Haiying Liang

Ancestral polyploidy in seed plants and angiosperms

Whole-genome duplication (WGD), or polyploidy, followed by gene loss and diploidization has long been recognized as an important evolutionary force in animals, fungi and other organisms, especially plants. The success of angiosperms has been attributed, in part, to innovations associated with gene or whole-genome duplications, but evidence for proposed ancient genome duplications pre-dating the divergence of monocots and eudicots remains equivocal in analyses of conserved gene order. Here we use comprehensive phylogenomic analyses of sequenced plant genomes and more than 12.6 million new expressed-sequence-tag sequences from phylogenetically pivotal lineages to elucidate two groups of ancient gene duplications—one in the common ancestor of extant seed plants and the other in the common ancestor of extant angiosperms. Gene duplication events were intensely concentrated around 319 and 192 million years ago, implicating two WGDs in ancestral lineages shortly before the diversification of extant seed plants and extant angiosperms, respectively. Significantly, these ancestral WGDs resulted in the diversification of regulatory genes important to seed and flower development, suggesting that they were involved in major innovations that ultimately contributed to the rise and eventual dominance of seed plants and angiosperms.

Transgenic American elm shows reduced Dutch elm disease symptoms and normal mycorrhizal colonization

The American elm (Ulmus americana L.) was once one of the most common urban trees in eastern North America until Dutch-elm disease (DED), caused by the fungus Ophiostoma novo-ulmi, eliminated most of the mature trees. To enhance DED resistance, Agrobacterium was used to transform American elm with a transgene encoding the synthetic antimicrobial peptide ESF39A, driven by a vascular promoter from American chestnut. Four unique, single-copy transgenic lines were produced and regenerated into whole plants. These lines showed less wilting and significantly less sapwood staining than non-transformed controls after O. novo-ulmi inoculation. Preliminary observations indicated that mycorrhizal colonization was not significantly different between transgenic and wild-type trees. Although the trees tested were too young to ensure stable resistance was achieved, these results indicate that transgenes encoding antimicrobial peptides reduce DED symptoms and therefore hold promise for enhancing pathogen resistance in American elm.

Agrobacterium-mediated transformation of American chestnut (Castanea dentata (Marsh.) Borkh.) somatic embryos

These studies were designed to test if a binary vector containing the gfp, bar and oxalate oxidase genes could transform American chestnut somatic embryos; to see if a desiccation treatment during co-cultivation would affect the transformation frequency of different American chestnut somatic embryo clones; to explore the effects of more rapid desiccation; and to see if the antibiotics used to kill the Agrobacterium were interfering with the regeneration of the somatic embryos. Two days of gradual desiccation was found to significantly enhance transient GFP expression frequency. When this treatment was tested on six American chestnut clones, five were transformed and four of these remained embryogenic. Transformation was confirmed by Southern hybridization. Phenotypically normal transgenic shoots were regenerated and rooted. Vascular tissue specific expression of the oxalate oxidase gene was detected in one transgenic line. Carbenicillin, cefotaxime, and tricarcillin were found to not interfere with the regeneration of transformed embryos.

Overexpression of Constans Homologs CO1 and CO2 Fails to Alter Normal Reproductive Onset and Fall Bud Set in Woody Perennial Poplar

CONSTANS (CO) is an important flowering-time gene in the photoperiodic flowering pathway of annual Arabidopsis thaliana in which overexpression of CO induces early flowering, whereas mutations in CO cause delayed flowering. The closest homologs of CO in woody perennial poplar (Populus spp.) are CO1 and CO2. A previous report [1] showed that the CO2/FLOWERING LOCUS T1 (FT1) regulon controls the onset of reproduction in poplar, similar to what is seen with the CO/FLOWERING LOCUS T (FT) regulon in Arabidopsis. The CO2/FT1 regulon was also reported to control fall bud set. Our long-term field observations show that overexpression of CO1 and CO2 individually or together did not alter normal reproductive onset, spring bud break, or fall dormancy in poplar, but did result in smaller trees when compared with controls. Transcripts of CO1 and CO2 were normally most abundant in the growing season and rhythmic within a day, peaking at dawn. Our manipulative experiments did not provide evidence for transcriptional regulation being affected by photoperiod, light intensity, temperature, or water stress when transcripts of CO1 and CO2 were consistently measured in the morning. A genetic network analysis using overexpressing trees, microarrays, and computation demonstrated that a majority of functionally known genes downstream of CO1 and CO2 are associated with metabolic processes, which could explain their effect on tree size. In conclusion, the function of CO1 and CO2 in poplar does not appear to overlap with that of CO from Arabidopsis, nor do our data support the involvement of CO1 and CO2 in spring bud break or fall bud set.

