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Anthocyanins are the visual pigments that present most of the colors in plants. Its biosynthesis requires the coordinated expression of structural genes and regulatory genes. Pericarps are the rich sources of anthocyanins in maize seeds. In the experiment, the transcriptomes of transparent and anthocyanins-enriched pericarps at 15, 20, and 25 DAP were obtained. The results output 110.007 million raw reads and 51407 genes’ expression matrix. Using data filtration in R language, 2057 genes were eventually identified for weighted gene co-expression network analysis. The results showed that 2057 genes were classified into ten modules. The cyan module containing 183 genes was confirmed to be the key module with the highest correlation value of 0.98 to the anthocyanins trait. Among 183 genes, seven structural genes were mapped the flavonoid biosynthesis pathway, and a transcription factor Lc gene was annotated as an anthocyanin regulatory gene. Cluster heatmap and gene network analysis further demonstrated that Naringenin, 2-oxoglutarate 3-dioxygenase (Zm00001d001960), Dihydroflavonol 4-reductase (Zm00001d044122), Leucoanthocyanidin dioxygenase (Zm00001d014914), anthocyanin regulatory Lc gene (Zm00001d026147), and Chalcone synthase C2 (Zm00001d052673) participated in the anthocyanins biosynthesis. And the transcription factor anthocyanin regulatory Lc gene Zm00001d026147 may act on the genes Chalcone synthase C2 (Zm00001d052673) and Dihydroflavonol 4-reductase (Zm00001d044122). The yeast one-hybrid assays confirmed that the Lc protein could combine with the promoter region of C2 and directly regulate the anthocyanin biosynthesis in the pericarp. These results may provide a new sight to uncover the module and hub genes related to anthocyanins biosynthesis in plants.

The endosperm of corn kernel consists of two components, a horny endosperm, and a floury endosperm. In the experiment, a kind of floury endosperm corn was identified. The result of phenotypic trait analysis and determination of amino acid content showed that the floury endosperm filled with the small, loose, and scattered irregular spherical shape starch granules and contained higher content of amino acid. The starch biochemical properties are similar between floury corns and regular flint corn. By using dynamically comparative transcriptome analysis of endosperm at 20, 25, and 30 DAP, a total of 113.42 million raw reads and 50.508 thousand genes were obtained. By using the weighted gene co-expression network analysis, 806 genes and six modules were identified. And the turquoise module with 459 genes was proved to be the key module closely related to the floury endosperm formation. Nine zein genes in turquoise module, including two zein-alpha A20 (Zm00001d019155 and Zm00001d019156), two zein-alpha A30 (Zm00001d048849 and Zm00001d048850), one 50 kDa gamma-zein (Zm00001d020591), one 22 kDa alpha-zein 14 (Zm00001d048817), one zein-alpha 19D1 (Zm00001d030855), one zein-alpha 19B1 (Zm00001d048848), and one FLOURY 2 (Zm00001d048808) were identified closely related the floury endosperm formation. Both zein-alpha 19B1 (Zm00001d048848) and zein-alpha A30 (Zm00001d048850) function as source genes with the highest expression level in floury endosperm. These results may provide the supplementary molecular mechanism of structure and nutrient formation for the floury endosperm of maize.

Streptococcus pyogenes Cas9 (SpCas9) is the most widely used genome editing tool in plants. The editing induced by SpCas9 strictly requires a canonical NGG protospacer-adjacent motif (PAM), significantly limiting its scope of application. Recently, five SpCas9 variants, SpCas9-NRRH, SpCas9-NRCH, SpCas9-NRTH, SpG, and SpRY, were developed to recognize non-canonical PAMs in human cells. In this study, these variants were engineered for plant genome editing, and their targeted mutagenesis capabilities were comprehensively examined at various canonical and non-canonical PAM sites in rice (Oryza sativa) by stable transformation. Moreover, both cytosine base editors using a rat APOBEC1 or a human APOBEC3a and adenine base editors using a directly evolved highly compatible TadA∗-8e deaminase were developed from these SpCas9 variants. Our results demonstrated that the developed SpCas9 variants-based base editors readily generated conversions between C∙G and T∙A in the target sites with non-canonical PAMs in transgenic rice lines. Collectively, the toolbox developed in this study substantially expands the scope of SpCas9-mediated genome editing and will greatly facilitate gene disruption and precise editing in plants.

