The development and utilization of genetic markers play a pivotal role in marker-assisted breeding of rice cultivars during pyramiding of valuable genes. Among molecular markers, SNPs have become the most promising due to their wide distribution within genomes and suitability for high -throughput automated genotyping. Although metadata of SNPs have been identified via next generation sequencing in rice, a large gap between the development of SNP markers and the application in breeding still exists. To promote the application of SNP markers based on the KASP (Kompetitive Allele-Specific PCR) method in rice breeding, a set of core SNP arrays was built via the screening of SNP databases and literature resources based on the KASP method. Five hundred and ninety six SNPs classified into eight subsets including quality control, indica-indica variation, highly polymorphic, functional genes, key genes targeting sites, gene cloned region, important trait associated and gap filling sites were chosen to design KASP primers and 565 out of them were successfully designed, and the assay design success rate was 94.8%. Finally, 467 out of the 565 successfully-designed SNPs can display diversity at the loci were used to develop a set of core SNP arrays. To evaluate the application value of the core SNP markers in rice breeding, 481 rice germplasms were genotyped with three functional KASP markers designed from the sequences of GBSSI, SSIIa, and Badh2 from the core SNP arrays for estimation of their grain quality performance. Eighteen rice lines, including Xiangwanxian 13, Basmati 370, Ruanhua A, and PR 33319–9–1-1-5-3-5-4-1, harbor all three favorable alleles. The core KASP arrays were also used for rice germplasm assessment, genetic diversity and population evaluation. Four hundred and eighty-one rice germplasms were divided into 3 groups: POP1, POP2 and POP3. POP1 and POP2 were indica rice subgroups consisting of 263 and 186 rice germplasms, respectively. POP3 was a japonica rice subgroup consisting of 32 rice germplasms. The average FST value for the three subgroups was 0.3501; the FST value of POP1 and POP3 was the largest (0.5482), while that of POP1 and POP2 was the smallest (0.0721). The results showed that the genetic distance between the japonica and indica rice subspecies was large, indicating that the core SNP markers were effective at discriminating the population structure of the germplasms. Finally, the core KASP arrays were used for association analysis with milled grain traits. A total of 31 KASP markers were significantly associated (P < 0.01) with ML and the LWR. Among the 31 markers, 13 were developed based on cloned genes or on identified loci related to yield traits. Notably, several KASP markers associated with grain quality were also found to be associated with brown planthopper resistance or green leafhopper resistance simultaneously. The core KASP arrays developed in our study were efficient and versatile for rice germplasm assessment, genetic diversity and population evaluation and are valuable for promoting SNP molecular breeding in rice. Our study demonstrated that useful assays combined with molecular breeding can be exploited for important economic trait improvement in rice breeding.

Grain appearance quality is a main determinant of market value in rice and one of the highly important traits requiring improvement in breeding programs. The genetic basis of grain shape and endosperm chalkiness have been given significant attention because of their importance in affecting grain quality. Meanwhile, the introduction of NGS (Next Generation Sequencing) has a significant part to play in the area of genomics, and offers the possibility for high-resolution genetic map construction, population genetics analysis and systematic expression profile study. A RIL population derived from an inter-subspecific cross between indica rice PYZX and japonica rice P02428 was generated, based on the significant variations for the grain morphology and cytological structure between these two parents. Using the Genotyping-By-Sequencing (GBS) approach, 2711 recombination bin markers with an average physical length of 137.68 kb were obtained, and a high-density genetic map was constructed. Global genetic mapping of QTLs affecting grain shape and chalkiness traits was performed across four environments and the newly identified stable loci were obtained. Twelve important QTL clusters were detected, four of which were coincident with the genomic regions of cloned genes or fine mapped QTL reported. Eight novel QTL clusters (including six for grain shape, one for chalkiness, and one for both grain shape and chalkiness) were firstly obtained and highlighted the value and reliability of the QTL analysis. The important QTL cluster on chromosome 5 affects multiple traits including circularity (CS), grain width (GW), area size of grain (AS), percentage of grains with chalkiness (PGWC) and degree of endosperm chalkiness (DEC), indicating some potentially pleiotropic effects. The transcriptome analysis demonstrated an available gene expression profile responsible for the development of chalkiness, and several DEGs (differentially expressed genes) were co-located nearby the three chalkiness-related QTL regions on chromosomes 5, 7, and 8. Candidate genes were extrapolated, which were suitable for functional validation and breeding utilization. QTLs affecting grain shape (grain width, grain length, length-width ratio, circularity, area size of grain, and perimeter length of grain) and chalkiness traits (percentage of grains with chalkiness and degree of endosperm chalkiness) were mapped with the high-density GBS-SNP based markers. The important differentially expressed genes (DEGs) were co-located in the QTL cluster regions on chromosomes 5, 7 and 8 affecting PGWC and DEC parameters. Our research provides a crucial insight into the genetic architecture of rice grain shape and chalkiness, and acquired potential candidate loci for molecular cloning and grain quality improvement.

