Meiping Zhang

Preparation of megabase-sized DNA from a variety of organisms using the nuclei method for advanced genomics research

Megabase-sized DNA is crucial to modern genomics research of all organisms. Among the preparation methods developed, the nuclei method is the simplest and most widely used for preparing high-quality megabase-sized DNA from divergent organisms. In this method, nuclei are first isolated by physically grinding the source tissues. The nontarget cytoplast organellar genomes and metabolites are removed by centrifugation and washing, thus maximizing the utility of the method and substantially improving the digestibility and clonability of the resultant DNA. The nuclei are then embedded in an agarose matrix containing numerous pores, allowing the access of restriction enzymes while preventing the DNA from physical shearing. DNA is extracted from the nuclei, purified and subsequently manipulated in the agarose matrix. Here we describe the nuclei method that we have successfully used to prepare high-quality megabase-sized DNA from hundreds of plant, animal, fish, insect, algal and microbial species. The entire protocol takes ∼3 d.

A BAC/BIBAC-based physical map of chickpea, Cicer arietinum L

BackgroundChickpea (Cicer arietinum L.) is the third most important pulse crop worldwide. Despite its importance, relatively little is known about its genome. The availability of a genome-wide physical map allows rapid fine mapping of QTL, development of high-density genome maps, and sequencing of the entire genome. However, no such a physical map has been developed in chickpea.ResultsWe present a genome-wide, BAC/BIBAC-based physical map of chickpea developed by fingerprint analysis. Four chickpea BAC and BIBAC libraries, two of which were constructed in this study, were used. A total of 67,584 clones were fingerprinted, and 64,211 (~11.7 ×) of the fingerprints validated and used in the physical map assembly. The physical map consists of 1,945 BAC/BIBAC contigs, with each containing an average of 28.3 clones and having an average physical length of 559 kb. The contigs collectively span approximately 1,088 Mb. By using the physical map, we identified the BAC/BIBAC contigs containing or closely linked to QTL4.1 for resistance to Didymella rabiei (RDR) and QTL8 for days to first flower (DTF), thus further verifying the physical map and confirming its utility in fine mapping and cloning of QTL.ConclusionThe physical map represents the first genome-wide, BAC/BIBAC-based physical map of chickpea. This map, along with other genomic resources previously developed in the species and the genome sequences of related species (soybean, Medicago and Lotus), will provide a foundation necessary for many areas of advanced genomics research in chickpea and other legume species. The inclusion of transformation-ready BIBACs in the map greatly facilitates its utility in functional analysis of the legume genomes.

A comparative physical map reveals the pattern of chromosomal evolution between the turkey ( Meleagris gallopavo ) and chicken ( Gallus gallus ) genomes

BackgroundA robust bacterial artificial chromosome (BAC)-based physical map is essential for many aspects of genomics research, including an understanding of chromosome evolution, high-resolution genome mapping, marker-assisted breeding, positional cloning of genes, and quantitative trait analysis. To facilitate turkey genetics research and better understand avian genome evolution, a BAC-based integrated physical, genetic, and comparative map was developed for this important agricultural species.ResultsThe turkey genome physical map was constructed based on 74,013 BAC fingerprints (11.9 × coverage) from two independent libraries, and it was integrated with the turkey genetic map and chicken genome sequence using over 41,400 BAC assignments identified by 3,499 overgo hybridization probes along with > 43,000 BAC end sequences. The physical-comparative map consists of 74 BAC contigs, with an average contig size of 13.6 Mb. All but four of the turkey chromosomes were spanned on this map by three or fewer contigs, with 14 chromosomes spanned by a single contig and nine chromosomes spanned by two contigs. This map predicts 20 to 27 major rearrangements distinguishing turkey and chicken chromosomes, despite up to 40 million years of separate evolution between the two species. These data elucidate the chromosomal evolutionary pattern within the Phasianidae that led to the modern turkey and chicken karyotypes. The predominant rearrangement mode involves intra-chromosomal inversions, and there is a clear bias for these to result in centromere locations at or near telomeres in turkey chromosomes, in comparison to interstitial centromeres in the orthologous chicken chromosomes.ConclusionThe BAC-based turkey-chicken comparative map provides novel insights into the evolution of avian genomes, a framework for assembly of turkey whole genome shotgun sequencing data, and tools for enhanced genetic improvement of these important agricultural and model species.

