Genetic diversity analysis and fingerprinting of 175 Chimonanthus praecox germplasm based on SSR molecular marker / 生物工程学报

The elucidation of resources pertaining to the Chimonanthus praecox varieties and the establishment of a fingerprint serve as crucial underpinnings for advancing scientific inquiry and industrial progress in relation to C. praecox. Employing the SSR molecular marker technology, an exploration of the genetic diversity of 175 C. praecox varieties (lines) in the Yanling region was conducted, and an analysis of the genetic diversity among these varieties was carried out using the UPDM clustering method in NTSYSpc 2.1 software. We analyzed the genetic structure of 175 germplasm using Structure v2.3.3 software based on a Bayesian model. General linear model (GLM) association was utilized to analyze traits and markers. The genetic diversity analysis revealed a mean number of alleles (Na) of 6.857, a mean expected heterozygosity (He) of 0.496 3, a mean observed heterozygosity (Ho) of 0.503 7, a mean genetic diversity index of Nei՚s of 0.494 9, and a mean Shannon information index of 0.995 8. These results suggest that the C. praecox population in Yanling exhibits a rich genetic diversity. Additionally, the population structure and the UPDM clustering were examined. In the GLM model, a total of fifteen marker loci exhibited significant (P < 0.05) association with eight phenotypic traits, with the explained phenotypic variation ranging from 14.90% to 36.03%. The construction of fingerprints for C. praecox varieties (lines) was accomplished by utilizing eleven primer pairs with the highest polymorphic information content, resulting in the analysis of 175 SSR markers. The present study offers a thorough examination of the genetic diversity and SSR molecular markers of C. praecox in Yanling, and establishes a fundamental germplasm repository of C. praecox, thereby furnishing theoretical underpinnings for the selection and cultivation of novel and superior C. praecox varieties, varietal identification, and resource preservation and exploitation.

Analysis of the chloroplast genome of Incarvillea younghusbandii Sprague / 生物工程学报

Incarvillea younghusbandii Sprague is a traditional tonic herb. The roots are used as herbal medicine for nourishing and strengthening, as well as treating postpartum milk deficiency and weakness. In this study, the chloroplast genome of I. younghusbandii was sequenced and assembled by the high-throughput sequencing technology. The sequence characteristics, sequence repeats, codon usage bias, phylogenetic relationships and estimated divergence time of I. younghusbandii were analyzed. The 159 323 bp sequence contained a large single copy (80 197 bp), a small single copy (9 030 bp) and two inverted repeat sequences (35 048 bp). It contained 120 genes, including 77 protein coding genes, 8 ribosomal RNA genes and 35 transfer RNA genes. AAA was the most frequent codon in the chloroplast coding sequence of I. younghusbandii. A total of 42 simple sequence repeats were identified in the chloroplast genome. Phylogenetic analysis revealed I. younghusbandii was mostly like its taxonomically close relative Incarvillea compacta. The divergence between I. younghusbandii and I. compacta was dated to 4.66 million years ago. This study was significant for the scientific conservation and development of resources related to I. compacta. It also provides a basic genetic resource for the subsequent species identification of the genus Incarvillea, and the population genetic diversity study of Bignoniaceae.

Establishment of DNA Fingerprints and Genetic Similarities Analysis of Dendrobium officinale / 中国实验方剂学杂志

This study adopted the 4 pairs of simple sequence repeats(SSR) primers selected by National Resource Center for Chinese Meteria Medica (CACMS) to detect 29 populations of Dendrobium officinale samples from 9 main places of production; 5,7,4 and 3 polymorphism bands were amplified from these 4 pairs of SSR primers. The DNA identities of different populations were constructed by SSR. The 29 D. officinale populations could be divided into 4 classes. The clustering result was related to the places of production. Samples from Yunnan,Guizhou,Sichuan provinces were classified into one category,while samples from Anhui and Guangxi provinces were classified into another category. Samples from Guangdong Danxia,Zhejiang Yongkang,Zhejiang Leqing and Taining belonged to a category. PopGene (version 1.32) software was applied to calculate the genetic similarity of the 29 D. officinale populations. The similarities was between 0.403 4 and 1.0.Based on the genetic similarity,the genetic consistency included three classes,A,B and C. Samples with a similar geographical location and landform environment have higher genetic similarities,which indicate the same genetic background. This paper provides reference information to study the identification, selection and breeding of good varieties.

