How Specific is Non-Hypersensitive Host and Nonhost Resistance of Barley to Rust and Mildew Fungi？

Full nonhost resistance can be defined as immunity, displayed by an entire plant species against all genotypes of a plant pathogen. Interesting biological questions are, whether the genes responsible for the nonhost status of a plant species have a general or a specific effectiveness to heterologous （＂nonhost＂） pathogens？ Is the nonhost resistance to pathogens of plant species that are related to the nonhost based on R-genes or on other types of genes？ We study this question in barley （Hordeum vulgare L.）, which is a near-nonhost to several rusts （Puccinia） of cereals and grasses. By crosses and selection we accumulated susceptibility and developed an experimental line, SusPtrit, with high susceptibility to at least nine different heterologous rust taxa such as the wheat and Agropyron leaf rusts （P. triticina and P. persistens, respectively）. At the microscopic level there is also some variation among barley accessions in the degree that the heterologous wheat powdery mildew （Blumeria graminis f.sp. tritici） is able to form haustoria in epidermal cells. So, also the genetics of the variation in level of nonhost resistance to heterologous mildew fungi can be studied in barley. Our data obtained on mapping populations involving three regular nonhost-immune accessions （Vada, Cebada Capa and Golden Promise） suggest that nonhost resistance is the joined effect of multiple, quantitative genes （QTLs） and very occasionally a major gene （R-gene？） is involved. Most QTLs have effect to only one or two heterologous rusts, but some have a wider spectrum. This was confirmed in a set of QTL-NILs. Those QTL-NILs are used to fine-map the effective genes. In some cases, a QTL region with effectiveness to several heterologous rusts might be a cluster of genes with a more narrow spectrum of effectiveness. Our evidence suggests that nonhost resistance in barley to rust and powdery mildew fungi of related Gramineae is not due to R-genes, but to pathogen species-specific quantitative resistance genes.     

SNP Analysis and Haplotype Identification in Chymotrypsin Inhibitor-2 （CI-2） Gene of Barley

Barley chymotrypsin inhibitor-2 （CI-2） was considered to be a promising candidate for enhancing the nutritional value of other cereals by increasing its concentration as it is rich in lysine than any other storage protein. Also, it was proposed that CI-2 might play an important role in the inhibition of proteolytic enzymes from pests or pathogens as CI-2 can strongly inhibit chymotrypsin and subtilisin. In this study, a total of 93 CI-2 gene sequences were isolated from wild and cultivated barley. 48 SNPs and 4 indels were detected across the entire sequences. The frequency of SNPs in the noncoding region （1 out of 9 bases） was slightly higher than that in the coding region （1 out of 10.7 bases）. In all, 33.3% of the candidate cSNPs resulted in amino acid changes. As a total, the 24 cSNPs resulted in 15 amino acid changes. Ten distinguishable haplotypes were detected, among which 3 haplotypes were shared in the most barley accessions, whereas the rest of the haplotypes appeared at a lower frequency. In addition, three haplotypes （haplotype 4, 8, and 9） were unique for single accessions. These results suggested that low diversity at the CI-2 locus was detected among the cultivated and wild barley.     

Mapping of Mutant Gene prbs Controlling Poly-Row-and-Branched Spike in Barley （Hordeum vulgare L.）

A row-type mutant of barley named poly-row-and-branched spike (prbs) was previously obtained from a two-rowed cultivar Pudamai-2 after treated by inflorescence soaking in maize total DNA solution.The mutant produces branched spikes with irregular multiple rows.Genetic analysis indicated that the mutant phenotype was caused by a recessive gene prbs,and the PRBS locus had a recessive epistatic effect on an independent locus (denoted as Vrsx) conferring the variation of two-rowed spike vs.six-rowed spike.This study aimed to map PRBS as well as Vrsx using simple sequence repeats (SSR) markers.We developed an F2 population from a cross between the prbs mutant and a six-rowed cultivar Putianwudu for the gene mapping.As the two target loci interacted to result in a segregation ratio of two-rowed type:sixrowed type:prbs=9:3:4 in the population,we adopted a special strategy to map the two loci.PRBS was mapped between SSR markers HvLTPPB and Bmag0508A on the short arm of chromosome 3H,with distances of 24.7 and 14.3 cM to the two markers,respectively.Vrsx was mapped between SSR markers Bmag0125 and Bmag0378 on chromosome 2H,with distances of 6.9 and 15.3 cM to the two markers,respectively.This suggests that Vrsx should be the known locus Vrsl,which predominantly controls row-type variation in barley cultivars,and PRBS is a new locus related to the row type of spikes in barley.     

