A Chromosome Segment Substitution Library of Weedy Rice for Genetic Dissection of Complex Agronomic and Domestication Traits

Chromosome segment substitution lines (CSSLs) are a powerful alternative for locating quantitative trait loci (QTL), analyzing gene interactions, and providing starting materials for map-based cloning projects. We report the development and characterization of a CSSL library of a U.S. weedy rice accession ‘PSRR-1’ with genome-wide coverage in an adapted rice cultivar ‘Bengal’ background. The majority of the CSSLs carried a single defined weedy rice segment with an average introgression segment of 2.8 % of the donor genome. QTL mapping results for several agronomic and domestication traits from the CSSL population were compared with those obtained from two recombinant inbred line (RIL) populations involving the same weedy rice accession. There was congruence of major effect QTLs between both types of populations, but new and additional QTLs were detected in the CSSL population. Although, three major effect QTLs for plant height were detected on chromosomes 1, 4, and 8 in the CSSL population, the latter two escaped detection in both RIL populations. Since this was observed for many traits, epistasis may play a major role for the phenotypic variation observed in weedy rice. High levels of shattering and seed dormancy in weedy rice might result from an accumulation of many small effect QTLs. Several CSSLs with desirable agronomic traits (e.g. longer panicles, longer grains, and higher seed weight) identified in this study could be useful for rice breeding. Since weedy rice is a reservoir of genes for many weedy and agronomic attributes, the CSSL library will serve as a valuable resource to discover latent genetic diversity for improving crop productivity and understanding the plant domestication process through cloning and characterization of the underlying genes.     

Functional Loss of Semaphorin 3C and/or Semaphorin 3D and Their Epistatic Interaction with Ret Are Critical to Hirschsprung Disease Liability

Innervation of the gut is segmentally lost in Hirschsprung disease (HSCR), a consequence of cell-autonomous and non-autonomous defects in enteric neuronal cell differentiation, proliferation, migration, or survival. Rare, high-penetrance coding variants and common, low-penetrance non-coding variants in 13 genes are known to underlie HSCR risk, with the most frequent variants in the ret proto-oncogene (RET). We used a genome-wide association (220 trios) and replication (429 trios) study to reveal a second non-coding variant distal to RET and a non-coding allele on chromosome 7 within the class 3 Semaphorin gene cluster. Analysis in Ret wild-type and Ret-null mice demonstrates specific expression of Sema3a, Sema3c, and Sema3d in the enteric nervous system (ENS). In zebrafish embryos, sema3 knockdowns show reduction of migratory ENS precursors with complete ablation under conjoint ret loss of function. Seven candidate receptors of Sema3 proteins are also expressed within the mouse ENS and their expression is also lost in the ENS of Ret-null embryos. Sequencing of SEMA3A, SEMA3C, and SEMA3D in 254 HSCR-affected subjects followed by in silico protein structure modeling and functional analyses identified five disease-associated alleles with loss-of-function defects in semaphorin dimerization and binding to their cognate neuropilin and plexin receptors. Thus, semaphorin 3C/3D signaling is an evolutionarily conserved regulator of ENS development whose dys-regulation is a cause of enteric aganglionosis.     

Role of the Cytosolic Loop C2 and the C Terminus of YidC in Ribosome Binding and Insertion Activity

Members of the YidC/Oxa1/Alb3 protein family mediate membrane protein insertion, and this process is initiated by the assembly of YidC·ribosome nascent chain complexes at the inner leaflet of the lipid bilayer. The positively charged C terminus of Escherichia coli YidC plays a significant role in ribosome binding but is not the sole determinant because deletion does not completely abrogate ribosome binding. The positively charged cytosolic loops C1 and C2 of YidC may provide additional docking sites. We performed systematic sequential deletions within these cytosolic domains and studied their effect on the YidC insertase activity and interaction with translation-stalled (programmed) ribosome. Deletions within loop C1 strongly affected the activity of YidC in vivo but did not influence ribosome binding or substrate insertion, whereas loop C2 appeared to be involved in ribosome binding. Combining the latter deletion with the removal of the C terminus of YidC abolished YidC-mediated insertion. We propose that these two regions play an crucial role in the formation and stabilization of an active YidC·ribosome nascent chain complex, allowing for co-translational membrane insertion, whereas loop C1 may be involved in the downstream chaperone activity of YidC or in other protein-protein interactions.