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Crop Biotechnology Research

(1). Marker-assisted breeding on rice bacterial blight resistance

T he study of marker-assisted bacterial blight resistance breeding has revealed the distribution of xa5, Xa7, and Xa21 genes in the F 2 populations derived from the two corsses of Tai-Ken 9 × IRBB66 and Taichung-Shien 10 × IRBB66. The three genes were dectected by closely linked molecular markers blonging to STS, SSR, and CAPS types. The results show that the above molecular markers worked efficiently in discrimination of various genotypes in progenies. The proportion of homozygous resistant genotypes of xa5, Xa7, and Xa21 loci are 27.95%, 20.88%, and 24.08% respectively in the F 2 population of TCS10 × IRBB66. On the other hand, the proportion of homozygous resistant genotypes of xa5, Xa7, and Xa21 loci are 25.0%, 18.95%, and 24.28% respectively in the F2 population of TK9 × IRBB66. The distributions of most examined genotypes are following Mendel segregation law except Xa7 locus in the F 2 population of TK9 × IRBB66, which showing bias distritubion. The results of our study provide the information of inheritance behaviors of these three resistance genes, and the information will be helpful for the proper strategies design of breeding.

(2). Detection of tissue culture variation and cloning of mutation-related genes on Phalaenopsis spp.

The mutant plants of three varieties have be collected, including 1.Fuller's Sunset;2.Little Emperor;3.Sogo Gold Tris. Two hundred RAPD primer pairs have been tested on normal and mutant plants, and the markers have amplified by the cDNA-PCR examination. Totally 716 RAPD molecular markers have been generated, 55 markers were differently expressed. Thirty five markers represent over-expressed loci and 20 markers represent down-regulated loci. These loci could be the key mutant genes related to tissue culture-variation and these loci could be the candidate for developing mutation-detection genes. Forty-one differently expressed fragments have been sequenced.

(3). Establishment of DNA barcode and biochemical fingerprints of buckwheat and Job’s tears

On buckwheat study, we use buckwheat Taichung No.5 and Tartary buckwheat Taichung No. 2 as research materials. Total RNA of leaves and developing seeds were extracted to compare their profiles. This year, we complete the analysis of seed transcriptome, gene ontology and annotation. For seed transcriptomes, there are 97,200 and 74,544 contigs are assembled respectively. Contig analysis report is also completed. In order to understand the functional components in seeds and plants, we extract buckwheat samples, concentrated and then subjected to HPLC-DAD analysis. Finally, samples are analyzed by LC-MS. The principle components are different between fruits and leaves, and that rutin content is higher in Tartary buckwheat than common buckwheat.

On Job’s tears study, we follow literatures on chloroplast genome study to design 20 sets of primers, amplified selected regions of chloroplast genes and rRNA ITS gene. The chloroplast genes cover 1/10 of chloroplast genome but polymorphism is lower than the rRNA ITS gene. We suggest to use rRNA ITS as DNA barcode for Coix lacryma-jobi L. On chemical fingerprinting, we have extracted seed coats of Job’s tears Taichung No. 3 using ethanol and concentrated for further analysis.

(4). Study on the effects of buckwheat extracts on modulating blood sugars

There are many beneficial components in buckwheat seeds and plants including rutin, quercetin, phenolic acids and dietary fiber. Research has shown that buckwheat or its extracts are beneficial in the regulation of blood sugar and can ameliorate adverse symptoms associated with Diabetes mellitus. In addition, buckwheat is rich of D-chiro-inositol (DCI), which is regarded as effective insulin sensitizing agent. Buckwheat extracts can be taken as dietary supplement to stabilize blood sugar. In this project, buckwheat seeds and plant components were extracted and concentrated. The root extracts are shown to inhibit action of α-amylase. The extracts of root, leaves and seeds will be subjected to chemical fingerprinting by HPLC. Seed mills of buckwheat, Tartary buckwheat and other grains including rice and wheat, are digested by α-amylase hydrolysis, the results shows that both buckwheat seed mills are digested slower than other grains. The absorption spectrum of the digest solution will be subjected to analysis later to compare study the types and quantity of polyphenols. Seeds mill of both buckwheat are compared and confirmed that rutin content is significantly higher in Tartary buckwheat than in common buckwheat.