Genetic reprogramming of differentiated cells is studied broadly in multicellular Viridiplantae as an adaptation to herbivory or damage; however, mechanisms underlying cell development and redifferentiation are largely unknown in red algae, their nearest multicellular relatives. Here we investgate cell reprogramming in the widely cultivated, edible seaweed Neopyropia yezoesis (“nori”), where vegetative cells in wounded blades differentiate and release as large numbers of asexual spores. Based upon physiological changes and transcriptomic dynamics after wound stress in N. yezoensis and its congener Neoporphyra haitanensis, another cultivar that does not differentiate spores after wounding, we propose a three-phase model of wound-induced spore development in N. yezoensis. In Phase I, propagation of ROS by RBOH and SOD elicites systematic transduction of the wound signal, while Ca2+ dependent signaling induces cell reprogramming. In Phase II, a TOR signaling pathway and regulation of cyclin and CDK genes result in cell divisions that spread inward from the wound edge. Once sporangia form, Phase III involves expression of proteins required for spore maturation and cell wall softening. Our analyses not only provide the first model for core molecular processes controlling cellular reprogramming in rhodophytes, but also have practical implications for achieving greater control over seeding in commercial nori farming.

An eCDAL was established from Japanese lamprey CDA1-like 4 to achieve a high editing frequency in a broad region as a C-terminal cytosine base editors (CT-CBE). Then, a novel plant dual-base editors DuBE (pDuBE1) was developed by integrating TadA-8e into eCDAL. The editing efficiency of pDuBE1 could reach to 87.6%, with frequencies of concurrent A-to-G and C-to-T conversions as high as 49.7% in stably transformed plant cells. Our results showed that pDuBE1 could mediate robust dual editing in plant genome, providing a powerful manipulation tool for precise crop breeding and screening platforms for in planta direct evolution. This article is protected by copyright. All rights reserved.

Pyropia yezoensis, commonly known as “Nori” or “Laver” is an economically important marine crop. In natural or selected populations of P. yezoensis, coloration mutants are frequently observed. Various coloration mutants are excellent materials for genetic research and study photosynthesis. However, the candidate gene controlling the Pyropia coloration phenotype remains unclear to date. QTL-seq, in combination with kompetitive allele-specific PCR (KASP) and RNA-seq, can be generally applied to population genomics studies to rapidly identify genes that are responsible for phenotypes showing extremely opposite traits. Through cross experiments between the wild line RZ and red-mutant HT, offsprings with 1–4 sectors chimeric blade were generated. Statistical analyses revealed that the red thallus coloration phenotype is conferred by a single nuclear allele. Two-pair populations, consisting of 24 and 56 wild-type/red-type single-genotype sectors from F1 progeny, were used in QTL-seq to detect a genomic region in P. yezoensis harboring the red coloration locus. Based on a high-quality genome, we first identified the candidate region within a 3.30-Mb region at the end of chromosome 1. Linkage map-based QTL analysis was used to confirm the candidate region identified by QTL-seq. Then, four KASP markers developed in this region were used to narrow down the candidate region to a 1.42-Mb region. Finally, we conducted RNA-seq to focus on 13 differentially expressed genes and further predicted rcl-1, which contains one non-synonymous SNP [A/C] in the coding region that could be regulating red thallus coloration in P. yezoensis. Our results provide novel insights into the underlying mechanism controlling blade coloration, which is a desirable trait in algae.

Pyropia yezoensis, commonly known as “Nori” or “Laver” is an economically important marine crop. In natural or selected populations of P. yezoensis, coloration mutants are frequently observed. Various coloration mutants are excellent materials for genetic research and study photosynthesis. However, the candidate gene controlling the Pyropia coloration phenotype remains unclear to date. QTL-seq, in combination with kompetitive allele-specific PCR (KASP) and RNA-seq, can be generally applied to population genomics studies to rapidly identify genes that are responsible for phenotypes showing extremely opposite traits. Through cross experiments between the wild line RZ and red-mutant HT, offsprings with 1–4 sectors chimeric blade were generated. Statistical analyses revealed that the red thallus coloration phenotype is conferred by a single nuclear allele. Two-pair populations, consisting of 24 and 56 wild-type/red-type single-genotype sectors from F1 progeny, were used in QTL-seq to detect a genomic region in P. yezoensis harboring the red coloration locus. Based on a high-quality genome, we first identified the candidate region within a 3.30-Mb region at the end of chromosome 1. Linkage map-based QTL analysis was used to confirm the candidate region identified by QTL-seq. Then, four KASP markers developed in this region were used to narrow down the candidate region to a 1.42-Mb region. Finally, we conducted RNA-seq to focus on 13 differentially expressed genes and further predicted rcl-1, which contains one non-synonymous SNP [A/C] in the coding region that could be regulating red thallus coloration in P. yezoensis. Our results provide novel insights into the underlying mechanism controlling blade coloration, which is a desirable trait in algae.

