine nonidentical CHS gene coding regions, including those linked and those not linked to the long inverted repeat in Chromosome Gm8; and (6), the derivation of CHS7- and CHS8-specific siRNAs from both strands. The latter implies that such siRNAs are processed from dsRNA substrates produced by the pairing of sense transcripts with antisense copies derived from RdRP action. Figure 7 depicts the plausible succession of these steps schematically.
As with the transacting-siRNAs of Arabidopsis (Yoshikawa et al., 2005
Tissue-Specific Biogenesis of the CHS siRNAs from the Inverted Repeat I Locus Clusters in Seed Coats Is More Plausible Than Lack of Signal Amplification in Other Tissues
Conversely, the primary CHS siRNAs could potentially be generated from a dsRNA molecule produced in all tissues, but possibly they are not being amplified to detectable levels for lack of an RdRP enzyme in other tissues. RdRPs are involved in RNA amplification of primary siRNAs and generate more dsRNAs that are subsequently processed into the secondary siRNAs (Zamore and Haley, 2005
Likewise, we have sequenced libraries from other tissue and organ systems, including leaves and stems that also produce large numbers of small RNAs but only a handful of CHS-specific siRNAs similar to the very low percentages shown for the cotyledon library in Table 3. We have previously demonstrated that CHS transcripts in the leaves of Williams (ii), including those for CHS1, 3, 6, 7, and 8 in soybean leaves, are induced >1000-fold within 8 h after infection with the bacterial pathogen Pseudomonas syringae (Zabala et al., 2006
The CHS siRNAs Are Not Transported from the Seed Coat to the Developing Cotyledons or Other Tissues
The soybean seed coat, derived from the maternal ovular integuments, encloses the filial tissues (the embryo and the cotyledons) and includes two vascular bundles (the phloem and xylem elements) at the hilum, the point of attachment to the pod (Thorne, 1981
Regulation of an Important Pathway by Tissue-Specific siRNA Biogenesis
While much emphasis has been placed to date on the evolutionarily ancient and highly conserved miRNAs, examples of siRNAs more uniquely tied to a particular species are likely to arise. As illustrated by the CHS siRNA system, expansion of duplicate genes can potentially spawn a unique regulatory system in a physiological process during natural selection and evolution or during domestication of a plant species. Thus, siRNA regulation could be an important addition to our knowledge of plant allelic diversity and short-term evolutionary mechanisms. Allen et al. (2004) The small RNA sequencing populations from the seed coat and cotyledons have revealed a vast number of additional small RNAs (miRNAs or siRNAs) varying greatly in normalized sequence counts. Many have much higher occurrence than the CHS siRNAs characterized here and some also show tissue specificity. We have clearly shown that the CHS siRNAs are physiologically functional to downregulate a pathway and produce a visible trait difference, lack of seed coat pigmentation. Thus, we anticipate that continued investigation of the novel sequences revealed in these populations will lead to similar examples of regulation of other pathways in seed development as demonstrated here for the CHS siRNAs.
Plant Materials and Genetic Nomenclature The two isoline pairs of Glycine max used for this study were obtained from the USDA Soybean Germplasm Collections (Department of Crop Sciences, USDA/Agricultural Research Service University of Illinois, Urbana, IL). The genotypes of the four lines are described in Table 1. All lines are homozygous for the loci indicated, and only one of the alleles is shown for brevity in the tables and text. Plants were grown in the greenhouse and tissues harvested from at least four plants of each isoline. Leaves and roots were harvested from 4-week-old plants and quick frozen in liquid nitrogen. Seed coats and cotyledons were dissected from seeds at varying stages of development based on the fresh weight of the entire seed: 10 to 25 mg, 25 to 50 mg, 50 to 75 mg, 75 to 100 mg, and 100 to 200 mg. Dissected seed coats and cotyledons from seeds of the 50 to 75 mg weight range were fast frozen in liquid nitrogen. All tissues were stored at –70°C till further use.
