Progress on plant orphan genes.

Orphan genes are located in a special evolutionary branch and have no significant sequence similarity with any other identified genes. Orphan genes are prevalent in every species, comparative genomics analyses found that all sequenced species contained a portion of orphan genes, and the number of orphan genes obtained by distinct screening conditions is different. Orphan genes are often associated with various stress responses, species-specific evolution and substance metabolism regulation. However, most of the orphan genes have not been well annotated or even have no recognizable functional domains, which brings some difficulties to the functional characterization of orphan genes. Compared with conserved genes, there is less research on orphan genes, which leads to the possibility that the importance of orphan genes may be "unrewarded". In this review, we summarize the origin and evolution of orphan genes, plant orphan gene screening and functions, and analyse the existing challenges and future research priorities and solutions, which provide theoretical basis for the study of orphan gene function and action mechanisms.

[Comparative Analysis of Different Inonotus obliquus].

OBJECTIVE: To screen optimal Inonotus obliquus strains that is suitable to cultivation, extracting effective components on industrialized production, genetics and breeding. METHODS: Inonotus obliquus mycelia from different countries were used. High quality strain was screened by comparing the antagonism between Inonotus obliquus and penicillium. The contents of crude fat, crude ash, crude protein and crude fiber were analyzed. RESULTS: It was showed that antagonism ability of Finnish birch was the strongest, its mycelia diameter was up to 53. 27 mm. It was also showed that nutritional ingredient was abundant in artificial cultured Inonotus obliquus strains. The contents of crude fat and crude protein in Finnish birch were higher, which was 1. 93% and 20. 23%, respectively. The contents of crude fat and crude protein in NBRC8681 strains were lower, which was 1. 55% and 19. 85%, respectively. The content of crude ash of JL04 strains was higher(8. 76%). The content of crude ash of NBRC8681 strains was lower(6. 8%). Crude fiber content was higher in HLJ01 strains, its content reached to 45. 09%. The least content appeared in MAFF420101 strains, whose crude fiber content was only 38. 27%. CONCLUSION: The strains of Finnish birch, HLJ01 and JL04 are suitable to exploitation.

[Comparison of component from different species of Inonotus obliquus].

In this paper, water content, mineral element and active ingredient concentration on fifteen Inonotus obliquus which comes from all over the world were studied. In the aspect of water content, the free water content of Finnish birch was the highest, reaching 77.21%, significantly better than that of other strains. The free water content of JL04 is the lowest, only 54.6%. The bound water content of HLJ01 which from Heilongjiang is the highest, reaching 10. 74% , significant differences among other strains. The bound water content of Birch Russia was the lowest. In the aspect of mineral element, the calcium content of NBRC9788 was the high- est (3.49 mg · g(-1)), significantly better than other strains. The second was Finnish birch. The lowest was CX02. The phosphorus content of NBRC9788 was the highest (210.12 µg · g(-1)), significantly superior to other strains. The lowest was JL04. In the aspect of active ingredient concentration, the triterpenoids content of HLJ01 was highest (23.7 mg · g(-1)), significantly better than other strains. It was good strains for biological products research and development and then was Finnish birch. The betulin content of MAFF420165 and MAFF420308 was low, they were not suitable for production. The polysaccharide content of Finnish birch was the highest (9.7%), significantly better than the other strains, it is one of the most ideal and good strains to develop polysaccharide. The polysaccharide content from MAFF420308 and MAFF420256 was 1.2%, lower than other strains. We suggest that avoid using these strains in the study of polysaccharide product development.

An improve method for somatic embryogenesis of Schisandra chinensis (Turcz.) Baillon.