The mitogen-activated protein kinase (MAPK) cascade is composed of three hierarchically organized protein kinases (MAP kinase kinase kinases, MAP kinase kinases, and MAPKs) and plays a vital role in regulating developmental processes and stress responses in plants. Pyropia yezoensis belongs to the old Rhodophyta and grows in intertidal zones. Although MAPKs have been well studied in some model plants, there is little information available concerning the P. yezoensis MAPK gene family and their roles in adaptation to intertidal environments. In this study, we identified 15 MAPKs from P. yezoensis (PyMAPKs) based on genome-wide analysis of the omics database. Conserved motif analysis indicated that all PyMAPKs had typical protein kinase domains; however, in addition to TEY/TDY activation loop motifs, some new types of activation loop motifs were identified: TTY, TAY, TQY, TSH, TAH, and TQA. Phylogenetic analysis showed that, with other red algae, PyMAPKs formed three groups independent from green plants, indicating that red algae retained older MAPK types than green plants. Transcriptomic analysis suggested that members of the PyMAPK gene family were involved in response to dehydration/rehydration and temperature stress and showed differential expression patterns. This work represents the first complete inventory of MAPKs from red algae and could help elucidate the mechanisms involving these proteins in P. yezoensis responses to abiotic stress.

Aquaporins are a class of ancient channel proteins. The role of aquaporin proteins (AQPs) has been extensively studied in land plants. However, the information on AQPs in seaweeds is unclear. Pyropia yezoensis (Rhodophyta) grows in the intertidal zone and is believed to be a model research organism for seaweeds in the intertidal zone. In this study, we isolated two AQPs from P. yezoensis (PyAQPs). They contained two conserved Asn-Pro-Ala (NPA) motifs and six transmembrane α-helices (H1–H6). Phylogenetic analysis showed that they are classified into two subfamilies, with much greater evolutionary distance with green plants. PyAQP1 belonged to the Glp group, whereas PyAQP2 belonged to a single small group. Subcellular localization showed that PyAQP1 was distributed on the plasma membrane of the cells, and PyAQP2 was mainly distributed in the plasma membrane and cytoplasm of the cells. The two genes displayed differential expression profiles for different abiotic stresses (desiccation, salinity, and temperature) at the mRNA and protein levels. Over-expression of PyAQPs in yeast cells indicated that PyAQPs improved the growth of yeast under osmotic stress. These results provide an insight towards understanding the molecular mechanisms of P. yezoensis responding to stress.

During photosynthesis, photosynthetic electron transport generates a proton motive force (pmf) across the thylakoid membrane, which is used for ATP biosynthesis via ATP synthase in the chloroplast. The pmf is composed of an electric potential (ΔΨ) and an osmotic component (ΔpH). Partitioning between these components in chloroplasts is strictly regulated in response to fluctuating environments. However, our knowledge of the molecular mechanisms that regulate pmf partitioning is limited. Here, we report a bestrophin-like protein (AtBest), which is critical for pmf partitioning. While the ΔpH component was slightly reduced in atbest, the ΔΨ component was much greater in this mutant than in the wild type, resulting in less efficient activation of nonphotochemical quenching (NPQ) upon both illumination and a shift from low light to high light. Although no visible phenotype was observed in the atbest mutant in the greenhouse, this mutant exhibited stronger photoinhibition than the wild type when grown in the field. AtBest belongs to the bestrophin family proteins, which are believed to function as chloride (Cl- ) channels. Thus, our findings reveal an important Cl- channel required for ion transport and homeostasis across the thylakoid membrane in higher plants. These processes are essential for fine-tuning photosynthesis under fluctuating environmental conditions.