An EST database for Liriodendron tulipifera L. floral buds: the first EST resource for functional and comparative genomics in Liriodendron

Liriodendron tulipifera L. was selected by the Floral Genome Project for identification of new genes related to floral diversity in basal angiosperms. A large, non-normalized cDNA library was constructed from premeiotic and meiotic floral buds and sequenced to generate a database of 9,531 high-quality expressed sequence tags. These sequences clustered into 6,520 unigenes, of which 5,251 were singletons, and 1,269 were in contigs. Homologs of genes regulating many aspects of flower development were identified, including those for organ identity and development, cell and tissue differentiation, and cell-cycle control. Almost 5% of the transcriptome consisted of homologs to known floral gene families. Homologs of most of the genes involved in cell-wall construction were also recovered. This provides a new opportunity for comparative studies in lignin biosynthesis, a trait of key importance in the evolution of land plants and in the utilization of fiber from economically important tree species, such as Liriodendron. Also of note is that 1,089 unigenes did not match any sequence in the public databases, including the complete genomes of Arabidopsis, rice, and Populus. Some of these novel genes might be unique in basal angiosperm species and, when better characterized, may be informative for understanding the origins of diverged gene families. Thus, the Liriodendron expressed sequence tag database and library will help bridge our understanding of the mechanisms of flower initiation and development that are shared among basal angiosperms, eudicots, and monocots, and provide new opportunities for comparative analysis of gene families across angiosperm species.

Comparative expression analysis of resistant and susceptible Populus clones inoculated with Septoria musiva

Septoria musiva is a major pathogen of Populus and can cause leaf spots and stem cankers in susceptible clones. In order to investigate defense mechanisms of Populus in response to S. musiva, differential gene expression in leaf tissues of two resistant (DN34, P. deltoides×nigra; NM6, P. nigra×maximowiczii) and two susceptible clones (DN164, P. deltoides×nigra; NC11505, P. maximowiczii×trichocarpa) was analyzed by RNA-Seq. Of the 511 million reads obtained, 78% and 0.01% were successfully aligned to the genomes of P. trichocarpa and S. musiva, respectively. Functional annotation of differentially expressed genes based on comparisons between resistant and susceptible clones revealed that there were significant differences in the expression of genes involved in disease/stress resistance and oxidation-reduction in mock-inoculated leaves. Four days post inoculation with S. musiva, 36 differentially expressed genes were found to be regulated in the same direction in both resistant clones. The 22 up-regulated loci in resistant clones included genes involved in protein fate, cell wall structure, and responsiveness to various biotic and abiotic stresses. In particular, Potri.008G187100 locus encodes a putative multi antimicrobial extrusion protein and Potri.006G272600 encodes a family1 glycosyltransferase required for pathogen resistance. The differentially expressed loci with increased expression in the susceptible clones corresponded to NB-ARC domain-containing disease resistance protein, phospholipase A 2A, MutT/nudix family protein, and an elicitor-activated gene 3-1 product. The results from this study indicate that strong defense mechanisms involved in oxidation-reduction, protein fate, secondary metabolism, and accumulation of defense-related gene products may contribute to Septoria resistance in DN34 and NM6, while increased expression of hypersensitive response-loci, particularly those encoding NB-ARC domain-containing disease resistance proteins, may contribute to the susceptibility of DN164 and NC11505 through interaction with pathogen effectors.