Construction of BIBAC and BAC libraries from a variety of organisms for advanced genomics research

Large-insert BAC (bacterial artificial chromosome) and BIBAC (binary BAC) libraries are essential for modern genomics research for all organisms. We helped pioneer the BAC and BIBAC technologies, and by using them we have constructed hundreds of BAC and BIBAC libraries for different species of plants, animals, marine animals, insects, algae and microbes. These libraries have been used globally for different aspects of genomics research. Here we describe the procedure with the latest improvements that we have made and used for construction of BIBAC libraries. The procedure includes the preparation of BIBAC vectors, the preparation of clonable fragments of the desired size from the source DNA, the construction and transformation of BIBACs and, finally, the characterization and assembly of BIBAC libraries. We also specify the modifications necessary for construction of BAC libraries using the protocol. The entire protocol takes ∼7 d.

The Spatial and Temporal Transcriptomic Landscapes of Ginseng, Panax ginseng C. A. Meyer

Ginseng, including Asian ginseng (Panax ginseng C. A. Meyer) and American ginseng (P. quinquefolius L.), is one of the most important medicinal herbs in Asia and North America, but significantly understudied. This study sequenced and characterized the transcriptomes and expression profiles of genes expressed in 14 tissues and four different aged roots of Asian ginseng. A total of 265.2 million 100-bp clean reads were generated using the high-throughput sequencing platform HiSeq 2000, representing >8.3x of the 3.2-Gb ginseng genome. From the sequences, 248,993 unigenes were assembled for whole plant, 61,912–113,456 unigenes for each tissue and 54,444–65,412 unigenes for different year-old roots. We comprehensively analyzed the unigene sets and gene expression profiles. We found that the number of genes allocated to each functional category is stable across tissues or developmental stages, while the expression profiles of different genes of a gene family or involved in ginsenoside biosynthesis dramatically diversified spatially and temporally. These results provide an overall insight into the spatial and temporal transcriptome dynamics and landscapes of Asian ginseng, and comprehensive resources for advanced research and breeding of ginseng and related species.

Genome physical mapping of polyploids: a BIBAC physical map of cultivated tetraploid cotton, Gossypium hirsutum L.

Polyploids account for approximately 70% of flowering plants, including many field, horticulture and forage crops. Cottons are a world-leading fiber and important oilseed crop, and a model species for study of plant polyploidization, cellulose biosynthesis and cell wall biogenesis. This study has addressed the concerns of physical mapping of polyploids with BACs and/or BIBACs by constructing a physical map of the tetraploid cotton, Gossypium hirsutum L. The physical map consists of 3,450 BIBAC contigs with an N50 contig size of 863 kb, collectively spanning 2,244 Mb. We sorted the map contigs according to their origin of subgenome, showing that we assembled physical maps for the A- and D-subgenomes of the tetraploid cotton, separately. We also identified the BIBACs in the map minimal tilling path, which consists of 15,277 clones. Moreover, we have marked the physical map with nearly 10,000 BIBAC ends (BESs), making one BES in approximately 250 kb. This physical map provides a line of evidence and a strategy for physical mapping of polyploids, and a platform for advanced research of the tetraploid cotton genome, particularly fine mapping and cloning the cotton agronomic genes and QTLs, and sequencing and assembling the cotton genome using the modern next-generation sequencing technology.

Functional differentiation and spatial-temporal co-expression networks of the NBS-encoding gene family in Jilin ginseng, Panax ginseng C.A. Meyer

