HLA-A*02 is the most prevalent and polymorphic major histocompatibility complex (MHC) allele family in humans. Functional differences have been revealed among subtypes, demanding further subtyping of HLA-A*02 in basic and clinical settings. However, the fast growing polymorphisms render traditional primer- or probe-based typing methods impractical and result in increasing ambiguities in direct sequence-based typing. In this study, we combined group-specific amplification and mono-allelic sequencing to design and validate a simple scheme for the complete screening and accurate subtyping of all 540 reported HLA-A*02 alleles. This scheme could be performed in routine labs to facilitate studies with an interest in HLA-A*02.

Plant somatic embryos are widely used in the fields of germplasm conservation, breeding for genetic engineering and artificial seed production. MicroRNAs (miRNAs) play pivotal roles in somatic embryogenesis (SE) regulation. However, their regulatory roles during various stages of SE remain unclear. In this study, six types of embryogenic samples of Lilium pumilum DC. Fisch., including organogenic callus, embryogenic callus induced for 4 weeks, embryogenic callus induced for 6 weeks, globular embryos, torpedo embryos and cotyledon embryos, were prepared for small RNA sequencing. The results revealed a total of 2,378,760 small RNA reads, among which the most common size was 24 nt. Four hundred and fifty-two known miRNAs, belonging to more than 86 families, 57 novel miRNAs and 40 miRNA*s were identified. The 86 known miRNA families were sorted according to an alignment with their homologs across 24 land plants into the following four categories: 23 highly conserved, 4 moderately conserved, 15 less conserved and 44 species-specific miRNAs. Differentially expressed known miRNAs were identified during various stages of SE. Subsequently, the expression levels of 12 differentially expressed miRNAs and 4 targets were validated using qRT-PCR. In addition, six samples were mixed in equal amounts for transcript sequencing, and the sequencing data were used as transcripts for miRNA target prediction. A total of 66,422 unigenes with an average length of 800 bp were assembled from 56,258,974 raw reads. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment indicated that 38,004 and 15,497 unigenes were successfully assigned to GO terms and KEGG pathways, respectively. Among the unigenes, 2,182 transcripts were predicted to be targets for 396 known miRNAs. The potential targets of the identified miRNAs were mostly classified into the following GO terms: cell, binding and metabolic process. Enriched KEGG analysis demonstrated that carbohydrate metabolism was the predominant pathway in Lilium SE. Thus, we performed systemic characterization, homology comparisons and profiling of miRNA expression, and we constructed an miRNA-target network during Lilium SE for the first time. Our findings establish a foundation for the further exploration of critical genes and elucidation of SE in Lilium.

Plant cell totipotency is one of the 25 major topics in current scientific research, and somatic embryos are good experimental material for studying cell totipotency. Polar auxin transport plays an important regulatory role in somatic embryogenesis (SE). However, little is known about the auxin transport genes and their regulatory mechanisms in Lilium SE. In this study, we applied single-molecule real-time (SMRT) sequencing to Lilium pumilum DC. Fisch. for the first time and obtained a total of 119,649 transcripts, of which 14 encoded auxin transport genes. Correlation analyses between somatic embryo induction and gene expression under different treatments revealed that auxin transport genes, especially ATP-binding cassette (ABC) transporter B family member 21 (ABCB21) and PIN-FORMED (PIN) LIKES 7 (PILS7), may be key players in SE, and the necessary duration of picloram (PIC) treatment to induce SE is as short as 3 days. Our research provides valuable genetic information on Lilium pumilum, elucidating the candidate auxin transport genes involved in SE and their influencing factors. This study lays a foundation for elucidating the regulatory mechanism of auxin transport in SE.

Auxin transport mediates the asymmetric distribution of auxin that determines the fate of cell development. Agrobacterium-mediated genetic transformation is an important technical means to study gene function. Our previous study showed that the expression levels of LpABCB21 and LpPILS7 are significantly up-regulated in the somatic embryogenesis (SE) of Lilium pumilum DC. Fisch. (L. pumilum), but the functions of both genes remain unclear. Here, the genetic transformation technology previously developed by our team based on the L.pumilum system was improved, and the genetic transformation efficiency increased by 5.7–13.0%. Use of overexpression and CRISPR/Cas9 technology produced three overexpression and seven mutant lines of LpABCB21, and seven overexpression and six mutant lines of LpPILS7. Analysis of the differences in somatic embryo induction of transgenic lines confirmed that LpABCB21 regulates the early formation of the somatic embryo; however, excessive expression level of LpABCB21 inhibits somatic embryo induction efficiency. LpPILS7 mainly regulates somatic embryo induction efficiency. This study provides a more efficient method of genetic transformation of L. pumilum. LpABCB21 and LpPILS7 are confirmed to have important regulatory roles in L. pumilum SE thus laying the foundation for subsequent studies of the molecular mechanism of Lilium SE.

Somatic embryogenesis (SE) is of great significance in Lilium bulb production, germplasm preservation and genetic improvement. miRNAs are important regulators of plant growth and development at the transcriptional level. Previous research by our group has shown that lpu-miR171 and its target gene SCARECROW-LIKE 6 (SCL6) play an important regulatory role in lily SE, and we predicted and identified that endogenous target mimics (eTMs) can regulate lpu-miR171. However, the associated mechanism and internal regulatory network are not yet clear. In the present study, lpu-miR171 was used as an entry point to explore the regulatory network between its upstream eTMs and its downstream target gene LpSCL6, as well as to identify the mechanism of this regulatory network in Lilium SE. Tobacco transient transformation confirmed that miRNA171 significantly inhibited the expression of LpSCL6. On this basis, the Lilium stable genetic transformation system was used to demonstrate that silencing lpu-miR171a and lpu-miR171b and overexpressing LpSCL6-II and LpSCL6-I promoted starch accumulation in calli and the expression of key cell cycle genes, thus providing energy to meet preconditions for SE and accelerate the formation and development of Lilium somatic embryos. LpSCL6-II and LpSCL6-I are nuclear proteins with self-activation activity in yeast cells. In addition, we confirmed in Lilium that lpu-eTM171 is the eTM of lpu-miR171 that binds lpu-miR171 to prevent cleavage of the target gene LpSCL6, thereby promoting SE. Therefore, the present study established a new mechanism whereby the eTM-miR171-SCL6 module regulates SE in Lilium pumilum DC. Fisch. and provided new insights clarifying the mechanism of SE.