Eduardo R. Benitez

Nonsense Mutation of an MYB Transcription Factor Is Associated with Purple-Blue Flower Color in Soybean

Previous studies revealed that the recessive allele of the W2 locus generated purple-blue color and high vacuolar pH of flower petals in soybean. The location of W2 gene was reportedly close to simple sequence repeat marker Satt318 in molecular linkage group B2. We used information from the soybean genome to clone a candidate gene for W2. An MYB transcription factor gene belonging to G20 group was found in the vicinity of Satt318. Full-length cDNAs were cloned from purple-flowered cultivar Harosoy (W2 allele) and purple-blue flowered cultivars, Nezumisaya and w2-20 (w2 allele), by reverse transcription-PCR and designated as GmMYB-G20-1. Its open reading frame was 1083 bp long that encoded 361 amino acids in Harosoy. GmMYB-G20-1 had 53.7% similarity in amino acid sequence with the PH4 gene of petunia controlling blueness and vacuolar pH of flower petals. GmMYB-G20-1 of Nezumisaya and w2-20 had 3 base substitutions compared with that of Harosoy. The first substitution generated a stop codon in the MYB domain, resulting in truncated polypeptides. Cleaved amplified polymorphic sequence (CAPS) marker was developed to detect the base substitution. The polymorphic CAPS marker co-segregated with alleles at the W2 locus in the F(2) population. These results suggest that GmMYB-G20-1 might correspond to the W2 gene.

Single-Base Substitution in P1B-ATPase Gene Is Associated with a Major QTL for Seed Cadmium Concentration in Soybean

Cadmium (Cd) is a pollutant metal present in soils and toxic to biologic organisms. Previous studies using recombinant inbred lines derived from a cross between soybean (Glycine max [L.] Merr.) cultivars Harosoy and Fukuyutaka revealed a major quantitative trait loci for seed Cd concentration (cd1) in chromosome 9. The genome sequence of Williams 82 suggested that a P(1B)-ATPase gene involved in the transport of metals was located in the vicinity of cd1. cDNA sequencing suggested existence of two types of transcripts: one (GmHMA1a) consisting of 9 exons and 8 introns and the other (GmHMA1b) consisting of 8 exons and 7 introns. The putative polypeptide, GmHMA1a, consisted of 885 amino acids, whereas premature termination of translation of GmHMA1b generated a putative polypeptide with 559 amino acids. GmHMA1a had a 49.8% similarity with AtHMA3, a P(1B)-ATPase of Arabidopsis. GmHMA1a of Fukuyutaka differed from that of Harosoy by a single-base substitution that led to an amino acid substitution from E to G at amino acid position 608. A derived cleaved amplified polymorphic sequence (dCAPS) marker was developed to detect the base substitution, and this dCAPS marker was successfully associated with seed Cd concentration. Transgenic experiments may be necessary to verify that GmHMA1 actually corresponds to cd1.

A Single-Nucleotide Polymorphism in an Endo-1,4-β-Glucanase Gene Controls Seed Coat Permeability in Soybean

Physical dormancy, a structural feature of the seed coat known as hard seededness, is an important characteristic for adaptation of plants against unstable and unpredictable environments. To dissect the molecular basis of qHS1, a quantitative trait locus for hard seededness in soybean (Glycine max (L) Merr.), we developed a near-isogenic line (NIL) of a permeable (soft-seeded) cultivar, Tachinagaha, containing a hard-seed allele from wild soybean (G. soja) introduced by successive backcrossings. The hard-seed allele made the seed coat of Tachinagaha more rigid by increasing the amount of β-1,4-glucans in the outer layer of palisade cells of the seed coat on the dorsal side of seeds, known to be a point of entrance of water. Fine-mapping and subsequent expression and sequencing analyses revealed that qHS1 encodes an endo-1,4-β-glucanase. A single-nucleotide polymorphism (SNP) introduced an amino acid substitution in a substrate-binding cleft of the enzyme, possibly reducing or eliminating its affinity for substrates in permeable cultivars. Introduction of the genomic region of qHS1 from the impermeable (hard-seeded) NIL into the permeable cultivar Kariyutaka resulted in accumulation of β-1,4-glucan in the outer layer of palisade cells and production of hard seeds. The SNP allele found in the NIL was further associated with the occurrence of hard seeds in soybean cultivars of various origins. The findings of this and previous studies may indicate that qHS1 is involved in the accumulation of β-1,4-glucan derivatives such as xyloglucan and/or β-(1,3)(1,4)-glucan that reinforce the impermeability of seed coats in soybean.