The reaction center (RC) of photosystem II (PSII), which is composed of D1, D2, PsbI, and cytochrome b559 subunits, forms at an early stage of PSII biogenesis. However, it is largely unclear how these components assemble to form a functional unit. In this work, we show that synthesis of the PSII core proteins D1/D2 and formation of the PSII RC is blocked specifically in the absence of ONE-HELIX PROTEIN1 (OHP1) and OHP2 proteins in Arabidopsis (Arabidopsis thaliana), indicating that OHP1 and OHP2 are essential for the formation of the PSII RC. Mutagenesis of the chlorophyll-binding residues in OHP proteins impairs their function and/or stability, suggesting that they may function in the binding of chlorophyll in vivo. We further show that OHP1, OHP2, and HIGH CHLOROPHYLL FLUORESCENCE244 (HCF244), together with D1, D2, PsbI, and cytochrome b559, form a complex. We designated this complex the PSII RC-like complex to distinguish it from the RC subcomplex in the intact PSII complex. Our data imply that OHP1, OHP2, and HCF244 are present in this PSII RC-like complex for a limited time at an early stage of PSII de novo assembly and of PSII repair under high-light conditions. In a subsequent stage of PSII biogenesis, OHP1, OHP2, and HCF244 are released from the PSII RC-like complex and replaced by the other PSII subunits. Together with previous reports on the cyanobacterium Synechocystis, our results demonstrate that the process of PSII RC assembly is highly conserved among photosynthetic species.

Highlight A novel CRY1-interacting protein, TCP2, regulated by blue light and involved in the CRY1-mediated blue light signal pathway to regulate photomorphogenesis, is described., Light regulates plant growth and development via multiple photoreceptors including phytochromes and cryptochromes. Although the functions of photoreceptors have been studied extensively, questions remain regarding the involvement of cryptochromes in photomorphogenesis. In this study, we identified a protein, TEOSINTE-LIKE1, CYCLOIDEA, and PROLIFERATING CELL FACTOR 2 (TCP2), which interacts with the cryptochrome 1 (CRY1) protein in yeast and plant cells via the N-terminal domains of both proteins. Transgenic plants overexpressing TCP2 displayed a light-dependent short hypocotyl phenotype, especially in response to blue light. Moreover, light affected TCP2 expression in a wavelength-dependent manner and TCP2 positively regulates mRNA expression of HYH and HY5. These results support the hypothesis that TCP2 is a transcription activator which acts downstream of multiple photoreceptors, including CRY1.

This study investigated a new fungal disease that attacks Pyropia yezoensis, an economically important red alga that is extensively cultured in China. An incidence was found in a P. yezoensis farm during mid to end November 2012 at Haizhou Bay, Jiangshu Province, China. Histopathology revealed that the naturally infected thalli were overwhelmed by a fungus, leading to progressive red rot symptoms. The causative agent was isolated, grown in pure culture, and identified as a member of the genus Alternaria by morphology and sequence analysis of the nuclear ribosomal DNA containing the internal transcribed spacer (ITS) region of ITS1 and ITS2. In artificial infection experiments on P. yezoensis blades, the fungal isolate was able to cause the same characteristic histopathology seen in natural infections. This fungal isolate grew well at a wide range of temperatures (8–36 °C) and at low salinities (5–50 ‰). In an orthogonal test used to determine the effects of environmental factors (temperature, salinity, and conidia concentration) on disease expansion, it was found that higher temperatures and lower salinities easily caused red rot disease, with the optimal conditions for disease development being 23 °C, 24 ‰ salinity, and a conidia concentration of 105 mL−1. This is the first report to show that Alternaria causes red rot disease in P. yezoensis.