Comparative Analysis of Hina Gene Sequences in Wild （Hordeum spontaneum） and Cultivated （H. vulgare） Barleys

The Hina gene is one of the two known Hin genes for hardness, and its RNA expression is correlated with grain hardness and dry matter digestibility variation. In this study, only one clone of Hina gene was obtained from one barley accession. A total of 121 Hina gene sequences were isolated from 121 wild barley {Hordeum spontaneum) accessions in Israel, Iran, and Turkey, and then their molecular characteristics were compared with 97 Hina gene sequences from 74 cultivated barley (H. vulgäre) lines in Europe and 23 landrace (H. vulgäre) with global distribution and other 26 Hina gene sequences from cultivated barleys (H. vulgäre) with unknown global distribution. C/s-acting regulatory element (CARE) searching revealed that there were different types of regulatory element for the Hina gene in wild and landrace/cultivated barleys. There were six consistent cw-acting binding sites in wild and landrace/cultivated barleys, whereas 8 to 16 inconsistent TATA-boxes were observed. In addition, three special elements (E2Fb, Spl, and boxS) were only observed in wild barley, while one (AT 1 -motif) was only found in landrace/cultivated barley. Forty-four deduced amino acid sequences of HINA from wild and landrace/cultivated barleys were obtained by deleting repetitive amino acid sequences, and they were clustered into two groups on the basis of Neighbor-Joining analysis. However, there was no obvious difference in the amino acid sequences of HINA between wild and landrace/cultivated barleys. Comparing to protein secondary structure of wheat PINA, it was indicated that HINA also existed a signal peptide. In addition, HINA was a hydrophilic protein on the basis of the protein properties and composition.     

Identiifcation and Molecular Mapping of the RsDmR Locus Conferring Resistance to Downy Mildew at Seedling Stage in Radish （Raphanus sativus L.）

Downy mildew （DM）, caused by the fungus Peronospora parasitica, is a destructive disease of radish （Raphanus sativus L.） worldwide. Host resistance has been considered as an attractive and environmentally friendly approach to control the disease. However, the genetic mechanisms of resistance in radish to the pathogen remain unknown. To determine the inheritance of resistance to DM, F1, F2 and BC1F1 populations derived from reciprocal crosses between a resistant line NAU-dhp08 and a susceptible line NAU-qtbjq-06 were evaluated for their responses to DM at seedling stage. All F1 hybrid plants showed high resistance to DM and maternal effect was not detected. The segregation for resistant to susceptible individuals statistically iftted a 3：1 ratio in two F2 populations （F2（SR） and F2（RS））, and 1：1 ratio in two BC1F1 populations, indicating that resistance to DM at seedling stage in radish was controlled by a single dominant locus designated as RsDmR. A total of 1 972 primer pairs （1 036 SRAP, 628 RAPD, 126 RGA, 110 EST-SSR and 72 ISSR） were screened, and 36 were polymorphic between the resistant and susceptible bulks, and consequently used for genotyping individuals in the F2 population. Three markers （Em9/ga24370, NAUISSR826700 and Me7/em10400） linked to the RsDmR locus within a 10.0 cM distance were identiifed using bulked segregant analysis （BSA）. The SRAP marker Em9/ga24370 was the most tightly linked one with a distance of 2.3 cM to RsDmR. These markers tightly linked to the RsDmR locus would facilitate marker-assisted selection and resistance gene pyramiding in radish breeding programs.     