Ginseng, Panax ginseng C.A. Meyer, is one of the most important medicinal plants for human health and medicine. It has been documented that over 80% of genes conferring resistance to bacteria, viruses, fungi and nematodes are contributed by the nucleotide binding site (NBS)-encoding gene family. Therefore, identification and characterization of NBS genes expressed in ginseng are paramount to its genetic improvement and breeding. However, little is known about the NBS-encoding genes in ginseng. Here we report genome-wide identification and systems analysis of the NBS genes actively expressed in ginseng (PgNBS genes). Four hundred twelve PgNBS gene transcripts, derived from 284 gene models, were identified from the transcriptomes of 14 ginseng tissues. These genes were classified into eight types, including TNL, TN, CNL, CN, NL, N, RPW8-NL and RPW8-N. Seven conserved motifs were identified in both the Toll/interleukine-1 receptor (TIR) and coiled-coil (CC) typed genes whereas six were identified in the RPW8 typed genes. Phylogenetic analysis showed that the PgNBS gene family is an ancient family, with a vast majority of its genes originated before ginseng originated. In spite of their belonging to a family, the PgNBS genes have functionally dramatically differentiated and been categorized into numerous functional categories. The expressions of the across tissues, different aged roots and the roots of different genotypes. However, they are coordinating in expression, forming a single co-expression network. These results provide a deeper understanding of the origin, evolution and functional differentiation and expression dynamics of the NBS-encoding gene family in plants in general and in ginseng particularly, and a NBS gene toolkit useful for isolation and characterization of disease resistance genes and for enhanced disease resistance breeding in ginseng and related species.

Structural Variation, Functional Differentiation, and Activity Correlation of the Cytochrome P450 Gene Superfamily Revealed in Ginseng

Most genes exist in a form of family or superfamily in higher organisms Cytochrome P450 (CYP) gene superfamily is involved in ginsenoside biosynthesis Genome‐wide identification of members of the CYP gene superfamily in ginseng Assessed its origin, evolution, and spatiotemporal expression Examined its functional differentiation and action correlation and regulation

Evolution, functional differentiation, and co-expression of the RLK gene family revealed in Jilin ginseng, Panax ginseng C.A. Meyer

Most genes in a genome exist in the form of a gene family; therefore, it is necessary to have knowledge of how a gene family functions to comprehensively understand organismal biology. The receptor-like kinase (RLK)-encoding gene family is one of the most important gene families in plants. It plays important roles in biotic and abiotic stress tolerances, and growth and development. However, little is known about the functional differentiation and relationships among the gene members within a gene family in plants. This study has isolated 563 RLK genes (designated as PgRLK genes) expressed in Jilin ginseng (Panax ginseng C.A. Meyer), investigated their evolution, and deciphered their functional diversification and relationships. The PgRLK gene family is highly diverged and formed into eight types. The LRR type is the earliest and most prevalent, while only the Lec type originated after P. ginseng evolved. Furthermore, although the members of the PgRLK gene family all encode receptor-like protein kinases and share conservative domains, they are functionally very diverse, participating in numerous biological processes. The expressions of different members of the PgRLK gene family are extremely variable within a tissue, at a developmental stage and in the same cultivar, but most of the genes tend to express correlatively, forming a co-expression network. These results not only provide a deeper and comprehensive understanding of the evolution, functional differentiation and correlation of a gene family in plants, but also an RLK genic resource useful for enhanced ginseng genetic improvement.

Characterization of the complete chloroplast genome of Jilin ginseng (Panax ginseng C. A. Meyer) using next generation sequencing

Abstract Jilin ginseng, Panax ginseng C. A. Meyer, belongs to the Araliaceae family. It is known as the number one medicinal herb and one of the three native treasures in Northern China and has been cultivated in China for over 2000 years. Jilin ginseng is the staple ginseng in China, which contributes to 85% of the ginseng production in the country, thus becoming one of the most important, rapidly increasing industries in both the Province of Jilin and China. In this study, the complete chloroplast genome of Panax ginseng C. A. Meyer was determined by the next generation sequencing. The complete chloroplast genome of Jilin ginseng (P. ginseng C. A. Meyer) was 156,286 bp in length and displays a typical quadripartite structure of the large (LSC, 87,127 bp) and small (SSC, 18,329 bp) single-copy regions, separated by a pair of inverted repeat regions (IRs, 25,415 bp each). It harbors 132 functional genes, including 132 protein-coding genes, 37 transfer RNA, and 8 ribosomal RNA genes species. The overall nucleotide composition was: 30.6% A, 31.3% T, 19.4% C, and 18.7% G, with a total G + C content of 38.1%. Phylogenetic relationship analysis shows that P. ginseng closely related to Panax quinquefolius L.