Bangia fuscopurpurea is a traditional mariculture crop having high nutritional value, eicosapntemacnioc acid (EPA) production, and protein content. As an intertidal species, it can tolerate drastic changes in abiotic factors such as temperature, hydration, and light radiation; however, genomic information on the evolutionary aspect and mechanism of EPA enrichment in B. fuscopurpurea and the role of EPA in cold tolerance in this species remain elusive. We conducted transcriptome profile analysis in B. fuscopurpurea to investigate the biological functions of genes associated with resistance to various environment factors. We identified 41,935 unigenes that were assembled and applied to public databases to define their functional annotation (NR, GO, KEGG, KOG, and SwissProt). We further identified genes that encoded key enzymes in EPA biosynthesis; five paralogous genes encoding delta5 desaturase were detected in B. fuscopurpurea. Fatty acid profiling and gene expression analysis of B. fuscopurpurea grown under cold stress were simultaneously performed. The EPA content was increased by 29.8% in the samples grown at 4°C, while the total amount of fatty acids remained unchanged. Moreover, all the EPA biosynthesis-related desaturase and elongase genes were upregulated under cold stress. Thus, we hypothesized that diverse EPA biosynthesis pathways and significant increase in gene copy numbers of fatty acid desaturases, together with the concomitant elevation in the transcriptional level of genes associated with fatty acid metabolism, lead to EPA accumulation and subsequently affect membrane fluidity, contributing to cold stress resistance in B. fuscopurpurea. Our findings not only provide a fundamental genetic background for further research in B. fuscopurpurea, but also have important implications for screening and genetic engineering of algae and plants for EPA production.

Quantitative real-time PCR (qRT-PCR) is frequently used for gene expression analysis. The selection of reference genes is required for normalization of the variation to avoid misinterpretation of experimental results and erroneous analyses. Pyropia yezoensis, growing in the intertidal zone, is an economically important seaweed. Although this intertidal seaweed has been an experimental system for understanding stress tolerance and developmental mechanisms, reference genes suitable for the normalization of qRT-PCR data have not previously been identified. In this study, expression stability of six candidate reference genes, including ACT, eIF4A, EF1α, GAPDH, TUA, and UBQ (traditional housekeeping genes), has been validated in a diverse set of samples representing different developmental stages and stress conditions of P. yezoensis. Three qRT-PCR analysis methods, geNorm, NormFinder, and BestKeeper, were evaluated systematically. The results indicated that ACT3, eIF4A, and EF1α were the optimal reference genes for P. yezoensis under stress conditions; UBQ, EF1α, and eIF4A were suitable for studying the expression of genes related to P. yezoensis development. TUA showed the lowest expression stability both under stress conditions and over developmental stages. Our results have provided a reference gene application guideline for P. yezoensis gene expression characterization using the qRT-PCR system.

High light-inducible proteins are critical for photosynthetic organisms when responding to high light stress. We cloned and characterized the HLIP gene from Porphyra yezoensis, which was designated as PyHLIP. Sequence analysis revealed that the open reading frame of PyHLIP was 387 bp in length that encoded a 128 amino acid-polypeptide containing a plastid transit peptide and an obvious membrane-spanning α-helix region. No intron existed in the genomic DNA of PyHLIP. The putative promoter of PyHLIP was TATA-less, and the initiator element (Inr) existed upstream of ATG. Analysis of genomic organization indicated there were two copies of PyHLIP gene in the P. yezoensis nuclear genome. The expression of PyHLIP can be induced by various stress conditions and is especially sensitive to high light.

The agarophyte Gracilariopsis lemaneiformis is both important for biological research and of significant economic value. However, the genetic diversity of wild populations of the alga has not been studied. We used amplified fragment length polymorphism (AFLP) PCR and simple sequence repeat (SSR) analysis to investigate diversity in four field populations, three from the coast of Qingdao and one from Weihai, China. Forty G. lemaneiformis isolates collected from the four different geographical groups were analyzed using 16 pairs of SSR primers for PCR amplification. However, no polymorphisms were detected, indicative of a degree of genetic homogeneity. A total of 347 reproducible bands were then amplified using eight AFLP primer pairs, and genetic indices of diversity within and between populations were calculated. This analysis revealed only low levels of genetic diversity both within and between the four geographical groups of G. lemaneiformis. The Weihai population showed a higher within-population genetic diversity than any of the Qingdao populations. The data suggest that there is only limited gene flow between populations. An UPGMA dendrogram revealed two main clusters, and one of these included all the Qingdao isolates. The order of clustering was in accordance with their geographical distribution. These results suggest that the wild G. lemaneiformis populations are closely related and that there is little genetic diversity within wild germplasm in the regions sampled.