Cloning and Characterization of Genes Coding for Fructan Biosynthesis Enzymes （FBEs）in Triticeae Plants

Fructan is not only a carbon source for storage but also plays an important role as anti-stress agents in many plant species. Complex fructans having both β-（2,1）- and β-（2,6）-linked fructosyl units accumulate in Triticeae plants commonly. Three enzymes （sucrose： sucrose 1-fructosyltransferase, 1-SST, EC： 2.4.1.99; sucrose： fructan 6-fructosyltransferase, 6- SFT, EC： 2.4.1.10; and fructan： fructan 1-fructosyltransferase, 1-FFT, EC： 2.4.1.100） were involved in fructan biosynthesis in Triticeae plant species. We successfully isolated these genes from tetraploid wheat （Triticum turgidum, genotype： AABB）, common wheat （Triticum aestivum L., genotype： AABBDD） and three wild relatives of common wheat, Triticum urartu Thum. （the origin of the AA genome）, Aegilops speltoides （Tausch） Gren. （the putative source of the SS genome） and Aegilops tauschii Coss. （the source of the DD genome）. Sequence analysis revealed that all the FBEs （fructan biosynthetic enzymes） had three highly conserved functional motifs except 1-SST （EU981912） from tetraploid wheat species only with conserved DPNG. Low pI （isoelectric point） and potential N-glycosylation sites were predicted, which were crucial for protein compartmentation and post-translational process. Analysis on subcelluar localization signals showed that only 6-SFT had vacuolar-directed signal. Sequences alignment result showed that 1-SST and 1-FFT were more conservative and had closer relationship each other, while 6-SFT was more active during the evolution processing. According to the syntenic relationship between wheat and rice genome, FBEs were predicated to be located on the homeologous group 6 and group 2 chromosomes. Expression profile confirmed that expression of all the three FBEs were drought-stress induced. This study can assist to establish a useful theoretical platform for cold- or drought-tolerant improvement of wheat by modulating FBEs expression.     

Isolation and Expression Analysis of Two Genes Encoding Cinnamate 4-Hydroxylase from Cotton （Gossypium hirsutum）

Two genes （GhC4H1 and GhC4H2） that encode putative cotton cinnamate 4-hydroxylases that catalyze the second step in the phenylpropanoid pathway were isolated from developing cotton fibers. GhC4H1 and GhC4H2 each contain open reading frames of 1 518 base pairs （bp） in length and both encode proteins consisting of 505 amino acid residues. They are 90.89% identical to each other at the amino acid sequence level and belong to class I of plant C4Hs. GhC4H1 and GhC4H2 genomic DNA are 2 247 and 2 161 bp long, respectively, and contain two introns located at conserved positions relative to the coding sequence. GhC4HI and GhC4H2 promoters were isolated and found to contain many cis-elements （boxes P, L and AC-1 element） previously identified in the promoters of other phenylpropanoid pathway genes. Histochemical staining showed GUS expression driven by the GhC4H1 and GhC4H2 promoters in ovules and fibers tissues. GhC4H1 and GhC4H2 were also widely expressed in other cotton tissues. GhC4H2 expression reached its highest level during the elongation stage of fiber development, whereas GhC4H1 expression increased during the secondary wall development period in cotton fibers. Our results contribute to a better understanding of the biochemical role of GhC4H1 and GhC4H2 in cotton fiber development.     

Identification of Self-Incompatibility Genotypes in Some Sand Pears （Pyrus pyrifolia Nakai） by PCR-RFLP Analysis

The identification of self-incompatibility genotype （S-genotype） will be useful for selection of pollinizers and design of crossing in cultivar improvement of sand pear. This paper reported the identification of self-incompatibility genotypes of seven Chinese and two Japanese sand pear cultivars using PCR-RFLP analysis and S-RNase sequencing. The Sgenotypes of these cultivars were determined as follows： Huali 1 S1S3, Shounan S1S3, Xizilti S1S4, Qingxiang S3S7, Sanhua S2S7, Huangmi （Imamuranatsu） S1S6, Huali 2 S3S4, Baozhuli S7S33, Cangxixueli S5S15. S-RNase alleles （S1 to S9） in sand pear could be identified effectively by PCR-RFLP analysis.     