A somatic embryogenesis protocol for plant regeneration of Schisandra chinensis (Turcz.) Baillon was established. Increased callus induction was obtained from mature zygotic embryos on Murashige and Skoog (MS) medium supplemented with 1.0 mg L(-1) N-phenyl-1,2,3-thidiazol-5-yl-urea (TDZ) or 2.0 mg L(-1) 2,4-dichlorophenoxyacetic acid (2,4-D). Addition of Zeatin (Zt) promoted the formation of embryogenic calli. To induce somatic embryogenesis, 2,4-D, TDZ and Zt were incorporated in the medium alone or in combination. Development of the maximum number of somatic embryos (81 globular, 37 heart, 52 torpedo and 37 cotyledon-stage) and germination of the highest number of embryos (50%) was observed on 1/2 MS medium supplemented with 1.0 mg L(-1) TDZ and 0.2 mg L(-1) Zt. Further development of somatic embryos into plantlets was completed in 1/2 MS medium free of plant growth regulators.

[Effects of nitrogen, phosphorus and potassium fertilizers on the yield, quality and nutrient uptake of Pulsatilla cernua].

OBJECTIVE: To study the effects of nitrogen (N), phosphorus (P2O5) and potassium (K2O) fertilizers on the growth, yield total saponins content and nutrient absorption of Pulsatilla cernua and provide a theoretical basis for good agriculture practice. METHODS: Field plot experiments was conducted, based on the D-saturation optimal design with three factors of nitrogen, phosphorus and potassium. Samples collected periodically were used for determination the contents of nutrient and total saponins, and for measurement of yield and agronomic characters. RESULTS: Nutrient contents in Pulsatilla cernua varied with growth stage and part under the same growth stage. Nutrient contents in aerial part were higher than that in root, while the proportion of nutritional absorption from seedling stage to the middle growth stage was larger than that at the late growth stage. Yield and total saponins content of Pulsatilla cernua were significantly influenced by the N1P2O5 and K2O applications, among three factors, N had the greatest effects, the next was K2O and P5O2. CONCLUSION: Pulsatilla cerntua under field cultivation should be fertilized properly, top-dressing with these fertilizers during the early growth stage and increasing the proportion of potassium. According to total saponin production of Pulsatilla cernua, the optimum fertilization model for high yield and good quality is 180 kg/hm2 of N, 79.74 kg/hm2 of P2O5, and 225 kg/hm2 of K20, with a N : P2O : K2O ratio of approximately 2.3 : 1 : 2.8.

Genic microsatellite markers in Brassica rapa: development, characterization, mapping, and their utility in other cultivated and wild Brassica relatives.

Genic microsatellite markers, also known as functional markers, are preferred over anonymous markers as they reveal the variation in transcribed genes among individuals. In this study, we developed a total of 707 expressed sequence tag-derived simple sequence repeat markers (EST-SSRs) and used for development of a high-density integrated map using four individual mapping populations of B. rapa. This map contains a total of 1426 markers, consisting of 306 EST-SSRs, 153 intron polymorphic markers, 395 bacterial artificial chromosome-derived SSRs (BAC-SSRs), and 572 public SSRs and other markers covering a total distance of 1245.9 cM of the B. rapa genome. Analysis of allelic diversity in 24 B. rapa germplasm using 234 mapped EST-SSR markers showed amplification of 2 alleles by majority of EST-SSRs, although amplification of alleles ranging from 2 to 8 was found. Transferability analysis of 167 EST-SSRs in 35 species belonging to cultivated and wild brassica relatives showed 42.51% (Sysimprium leteum) to 100% (B. carinata, B. juncea, and B. napus) amplification. Our newly developed EST-SSRs and high-density linkage map based on highly transferable genic markers would facilitate the molecular mapping of quantitative trait loci and the positional cloning of specific genes, in addition to marker-assisted selection and comparative genomic studies of B. rapa with other related species.

[Optimization of extraction technics of total saponins from Pulsatilla cernua].

OBJECTIVE: The extraction condition of total saponins from Pulsatilla cenua by ultrasonic wave was optimized by single factor and orthogonal experiments. METHODS: The largest absorbency of saponin was intended to be 470 nm by wavelength scan method with the pulchinenoside B4 as control sample, the linear relationship was observed between the absorbency and the content of saponin in the range of 0 - 0.040 mg/mL. RESULTS: The optimal conditions of extraction was as following: 80% of alcohol concentration, 40 min of ultrasonic time, 1: 20 of solid to liquid ratio, 80 W of ultrasonic power and one time for extraction. Among them, alcohol had the most significant effect on the extraction of total saponins. CONCLUSION: The content of total saponins in Pulsatilla cernua was 4. 32% under the optimal condition. The method developed here is efficient, stable, accurate and repeatable.