Microsatellites are repetitive genomic elements that show high levels of variation and therefore are useful tools for studying genetic polymorphism and constructing genetic linkage maps of eukaryotic organisms. Porphyra yezoensis Ueda is an economically important seaweed that is being targeted for genetic improvement using marker-assisted breeding. Hence, in an attempt to develop microsatellite markers for P. yezoensis, a microsatellite (or simple sequence repeat)-enriched library was constructed to identify (GA)n and (CA)n motifs. A total of 71 perfect microsatellite clones were identified, of which 30 simple sequence repeat primer pairs were developed. Of these, 24 (80%) amplified polymerase chain reaction products of expected sizes. Twelve primer pairs amplified two to four bands, whereas another 12 primer pairs produced monomorphic banding patterns. Data for 12 loci were analyzed using POPGENE software version 1.32. A total of 29 alleles were produced at 12 loci, with an average of 2.42 alleles (Na) and 1.81 effective alleles (Ne) per locus. These markers were used to analyze the genetic diversity within 11 geographically different lines of P. yezoensis. Overall, these lines were clustered into two divisions with those from close geographic locations clustering together. Further cloning and sequencing of size variant alleles at two microsatellite loci revealed that the variable numbers of motif repeats in different alleles were major sources of polymorphisms.

Dihydrodipicolinate synthase (DHDPS) is the main enzyme of a specific branch of the aspartate pathway leading to lysine biosynthesis in higher plants. We have cloned and characterized the DHDPS gene from Zizania latifolia Griseb, which was named ZlDHDPS. Sequence analysis indicates that it contains an ORF of 954 bp interrupted by two exons and one intron encoding a polypeptide of 317 amino acids lacking a plastid transit peptide and a stop codon. The sequence of ZlDHDPS has high identity with known plant DHDPS in GenBank. Southern blotting analysis indicates that there are two copies of Z. latifolia DHDPS (ZlDHDPS) gene in the Z. latifolia nuclear genome. RT-PCR analysis shows the expression of ZlDHDPS is tissue specific and high level expression is present in fast-growing tissue and reproductive tissue. The 5′-regulatory sequence of ZlDHDPS contains a GT-1 box and a (CA)n element, which may play a role in regulating the expression of ZlDHDPS. The fusion construct of the ZlDHDPS sequence with the reporter gene GUS was transiently expressed in the onion epidermal cells by particle gun-mediated bombardment suggesting that ZlDHDPS was located in the cytoplasm, different from DHDPS gene of other species. Functional complementary analysis showed that ZlDHDPS can recover the DHDPS-deleted mutant of Escherichia coli.

Erect panicle (EP) is one of the more important traits of the proposed ideotype of high-yielding rice. Several rice cultivars with the EP phenotype, which has been reported to be controlled by a dominant gene, have been successfully developed and released for commercial production in North China. To analyze the inheritance of the EP trait, we generated segregating F2 and BC1F1 populations by crossing an EP-type variety, Liaojing 5, and a curved-panicle-type variety, Fengjin. Our results confirmed that a dominant gene controls the EP trait. Simple-sequence repeat (SSR) and bulked segregant analyses of the F2 population revealed that the EP gene is located on chromosome 9, between two newly developed SSR markers, RM5833-11 and RM5686-23, at a genetic distance of 1.5 and 0.9 cM, respectively. Markers closer to the EP gene were developed by amplified fragment length polymorphism (AFLP) analysis with 128 AFLP primer combinations. Three AFLP markers were found to be linked to the EP gene, and the nearest marker, E-TA/M-CTC200, was mapped to the same location as SSR marker RM5686-23, 1.5 cM from the EP gene. A local map around the EP gene comprising nine SSR and one AFLP marker was constructed. These markers will be useful for marker-assisted selection (MAS) for the EP trait in rice breeding programs.

The tribe Oryzeae consists of 12 genera and 71 species with a world distribution.Zizania latifolia (Griseb.) Turcz. ex Stapf is included in this tribe and possesses numerous traits valuable for rice breeding, such as disease and insect resistance, cold and flood tolerance, and high grain quality. The genetics and breeding ofZ. latifolia are still in their infancy. To facilitate genomic studies ofZizania, a genomic DNA library was constructed using a transformation-competent artificial chromosome (TAC) vector system. The TAC library contains 91, 584 TAC clones with an average insert size of approximately 45 kb, covering six haploidZizania genome equivalents. Very low signals after hybridization with chloroplast and mitochondrial genes indicate that the TAC library is predominantly composed of nuclear DNA. The TAC clones were stable inE. coli for 100 generations. Clones containing thedihydrodipicolinate synthase (DHPS) gene were screened by pooled PCR. The positive clones can be used forZ. latifolia DHPS gene cloning and functional analysis. The library will be useful in studies of genome structure, gene cloning and evolution of rice.