Expression and Activity Analysis of Non-Structural Protein 2 （NS2） of Bombyx mori Densovirus Zhenjiang Strain

The gene of the non-structure protein 2 （NS2） was cloned by PCR from the genome ofBombyx mori densovirus Zhenjiang strain （BmDNV-Z）, inserted into prokaryotic expression vector pET28a to construct recombinant plasmid pET28a-NS2 and then expressed in bacteria Escherichia coli BL21 （DE3）. The expressed recombinant protein was identified by SDS-PAGE and Western blot analysis. Then, the recombinant protein was purified by Ni-NTA column, renatured and tested for enzyme activities. The purified NS2 protein exhibited a helicase activity unwinding double-stranded DNA substrates into single-strand primers, and higher unwinding activity to polarity substrate. Similarly, the purified NS2 protein possessed an ATPase activity and its enzyme activity was 0.276 μmol gg^-1 h^-1 in this study. The results indicated that the non- structure protein which encoded by the gene of BmDNV-Z NS2 possesses the biological activities of helicase and ATPase, and the helicase prefers to polarity substrates. Based on these results, it is speculated that the gene of BmDNV-Z NS2 plays an important role in the viral DNA replication.     

Identification and Expression Analysis of the Phosphoenolpyruvate Carboxylase Gene Family in Peanut （Arachis hypogaea L.）

Phosphoenolpyruvate carboxylase （PEPC） is widely distributed in plants and bacteria, and catalyzes the carboxylation of phosphoenolpyruvate to form oxaloacetate and inorganic phosphate. To investigate the molecular mechanisms of the regulation and control of peanut oil, with the degenerated primers and RACE-PCR approach, five PEPC genes were cloned from peanut, and designated as AhPEPC1, AhPEPC2, AhPEPC3, AhPEPC4, and AhPEPC5, respectively. The structure and phylogenetic analysis of PEPC protein indicated that AhPEPC1-4 genes encoded a typical plant-type PEPC-enzyme, and AhPEPC5 a bacterial-type. By real-time quantitative RT-PCR approach the expression pattern of each gene was detected in various tissues of normal and high oil-content peanut varieties. It was found that there was a lower expression level of AhPEPCs genes except for the AhPEPC2 in high-oil peanut than normal-oil peanut line. The results provide some fundamental information for the further investigation of plant PEPC proteins and their role in regulation of oil-content in peanut seeds.     

QTL Detection and Epistasis Analysis for Heading Date Using Single Segment Substitution Lines in Rice （Oryza sativa L.）

Heading date of rice is a key agronomic trait determining cultivated areas and seasons and affecting yield. In the present study, ifve primary single segment substitution lines with the same genetic background were used to detect quantitative trait loci （QTLs） for heading date in rice. Two QTLs, qHD3 and qHD6 on the short arm of chromosome 3 and the short arm of chromosome 6, respectively, were identiifed under natural long-day （NLD）. Nineteen secondary single segment substitution lines （SSSLs） and seven double segments pyramiding lines were designed to map the two QTLs and to evaluate their epistatic interaction between them. By overlapping mapping, qHD3 was mapped in a 791-kb interval between SSR markers RM3894 and RM569 and qHD6 in a 1 125-kb interval between RM587 and RM225. Results revealed the existence of epistatic interaction between qHD3 and qHD6 under natural long-day （NLD）. It was also found that qHD3 and qHD6 had signiifcant effects on plant height and yield traits, indicating that both of the QTLs have pleiotropic effects.     

Cloning and Characterization of a Somatic Embryogenesis Receptor-Like Kinase Gene in Cotton （Gossypium hirsutum）

A novel gene,GhSERK1,was identified in cotton.It encoded a protein belonging to the somatic embryogenesis receptorlike kinase (SERK) family.The genomic sequence of GhSERKI was 6920 bp in length,contain...