Expression of a self-processing, pathogen resistance-enhancing gene construct in Arabidopsis.

A gene cassette, p35S-CNO, was designed to express three gene products driven by a single constitutive CaMV 35S promoter. The individual coding regions were linked in frame to produce a single polyprotein, using spacer sequences encoding a specific heptapeptide cleavage recognition site (ENLYFQS) for the nuclear-inclusion-a (NIa) proteinase of tobacco etch virus (TEV). The protein coding sequences used were: a Trichoderma harzinum endochitinase, a truncated NIa proteinase of TEV, and a wheat oxalate oxidase. When p35S-CNO construct was tested in Arabidopsis thaliana, the polyprotein was properly cleaved after translation and the products exhibited functional enzymatic activity in vivo.

Preliminary Genomic Characterization of Ten Hardwood Tree Species from Multiplexed Low Coverage Whole Genome Sequencing

Forest health issues are on the rise in the United States, resulting from introduction of alien pests and diseases, coupled with abiotic stresses related to climate change. Increasingly, forest scientists are finding genetic/genomic resources valuable in addressing forest health issues. For a set of ten ecologically and economically important native hardwood tree species representing a broad phylogenetic spectrum, we used low coverage whole genome sequencing from multiplex Illumina paired ends to economically profile their genomic content. For six species, the genome content was further analyzed by flow cytometry in order to determine the nuclear genome size. Sequencing yielded a depth of 0.8X to 7.5X, from which in silico analysis yielded preliminary estimates of gene and repetitive sequence content in the genome for each species. Thousands of genomic SSRs were identified, with a clear predisposition toward dinucleotide repeats and AT-rich repeat motifs. Flanking primers were designed for SSR loci for all ten species, ranging from 891 loci in sugar maple to 18,167 in redbay. In summary, we have demonstrated that useful preliminary genome information including repeat content, gene content and useful SSR markers can be obtained at low cost and time input from a single lane of Illumina multiplex sequence.

Genetic characterization of Liriodendron seed orchards with EST-SSR markers

Liriodendron tulipifera L., is a wide-spread, fast-growing pioneering tree species native to eastern North America. Commonly known as yellow-poplar, tulip tree, or tulip-poplar, the species is valued, both ecologically and economically. It is perhaps the most commonly used utility hardwood in the USA, and is planted widely for reforestation and, in varietal forms, as an ornamental. Although most seedlings used for reforestation today derive from collections in natural populations, two known seed orchards, established from plus-tree selections, i.e. superior phenotypes, in the 1960’s and 1970’s have been used for local and regional planting needs in Tennessee and South Carolina. However, very little is known about the population genetics of yellow-poplar nor the genetic composition of the existing seed orchards. In this study, 194 grafted yellow-poplar trees from a Clemson, SC orchard and a Knoxville, TN orchard were genetically characterized with 15 simple sequence repeat (SSR) markers developed from expressed sequence tags (ESTs). Of the 15 EST-SSR markers, 14 had a polymorphic information content (PIC) of at least 0.5. There was no significant difference between the Clemson and Knoxville orchards in average effective number of alleles (5.93 vs 3.95), observed and expected heterozygosity (Ho: 0.64 vs 0.58; He: 0.74 vs 0.70), Nei’s expected heterozygosity (0.74 vs 0.58), or Shannon’s Information index (1.84 vs 1.51). The larger Clemson orchard exhibited a significantly greater number of observed alleles than the Knoxville orchard (15.3 vs7.4). Overall, substantial genetic diversity is captured in the Clemson and Knoxville orchards.

Selection and Screening Strategies

A number of transformation systems have been developed to insert foreign DNA into the appropriate plant genome (nuclear or plastid) (discussed in Chap. 3). However, only a small fraction of the treated cells become transgenic, while the majority of the cells remain untransformed using any of these methods. Thus, effective selection and screening strategies are needed to pick up the rare transgenic lines from a pool of nontransformed cells or plants. To date, more than 50 marker genes and a few molecular techniques have been developed to serve this essential purpose.