Jianhua Zhang

Statistic Analysis on Quantitative Characteristics for Developing the DUS Test Guideline of Ranunculus asiaticus L.

Abstract Selection of quantitative characteristics, division of their expression ranges, and selection of example varieties are key issues on developing DUS Test Guidelines, which are more crucial for quantitative characteristics since their expressions vary in different degrees. Taking the development of DUS Test Guideline of Ranunculus asiaticus L. as an example, this paper applied statistic-based approaches for the analyses of quantitative characteristics. We selected 9 quantitative characteristics from 18 pre-selected characteristics, based on within-variety uniformity, stability between different growing cycles, and correlation among characteristics, by the analyses of coefficient of variation, paired-samples t-test and partial correlation. The expression ranges of the 9 selected quantitative characteristics were divided into different states using descriptive statistics and distribution frequency of varieties. Eight of the 9 selected quantitative characteristics were categorized as standard characteristics as they showed one peak in distribution frequency of 120 varieties in various expressions of the characteristics, whereas, plant height can be categorized as grouping characteristic since it gave two peaks, and can group the varieties into pot and cut varieties. Finally, box-plot was applied to visually select the example varieties, and varieties 7, 12, and 28 were determined as the example varieties for plant height. The methods described in this paper are effective for the selection of quantitative characteristics, division of expression ranges, and selection of example varieties in Ranunculus asiaticus L. for DUS test, and may also be interest for other plant genera.

Diversity in flower colorations of Ranunculus asiaticus L. revealed by anthocyanin biosynthesis pathway in view of gene composition, gene expression patterns, and color phenotype

Anthocyanin biosynthesis is one of the best studied secondary metabolisms. However, related pathways were generally concluded based on anthocyanin components; most studies focused on the backbone forming of anthocyanidins (cyanidin, delphinidin, and pelargonidin) of model or commercial plants, while anthocyanin modification was less discussed, and non-model plants with abundant colorations were less researched either. Ranunculus asiaticus L. has great diversity in flower colorations, not only indicating its value in researching anthocyanin biosynthesis but also implying it is unique in this regard. Based on transcriptome sequencing and gene annotation of three varieties (10 samples) of Ranunculus asiaticus L., 176 unigenes from 151,136 unigenes were identified as involved in anthocyanin biosynthesis, among which, 74 unigenes were related to anthocyanin modification; 61 unigenes were responsible for glycosylation at C3 and C5 with 3-monosaccharides of glucose, 3-biosides of rutinose, sophorose, or sambubiose to form 3Gly-, 3Gly5Gly-, 3Gly3′Gly-, 3Gly2′′Gly-, 3Gly2′′Xly-, 3Gly2′′Rly-glycosylated anthocyanins, etc.; 2 unigenes transferred –CH3; 11 unigenes of BAHD family catalyzd the aromatic or malonyl acylation at 6′′ / 6′′′′position of 3/5-O-glucoside. Based on gene composition, a putative pathway was established. The pathway was validated by flower colorations, and gene expression patterns where F3H, F3′H, 3GT, 5GT, and FMT2 were highly expressed in varieties colored as lateritious and carmine, while variety with purple flowers had high expression of F3′5′H and 3MAT. In view of anthocyanin biosynthesis pathway of Ranunculus asiaticus L., great diversity in its flower colorations was illustrated via the complete branches (F3H, F3′H and F3′5′H) as well as complete modifications (glycosylation, methylation, and acylation), and besides, via the higher percentage of C3 glycosylation than C5 glycosylation.

Test of hirudin activity by tracking the binding of hirudin to thrombin in the presence of BS3 cross-linking.

Hirudin has a great potential in inhibiting thrombin, and its antithrombin activity has direct bearing on its clinical application. Using bovine alpha-thrombin and recombinant hirudin of Poecilobdella javanica purified from Phichia pastoris as materials, this study introduced a novel method to testing antithrombin activity of hirudin visually and dynamically by tracking the binding of hirudin to thrombin. After incubating the mixture of thrombin and hirudin at 37 °C for 5 min, the binding of hirudin to thrombin was cross-linked by bis[sulfosuccinimidyl] suberate for 30 min and visualized by SDS-polyacrylamide gel electrophoresis. With the aid of image analysis on the basis of INRA-Noésis E1D analysis software, antithrombin activity of hirudin was calculated through intensity variations of protein bands of either thrombin-hirudin compound, unbound thrombin, or unbound hirudin. In this regard, activity of the given hirudin was tested to be 5625 ATU/mg based on a single reaction, and 5675.3 ATU/mg based on a series of reactions in a stepwise manner, close to the result of 6000 ATU/mg concluded by titration method. The superiorities of the method include good accuracy (the minimum testable concentration of hirudin is 1.5 &mgr;g/ml) and little sample consumption (sample consumption of hirudin is generally 1–11.5 &mgr;l using the apparatus of Mini Protean 3 Cell). Easy operation, low input, and equipment requirement also grant it as an effective way.

Distinctness Determination of DUS Test on Some Quantitative Characteristics of Rice

When separately using the DUS Test Guideline to determine the distinctness of some quantitative characteristics, we found even the characteristics of the same variety had different “Notes” in different environments. So, the correlation of quantitative characteristics with environment factor, with variety factor, and characteristics themselves were discussed in this paper. Results showed that, in order to improve the reliability of DUS test, all the quantitative characteristics should be grouped according to their stability or correlation coefficients with environment changes; different groups can be given corresponding weighting coefficients according to their stability; the distinctness determination could be based on the weighted measurements of characteristic groups; variety factor should be considered in DUS test and also in the selection of standard varieties used in DUS test.