Pogostemon cablin (Blanco) Benth. (Patchouli) is an important aromatic and medicinal plant and widely used in traditional Chinese medicine as well as in the perfume industry. Patchoulol is the primary bioactive component in P. cablin, its biosynthesis has attracted widespread interests. Previous studies have surveyed the putative genes involved in patchoulol biosynthesis using next-generation sequencing method; however, technical limitations generated by short-read sequencing restrict the yield of full-length genes. Additionally, little is known about the expression pattern of genes especially patchoulol biosynthesis related genes in response to methyl jasmonate (MeJA). Our understanding of patchoulol biosynthetic pathway still remained largely incomplete to date. In this study, we analyzed the morphological character and volatile chemical compounds of P. cablin cv. ‘Zhanxiang’, and 39 volatile chemical components were detected in the patchouli leaf using GC-MS, most of which were sesquiterpenes. Furthermore, high-quality RNA isolated from leaves and stems of P. cablin were used to generate the first full-length transcriptome of P. cablin using PacBio isoform sequencing (Iso-Seq). In total, 9.7 Gb clean data and 82,335 full-length UniTransModels were captured. 102 transcripts were annotated as 16 encoding enzymes involved in patchouli alcohol biosynthesis. Accorded with the uptrend of patchoulol content, the vast majority of genes related to the patchoulol biosynthesis were up-regulated after MeJA treatment, indicating that MeJA led to an increasing synthesis of patchoulol through activating the expression level of genes involved in biosynthesis pathway of patchoulol. Moreover, expression pattern analysis also revealed that transcription factors participated in JA regulation of patchoulol biosynthesis were differentially expressed. The current study comprehensively reported the morphological specificity, volatile chemical compositions and transcriptome characterization of the Chinese-cultivated P. cablin cv. ‘Zhanxiang’, these results contribute to our better understanding of the physiological and molecular features of patchouli, especially the molecular mechanism of biosynthesis of patchoulol. Our full-length transcriptome data also provides a valuable genetic resource for further studies in patchouli.

Lysine succinylation is a novel, naturally occurring posttranslational modification (PTM) in living organisms. Global lysine succinylation identification has been performed at the proteomic level in various species; however, the study of lysine succinylation in plant species is relatively limited. Patchouli plant (P. cablin (Blanco) Benth., Lamiaceae) is a globally important industrial plant and medicinal herb. In the present study, lysine succinylome analysis was carried out in patchouli plants to determine the potential regulatory role of lysine succinylation in patchouli growth, development, and physiology. The global succinylation sites and proteins in patchouli plants were screened with an immunoprecipitation affinity enrichment technique and advanced mass spectrometry-based proteomics. Several bioinformatic analyses, such as function classification and enrichment, subcellular location predication, metabolic pathway enrichment and protein−protein interaction networking, were conducted to characterize the functions of the identified sites and proteins. In total, 1097 succinylation sites in 493 proteins were detected in patchouli plants, among which 466 succinylation sites in 241 proteins were repeatedly identified within three independent experiments. The functional characterization of these proteins indicated that the tricarboxylic acid (TCA) cycle, oxidative phosphorylation, photosynthesis processes, and amino acid biosynthesis may be regulated by lysine succinylation. In addition, these succinylated proteins showed a wide subcellular location distribution, although the chloroplast and cytoplasm were the top two preferred cellular components. Our study suggested the important role of lysine succinylation in patchouli plant physiology and biology and could serve as a useful reference for succinylation studies in other medicinal plants.

Many commercially important drug and flavor compounds are secondary metabolites of terpenoid origin. Pogostemon cablin, a commercially important industrial and medicinal crop, accumulates abundant patchouli oil comprised of more than 24 unique sesquiterpene compounds, with the most abundant being patchouli alcohol. In this study, we analyzed the P. cablin transcriptome library, obtaining 74 terpenoid biosynthesis-related genes, and identified their expression patterns in leaves, stems, and flowers. These genes are members of 15 different families, and we detected all the enzymes involved in the sesquiterpenes pathway that are responsible for patchoulol biosynthesis. Sequence structure, homology, conserved domain properties, and phylogeny of certain identified genes were systematically investigated. Color complementation assay was used to verify the functional activity of the MEP pathway proteins. Exogenous hormone treatment revealed that patchoulol synthesis is induced by methyl jasmonate (MeJA). Quantitative reverse-transcription PCR analysis indicated that the MVA pathway genes (acetoacetyl-CoA thiolase, 3-hydroxy-3-methylglutaryl-coenzyme A reductase, mevalonate diphosphate decarboxylase, and farnesyl diphosphate synthase) participate in patchoulol biosynthesis and are mediated by MeJA. Taken together, this is the first report of integrated analysis of P. cablin MVA and MEP pathway related genes, providing a better understanding of terpenoid and/or patchoulol biosynthesis in P. cablin, and the basis for improving patchoulol production through genetic engineering.