Transcriptome analysis of female and male flower buds of Idesia polycarpa Maxim. var. vestita Diels

Background: Idesia polycarpa Maxim. var. vestita Diels, a dioecious plant, is widely used for biodiesel due to the high oil content of its fruits. However, it is hard to distinguish its sex in the seedling stage, which makes breeding and production problematic as only the female tree can produce fruits, and the mechanisms underlying sex determination and differentiation remain unknown due to the lack of available genomic and transcriptomic information. To begin addressing this issue, we performed the transcriptome analysis of its female and male flower. Results: 28,668,977 and 22,227,992 clean reads were obtained from the female and male cDNA libraries, respectively. After quality checks and de novo assembly, a total of 84,213 unigenes with an average length of 1179 bp were generated and 65,972 unigenes (78.34%) could be matched in at least one of the NR, NT, Swiss-Prot, COG, KEGG and GO databases. Functional annotation of the unigenes uncovered diverse biological functions and processes, including reproduction and developmental process, which may play roles in sex determination and differentiation. The Kyoto Encyclopedia of Genes and Genomes pathway analysis showed many unigenes annotated as metabolic pathways, biosynthesis of secondary metabolites pathways, plant­ pathogen interaction, and plant hormone signal transduction. Moreover, 29,953 simple sequence repeats were identified using the microsatellite software. Conclusion: This work provides the first detailed transcriptome analysis of female and male flower of I. polycarpa and lays foundations for future studies on the molecular mechanisms underlying flower bud development of I. polycarpa.

Transcriptome analysis of Cinnamomum longepaniculatum by high-throughput sequencing

Background: Cinnamomum longepaniculatum is an important commercial crop and the main source of volatile terpenoids. The biosynthesis of key bioactive metabolites of C. longepaniculatum is not well understood because of the lack of available genomic and transcriptomic information. To address this issue, we performed transcriptome sequencing of C. longepaniculatum leaves to identify factors involved in terpenoid metabolite biosynthesis. Results: Transcriptome sequencing of C. longepaniculatum leaves generated over 56 million raw reads. The transcriptome was assembled using the Trinity software and yielded 82,061 unigenes with an average length of 879.43 bp and N50 value of 1387 bp. Furthermore, Benchmarking Universal Single-Copy Orthologs analysis indicated that our assembly is 91% complete. The unigenes were used to query the nonredundant database depending on sequence similarity; 42,809 unigenes were homologous to known genes in different species, with an annotation rate of 42.87%. The transcript abundance and Gene Ontology analyses revealed that numerous unigenes were associated with metabolism, while others were annotated in functional categories including transcription, signal transduction, and secondary metabolism. The Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that 19,260 unigenes were involved in 385 metabolic pathways, with 233 unigenes found to be involved in terpenoid metabolism. Moreover, 23,463 simple sequence repeats were identified using the microsatellite identification tool. Conclusion: This is the first detailed transcriptome analysis of C. longepaniculatum. The findings provide insights into the molecular basis of terpenoid biosynthesis and a reference for future studies on the genetics and breeding of C. longepaniculatum.

Genetic diversity analysis of common beans based on molecular markers

A core collection of the common bean (Phaseolus vulgaris L.), representing genetic diversity in the entire Mexican holding, is kept at the INIFAP (Instituto Nacional de Investigaciones Forestales, Agricolas y Pecuarias, Mexico) Germplasm Bank. After evaluation, the genetic structure of this collection (200 accessions) was compared with that of landraces from the states of Oaxaca, Chiapas and Veracruz (10 genotypes from each), as well as a further 10 cultivars, by means of four amplified fragment length polymorphisms (AFLP) +3/+3 primer combinations and seven simple sequence repeats (SSR) loci, in order to define genetic diversity, variability and mutual relationships. Data underwent cluster (UPGMA) and molecular variance (AMOVA) analyses. AFLP analysis produced 530 bands (88.5 percent polymorphic) while SSR primers amplified 174 alleles, all polymorphic (8.2 alleles per locus). AFLP indicated that the highest genetic diversity was to be found in ten commercial-seed classes from two major groups of accessions from Central Mexico and Chiapas, which seems to be an important center of diversity in the south. A third group included genotypes from Nueva Granada, Mesoamerica, Jalisco and Durango races. Here, SSR analysis indicated a reduced number of shared haplotypes among accessions, whereas the highest genetic components of AMOVA variation were found within accessions. Genetic diversity observed in the common-bean core collection represents an important sample of the total Phaseolus genetic variability at the main Germplasm Bank of INIFAP. Molecular marker strategies could contribute to a better understanding of the genetic structure of the core collection as well as to its improvement and validation.

PCR-mediated recombination in development of microsatellite markers: mechanism and implications

Protocols for microsatellite-enrichment libraries have been widely applied to several species in order to supply the most informative molecular markers for population and inbreeding studies. One drawback of these protocols is the ratio of designed primer pairs that fail to amplify the expected fragment, even after exhaustive optimization attempts. A possible cause of unsuccessful microsatellite primers may be that such loci are artifacts resulting from chimeric PCR products, instead of real genomic sequences. The microsatellite-enriched library constructed for Aegla longirostri (Crustacea, Decapoda, Anomura) showed that 29 percent of sequenced clones were chimeric products because these sequences shared one of the flanking regions around the same repeat motif but not the other. PCR-mediated recombination is a well-known event described for several procedures in which related sequences are used as a template. We have associated this phenomenon with microsatellite marker development. This study explained the high ratio of recombinant sequences generated in the A. longirostri microsatellite-enriched library. We discuss the mechanism and implications of PCR chimeric-product formation during microsatellite isolation.