Abstract
The structure of chalcone synthase (CHS) gene repeats in different alleles of the I (inhibitor) locus in soybean spawns endogenous RNA interference (RNAi) that leads to phenotypic change in seed coat color of this major agronomic crop. Here, we examined CHS gene copy number by digital PCR and single nucleotide polymorphisms (SNPs) through whole genome resequencing of 15 cultivars that varied in alleles of the I locus (I, ii, ik, and i) that control the pattern distribution of pigments in the seed coats. Lines homozygous for the ii allele had the highest copy number followed by the I and ik cultivars which were more related to each other than to the lines with ii alleles. Some of the recessive i alleles were spontaneous mutations, and each revealed a loss of copy number by digital PCR relative to the parent varieties. Amplicon sequencing and whole genome resequencing determined that the breakpoints of several ii to i mutations resulted from nonallelic homologous recombination (NAHR) events between CHS genes located in segmental duplications leading to large 138‐kilobase deletions that erase the structure generating the CHS siRNAs along with eight other non‐CHS genes. Functional hybrid CHS genes (designated CHS5:1) were formed in the process and represent rare examples of NAHR in higher plants that have been captured by examining spontaneous mutational events in isogenic mutant lines.
Highlights
Repetitive sequences are extensive in many organisms including plant genomes
Reduced Chalcone synthase (CHS) mRNAs and CHS activity were found in developing seed coats of soybean iso‐ lines with the dominant I or ii alleles compared to varieties that are homozygous for a recessive i allele that specifies fully pigmented seed coats (Wang, Todd, & Vodkin, 1994)
BAC sequencing and next‐generation sequence (NGS) sequencing of small RNA populations showed that short interfering RNAs related to CHS genes were produced in the Williams ii genotype by an unusual 27‐kb region that consists of two identical 10.91‐kb inverted repeats of CHS1, CHS3, CHS4 and CHS4, CHS3, CHS1 that are separated by 5.71 kb of inter‐ vening sequence that codes for one hypothetical protein (Clough et al, 2004; Tuteja, Clough, Chan, & Vodkin, 2004; Tuteja & Vodkin, 2008; Tuteja, Zabala, Varala, Hudson, & Vodkin, 2009)
Summary
Repetitive sequences are extensive in many organisms including plant genomes. For example, 80% of the maize genome consists of repeats (Schnable et al, 2009) and regions of genome duplica‐ tion have occurred in the progenitors of Arabidopsis (Arabidopsis Genome Initiative, 2000) and Glycine max (soybean), an inbred species of great agronomic importance for animal and human feed (Schmutz et al, 2010). All five lines with ii genotype (yellow seed coat with black hilum) that produce the CHS siRNAs had the high‐ est copy numbers, with an average median copy number for all five lines at 10.6 copies We sequenced the 8‐kb PCR amplicons derived from the two other spontaneous mutations, UC9 and W130, resulting in data sets of 228,668 and 213,458 reads, respectively These two ampl‐ icons were identical to W55 except for a single base difference at position 719 with a transition of C to T (Figure S4). All three CHS5:1 hybrid amplicons (W55, W130, and UC9) are similar to CHS5 at amino acid positions 9 and Targets
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