Small RNA Extraction and Gel Blot Analysis
To the precipitate dissolved in water, polyethylene glycol (molecular weight 8000) and sodium chloride were added to a final concentration of 5% and 0.5 M, respectively, followed by incubation on ice for 30 min. High molecular weight nucleic acids were precipitated by centrifugation at 11,000 rpm for 20 min, while the LMW nucleic acids in the supernatant were recovered by ethanol precipitation at –20°C overnight. LMW RNA concentrations were measured on the NanoDrop ND1000 spectrophotometer (Nanodrop Technologies) and samples stored at –70°C until further use. For diagnostic purposes, the LMW RNA fractions were separated on a 1.2% agarose/3% formaldehyde gel and stained with ethidium bromide. The predominant stainable species of these gels was a band that runs at
Seventy-five micrograms of LMW RNA concentrated in 16 µL 50% formamide was denatured at 70°C for 10 min. Denatured LMW RNAs were fractionated on 15% polyacrylamide 7 M urea denaturing gels, transferred to Hybond-NX membrane (Amersham) using a Bio-Rad Trans-Blot apparatus (Bio-Rad) at 100 V for 1 h. The membranes were equilibrated on 20x SSC saturated filters, air-dried, and UV cross-linked (Stratalinker; Stratagene). Prehybridization was performed in 50% formamide, 7% SDS, 0.05 M NaHPO4/NaH2PO4, pH 7.0, 0.3 M NaCl, 5x Denhardt's solution, and 100 µg/mL sheared denatured salmon sperm DNA at 40°C for at least 2 h. Hybridization was performed in the same solution by adding the hydrolyzed [
For accurate sizing of the siRNA species, an RNA ladder (10 to 150 nucleotides) was used and radiolabeled with [ The CHS antisense riboprobe used for LMW RNA analysis was transcribed in vitro from the T7 promoter of a BamHI cleaved CHS7 EST, AI437793, by means of the MAXIscript In Vitro Transcription Kit (Ambion). AI437793 contains the full-length CHS7 open reading frame. Riboprobes were treated with RNase free DNase to remove the DNA template, and the 20 µL probe was hydrolyzed to an average size of 50 nucleotides with 300 µL of 0.2 M carbonate buffer (0.08 M NaHCO3 and 0.120 M Na2CO3) by incubating at 60°C for 3 h. Subsequently, 20 µL of 3M NaOAc, pH 5.0, was added to the hydrolyzed probe before adding the probe to the hybridization solution.
The 5S rRNA oligoprobe was used as a loading control. A 27-mer oligo (5'-GGTGCATTAGTGCTGGTATGATCGCAC-3') antisense to the soybean 5S rRNA encoding gene was
Sequencing of Small RNA Libraries and Data Analysis A total of three to six million reads that were 33 bases long were obtained from the deep sequencing of the above-mentioned libraries. Adapter trimming was performed using the first occurrences of substring TCG as the unique identifier for the beginning of the adapter (5'-TCGTATGCCGTCTTCTGCTTG-3'). The sizes of the small RNAs after adapter trimming ranged from 14 to 33 nucleotides, with the majority in the range of 19 to 24 nucleotides. Adapter trimmed sequences were compared to obtain the number of unique sequences and occurrences of each. At this stage, all sequences present more than five times were carried forward for subsequent comparisons.
Alignments of these curated small RNAs to each individual BAC sequence were made using BLAST (Altschul et al., 1990
Accession Numbers
Supplemental Data
We thank Pam Long, Sean Bloomfield, and Martin Blistrabas for assistance with data analysis. This work was supported by grants from the University of Illinois Critical Research Initiative Program, the USDA, the Illinois Soybean Association, and the United Soybean Board.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Lila O. Vodkin (l-vodkin{at}illinois.edu).
[W] Online version contains Web-only data.
[OA] Open access articles can be viewed online without a subscription. www.plantcell.org/cgi/doi/10.1105/tpc.109.069856 Received July 10, 2009; Revision received September 3, 2009. accepted September 16, 2009.
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-32P]UTP-labeled riboprobe or the [
-32P]dATP-labeled oligoprobe at 40°C for 15 to 20 h. The filters were washed in 2x SSC and 0.2% SDS at 40°C for 15 min and exposed to Hyperfilm (Amersham). 