SSR and SCAR mapping of a multiple-allele male-sterile gene in Chinese cabbage (Brassica rapa L.).

The genic multiple-allele inherited male-sterile gene Ms in Chinese cabbage (Brassica rapa L.) was identified as a spontaneous mutation. Applying this gene to hybrid seed production, several B. rapa cultivars have been successfully bred in China. A BC(1) population (244 plants) was constructed for mapping the Ms gene. Screening 268 simple sequence repeat (SSR) markers which cover the entire genome of Chinese cabbage was performed with bulked segregant analysis (BSA). On the basis of linkage analysis, the Ms gene was located on linkage group R07. In addition, through the amplified fragment length polymorphism (AFLP) and the sequence-characterized amplified region (SCAR) techniques combining BSA, two SCAR markers which were converted from corresponding AFLP markers flanked the Ms gene. Finally, a genetic map of the Ms gene was constructed covering a total interval of 9.0 cM. Two SCAR markers, syau_scr01 and syau_scr04, flanked the Ms gene at distances of 0.8 and 2.5 cM, respectively. All the SSR markers (cnu_m273, cnu_m030, cnu_m295, and syau_m13) were mapped on the same side of the gene as syau_scr04, the nearest one of which, syau_m13, was mapped at a distance of 3.3 cM. These SSR and SCAR markers may be useful in marker-assisted selection and map-based cloning.

Genomic distribution of simple sequence repeats in Brassica rapa.

Simple Sequence Repeats (SSRs) represent short tandem duplications found within all eukaryotic organisms. To examine the distribution of SSRs in the genome of Brassica rapa ssp. pekinensis, SSRs from different genomic regions representing 17.7 Mb of genomic sequence were surveyed. SSRs appear more abundant in non-coding regions (86.6%) than in coding regions (13.4%). Comparison of SSR densities in different genomic regions demonstrated that SSR density was greatest within the 5'-flanking regions of the predicted genes. The proportion of different repeat motifs varied between genomic regions, with trinucleotide SSRs more prevalent in predicted coding regions, reflecting the codon structure in these regions. SSRs were also preferentially associated with gene-rich regions, with peri-centromeric heterochromatin SSRs mostly associated with retrotransposons. These results indicate that the distribution of SSRs in the genome is non-random. Comparison of SSR abundance between B. rapa and the closely related species Arabidopsis thaliana suggests a greater abundance of SSRs in B. rapa, which may be due to the proposed genome triplication. Our results provide a comprehensive view of SSR genomic distribution and evolution in Brassica for comparison with the sequenced genomes of A. thaliana and Oryza sativa.

The reference genetic linkage map for the multinational Brassica rapa genome sequencing project.

We describe the construction of a reference genetic linkage map for the Brassica A genome, which will form the backbone for anchoring sequence contigs for the Multinational Brassica rapa Genome Sequencing Project. Seventy-eight doubled haploid lines derived from anther culture of the F(1) of a cross between two diverse Chinese cabbage (B. rapa ssp. pekinensis) inbred lines, 'Chiifu-401-42' (C) and 'Kenshin-402-43' (K) were used to construct the map. The map comprises a total of 556 markers, including 278 AFLP, 235 SSR, 25 RAPD and 18 ESTP, STS and CAPS markers. Ten linkage groups were identified and designated as R1-R10 through alignment and orientation using SSR markers in common with existing B. napus reference linkage maps. The total length of the linkage map was 1,182 cM with an average interval of 2.83 cM between adjacent loci. The length of linkage groups ranged from 81 to 161 cM for R04 and R06, respectively. The use of 235 SSR markers allowed us to align the A-genome chromosomes of B. napus with those of B. rapa ssp. pekinensis. The development of this map is vital to the integration of genome sequence and genetic information and will enable the international research community to share resources and data for the improvement of B. rapa and other cultivated Brassica species.