Endonuclease heteroduplex mismatch cleavage for detecting mutation genetic variation of trypsin inhibitors in soybean

The objective of this work was to evaluate the genetic variation of trypsin inhibitor in cultivated (Glycine max L.) and wild (Glycine sofa Siebold & Zucc.) soybean varieties. Genetic variations of the Kunitz trypsin inhibitor, represented by a 21-kD protein (KTI), and of the Bowman-Birk trypsin chymotrypsin inhibitor (BBI) were evaluated in cultivated (G. max) and wild (G. sofa) soybean varieties. Endonuclease heteroduplex mismatch cleavage assays were performed to detect mutations in the KTI gene, with a single-stranded specific nuclease obtained from celery extracts (CEL I). The investigated soybean varieties showed low level of genetic variation in KTI and BBI. PCR-RFLP analysis divided the BBI-A type into subtypes A1 and A2, and showed that Tib type of KTI is the dominant type. Digestion with restriction enzymes was not able to detect differences between ti-null and other types of Ti alleles, while the endonuclease heteroduplex mismatch cleavage assay with CEL I could detect ti-null type. The digestion method with CEL I provides a simple and useful genetic tool for SNP analysis. The presented method can be used as a tool for fast and useful screening of desired genotypes in future breeding programs of soybean

APETALA 2‐

The spikelet is the basic unit of the grass inflorescence. In tetraploid (Triticum turgidum) and hexaploid wheat (Triticum aestivum), the spikelet is a short indeterminate branch with two proximal sterile bracts (glumes) followed by a variable number of florets, each including a bract (lemma) with an axillary flower. Varying levels of miR172 and/or its target gene Q (AP2L5) result in gradual transitions of glumes to lemmas, and vice versa. Here, we show that AP2L5 and its related paralog AP2L2 play critical and redundant roles in the specification of axillary floral meristems and lemma identity. AP2L2, also targeted by miR172, displayed similar expression profiles to AP2L5 during spikelet development. Loss-of-function mutants in both homeologs of AP2L2 (henceforth ap2l2) developed normal spikelets, but ap2l2 ap2l5 double mutants generated spikelets with multiple empty bracts before transitioning to florets. The coordinated nature of these changes suggest an early role of these genes in floret development. Moreover, the flowers of ap2l2 ap2l5 mutants showed organ defects in paleas and lodicules, including the homeotic conversion of lodicules into carpels. Mutations in the miR172 target site of AP2L2 were associated with reduced plant height, more compact spikes, promotion of lemma-like characters in glumes and smaller lodicules. Taken together, our results show that the balance in the expression of miR172 and AP2-like genes is crucial for the correct development of spikelets and florets, and that this balance has been altered during the process of wheat and barley (Hordeum vulgare) domestication. The manipulation of this regulatory module provides an opportunity to modify spikelet architecture and improve grain yield