Oxidosqualene Cyclases Involved in the Biosynthesis of Diverse Triterpenes in Camellia sasanqua.

Camellia sasanqua is an important economic plant that is rich in lipophilic triterpenols with pharmacological activities including antiallergic, anti-inflammatory, and anticancer activities. However, the key enzymes related to triterpene biosynthesis have seldom been studied in C. sasanqua. Oxidosqualene cyclases (OSCs) are the rate-limiting enzymes related to triterpene biosynthesis. In this study, seven putative OSC genes (CsOSC1-7) were mined from the C. sasanqua transcriptome. Six CsOSCs were characterized for the biosynthesis of diverse triterpene skeletons, including α-amyrin, ß-amyrin, δ-amyrin, dammarenediol-II, ψ-taraxasterol, taraxasterol, and cycloartenol by the heterologous expression system. CsOSC3 was a multiple functional α-amyrin synthase. Three key residues, Trp260, Tyr262, and Phe415, are critical to the catalytic performance of CsOSC3 judging from the results of molecular docking and site-directed mutagenesis. These findings provide important insights into the biosynthesis pathway of triterpenes in C. sasanqua.

Mapping causal genes and genetic interactions for agronomic traits using a large F2 population in rice.

Dissecting the genetic mechanisms underlying agronomic traits is of great importance for crop breeding. Agronomic traits are usually controlled by multiple quantitative trait loci (QTLs) and genetic interactions, and mapping the underlying causal genes is still labor-intensive and time-consuming. Here, we present a genetic tool for directly targeting the specific causal genes by using a single-gene resolution linkage map that was constructed from 3756 F2 rice plants via targeted sequencing technology and Tukey-Kramer multiple comparisons test. Three large- and moderate-effect QTLs, qHD6-2, qGL3-1, and qGW5-2, were successfully mapped to their specific causal genes, Hd1, GS3, and GW5, respectively. A complex genetic interaction network containing 30 QTL-QTL interactions was constructed, revealing that the alternative allele of hub QTL, qHD6-2, can hide or release the genetic contributions of the alleles at interacting loci. Moreover, arranging genetic interactions in the models lead to more accurate phenotypic predictions. These results provide a community resource and new feasible strategy for deciphering the genetic mechanisms of complex agronomic traits and accelerating crop breeding.

Improvement of multiplex semi-nested PCR system for screening of rare mutations by high-throughput sequencing.

The CRISPR/Cas9 system is an efficient gene-editing method, but it is difficult to obtain mutants for some specific species and special genome structures. A previously reported multiplexed, semi-nested PCR target-enrichment approach, which does not rely on transgenic technology, has been shown to be an effective and affordable strategy for the discovery of rare mutations in a large sodium azide-induced rice population. However, this strategy has the potential for further optimization. Here, we describe an improved multiplex semi-nested PCR target-enrichment strategy with simplified processing procedures, reduced false-positive rates and increased mutation detection frequency (1 mutation/73 Kb).

Construction of Commercial Sweet Cherry Linkage Maps and QTL Analysis for Trunk Diameter.

A cross between the sweet cherry (Prunus avium) cultivars 'Wanhongzhu' and 'Lapins' was performed to create a mapping population suitable for the construction of a linkage map. The specific-locus amplified fragment (SLAF) sequencing technique used as a single nucleotide polymorphism (SNP) discovery platform and generated 701 informative genotypic assays; these, along with 16 microsatellites (SSRs) and the incompatibility (S) gene, were used to build a map which comprised 8 linkage groups (LGs) and covered a genetic distance of 849.0 cM. The mean inter-marker distance was 1.18 cM and there were few gaps > 5 cM in length. Marker collinearity was maintained with the established peach genomic sequence. The map was used to show that trunk diameter (TD) is under the control of 4 loci, mapping to 3 different LGs. Different locus influenced TD at a varying stage of the tree's development. The high density 'W×L' genetic linkage map has the potential to enable high-resolution identification of QTLs of agronomically relevant traits, and accelerate sweet cherry breeding.

Discovery of rare mutations in extensively pooled DNA samples using multiple target enrichment.

Chemical mutagenesis is routinely used to create large numbers of rare mutations in plant and animal populations, which can be subsequently subjected to selection for beneficial traits and phenotypes that enable the characterization of gene functions. Several next-generation sequencing (NGS)-based target enrichment methods have been developed for the detection of mutations in target DNA regions. However, most of these methods aim to sequence a large number of target regions from a small number of individuals. Here, we demonstrate an effective and affordable strategy for the discovery of rare mutations in a large sodium azide-induced mutant rice population (F2 ). The integration of multiplex, semi-nested PCR combined with NGS library construction allowed for the amplification of multiple target DNA fragments for sequencing. The 8 × 8 × 8 tridimensional DNA sample pooling strategy enabled us to obtain DNA sequences of 512 individuals while only sequencing 24 samples. A stepwise filtering procedure was then elaborated to eliminate most of the false positives expected to arise through sequencing error, and the application of a simple Student's t-test against position-prone error allowed for the discovery of 16 mutations from 36 enriched targeted DNA fragments of 1024 mutagenized rice plants, all without any false calls.

Identification and fine mapping of quantitative trait loci for seed vigor in germination and seedling establishment in rice.

Seed vigor is an index of seed quality that is used to describe the rapid and uniform germination and the establishment of strong seedlings in any environmental conditions. Strong seed vigor in low-temperature germination conditions is particularly important in direct-sowing rice production systems. However, seed vigor has not been selected as an important breeding trait in traditional breeding programs due to its quantitative inherence. In this study, we identified and mapped eight quantitative trait loci (QTLs) for seed vigor by using a recombinant inbred population from a cross between rice (Oryza sativa L. ssp. indica) cultivars ZS97 and MH63. Conditional QTL analysis identified qSV-1, qSV-5b, qSV-6a, qSV-6b, and qSV-11 influenced seedling establishment and that qSV-5a, qSV-5c, and qSV-8 influenced only germination. Of these, qSV-1, qSV-5b, qSV-6a, qSV-6b, and qSV-8 were low-temperature-specific QTLs. Two major-effective QTLs, qSV-1, and qSV-5c were narrowed down to 1.13-Mbp and 400-kbp genomic regions, respectively. The results provide tightly linked DNA markers for the marker-assistant pyramiding of multiple positive alleles for increased seed vigor in both normal and low-temperature germination environments.