In the present study we propose and test the concept of R-ISSR, a new tool for genomic fingerprinting, mapping, and gene tagging. The concept is based on the fact that primers for inter-simple sequence repeat (ISSR) and random-amplified polymorphic DNA (RAPD) analysis elicit different genomic information, and the combined use of these 2 kinds of primers in the same polymerase chain reaction (PCR) reactions might reveal new genomic loci that could not be detected with either technique alone. The feasibility of this tool was first electronically simulated with sequence analysis software andArabidopsis chromosome sequence. Next, different combinations of ISSR and RAPD primers were applied in real PCR reactions to detect new genomic loci in 2 maize lines (Q319 and 1145). Sequencing gels were used to separate PCR products and showed good resolving ability in comparison with agarose gels. RAPD primers could be successfully used with ISSR primers for the detection of new genomic loci and applied in a new way for genomic mapping, fingerprinting, and gene tagging.

Polygalacturonase-inhibiting Proteins (PGIPs) are plant proteins that counteract fungal Polygalacturonases (PGs), which are important virulence factors. As defense proteins, PGIP can efficiently limit fungal invasion. In this study, a full-length cDNA of PPGIP1 gene (peach PGIP gene copy no.1) has been cloned from peach [prunus persica (L.) Batch] variety ‘Okubo’, which is resistant to fungal diseases such as peach scab. Sequence analysis revealed that the cDNA of PPGIP1 is 1380 bp in length and that its containing open reading frame (ORF) encodes for a polypeptide of 330 amino acids. The PPGIP1 gene from genomic DNA shows a single 147 bp intron that is efficiently spliced out of the PPGIP1 pre-mRNA transcript. The PPGIP1 has a high degree of identify with previously isolated PGIP genes and the encoded polypeptide shows all the characteristic features of PGIP peptides. Analysis of genomic organization and expression indicate that the PPGIP1 gene is one of a low copy number gene family with three copies in peach genome and its expression can be induced by exogenous salicylic acid (SA).

Background Pyropia haitanensis and P. yezoensis are two economically important marine crops that are also considered to be research models to study the physiological ecology of intertidal seaweed communities, evolutionary biology of plastids, and the origins of sexual reproduction. This plastid genome information will facilitate study of breeding, population genetics and phylogenetics. Principal Findings We have fully sequenced using next-generation sequencing the circular plastid genomes of P. hatanensis (195,597 bp) and P. yezoensis (191,975 bp), the largest of all the plastid genomes of the red lineage sequenced to date. Organization and gene contents of the two plastids were similar, with 211–213 protein-coding genes (including 29–31 unknown-function ORFs), 37 tRNA genes, and 6 ribosomal RNA genes, suggesting a largest coding capacity in the red lineage. In each genome, 14 protein genes overlapped and no interrupted genes were found, indicating a high degree of genomic condensation. Pyropia maintain an ancient gene content and conserved gene clusters in their plastid genomes, containing nearly complete repertoires of the plastid genes known in photosynthetic eukaryotes. Similarity analysis based on the whole plastid genome sequences showed the distance between P. haitanensis and P. yezoensis (0.146) was much smaller than that of Porphyra purpurea and P. haitanensis (0.250), and P. yezoensis (0.251); this supports re-grouping the two species in a resurrected genus Pyropia while maintaining P. purpurea in genus Porphyra. Phylogenetic analysis supports a sister relationship between Bangiophyceae and Florideophyceae, though precise phylogenetic relationships between multicellular red alage and chromists were not fully resolved. Conclusions These results indicate that Pyropia have compact plastid genomes. Large coding capacity and long intergenic regions contribute to the size of the largest plastid genomes reported for the red lineage. Possessing the largest coding capacity and ancient gene content yet found reveal that Pyropia are more primitive multicellular red algae.