(5). Study on the hybridization barriers and improving breeding efficiency of Paphiopedilum

Paphiopedilum is regarded as a promising genus that can succeed the moth orchids and Oncidium industry in Taiwan. New Paphiopedilum varieties is developed through hybridization, however there are few reports addressed on factors related to hybridization barriers. In addition, few seeds or difficulty of hybridization often occur on some elite varieties or combinations of hybridizations. The aims of this project are to investigate factors influencing the success of hybridization of Paphiopedilum, including characteristics of pollinia, time of pollination and growth of hybridized seedlings, in order to obtain new varieties. From observation of the reproductive organs, pollinia is located at both sides of gynandrium below stigma. The ovary includes three fused carpels, there is no obvious septum on stigma. The anther is composed of 4 pollinia, each pollinium is flat round or oval shape, containing numerous pollens. Anther wall can be observed from paraffin-embedding section of pollinium. At the beginning of flower opening, microspores are undergoing meiosis rapidly. After 2-5 days the anther wall starts to collapse and release cell components. We suggest that the anther wall undergo apoptosis and autophagy process to release cell nutrients for subsequent pollination process. The pollinia can absorb nutrients from the broken down anther wall, become sticky on the surface that enable the pollinia to adhere on the surface of stigma. The period is optimal for pollination. At the same time, the there are numerous finger-like archesporial tissue arising from placenta, in which megaspore mother cell undergoes meiosis inside. We suggest that fertilization can be completed soon after pollination and begins the development of seeds.

We also conducted cross hybridization of different species and pollinia storage experiment. Among the cross combination, pollen can germinate, pass through stigma and enter ovary. At -20 ℃ , pollinia can be stored for at least 4 weeks and still maintain viability.

(6). The chemical fingerprints of seed extracts of Coix lacryma-jobi

Job’s tears ( Coix lacryma-jobi) is traditional Chinese medicinal foods that is nutritious and contains rich functional metabolites. It has been proved to improve hyperlipidemia and hyperglycemia, inhibit tumor grwoth, enhance functions of immune system, anti-inflammation and regulate endocrine hormones. In order to understand the variation of seed extracts of different varieties and grown area. Ultrasonic assisted methanol extraction was employed for all samples included in this study. The extracts were analyzed by HPLC/UV 260 nm to compare their fingerprints. Using present chromatographic condition, 5 major peaks stably representing more than 90% of the total absorption area are identified. After seeds polishing, the absorption peak area lost 85-97%, indicating that the most UV absorption metabolites exist in bran. The chemical fingerprints and amounts of each metabolite are very similar between varieties Taichung No. 3 and Taichung Selection No. 4 grown at Da-Tsueng, Da-Ya and Erh-Lin. In the LC chromatogram, 3 peaks showed stability and reproducibility which can be employed for quality control in the future. The content of coixol can be detected by HPLC. With GCMS, 5 reported coix lactams and coixol can be detected and relatively quantified. The chemical fingerprints of seed extracts can be employed as basis for comparing samples of different origin.

(7). Analysis of rutin and quercetin in seed and leaf of buckwheat and Tartary buckwheat

Buckwheat is an important miscellaneous crop in central Taiwan, it contains abundant nutraceutical components and rutin. The content of rutin in buckwheat Taichung No. 2 is the highest at 7th week after sowing, which is 38397 ppm, approximately equal to cited value (38752 ppm) in literatures. It is also the highest at 7th week after sowing for Taichung No. 5, which is 46348 ppm, that is approximately 60% higher than that of published data. The overall biomass was the highest at 9th week after sowing. Plant harvested at this stage can yield 120 kg and 56kg of rutin for buckwheat and Tartary buckwheat, respectively. The extraction of rutin is the best using alcohol, followed by alcohol-acetic acid-water mixture, the least by water. It is the same for seeds extraction, by alcoholic extraction can yield the most rutin and soluble sugars.

(8). The analysis of buckwheat seed and leaf transcriptome

Buckwheat ( Fagopyrum esculentum Moench) is an annual crop belonging to the genus Fagopyrum, Polygonum. Buckwheat contains beneficial components and yield considerable amounts of nectar, therefore it has become one of the most important crops in the world. In this study we sequenced the transcriptome of buckwheat seeds and leaves, 143 M and 247 M sequence are read and assembled into 97,200 and 74,544 contigs, the average coverage is 1474 and 3340, respectively. Average length of contigs is 565 and 607 bp, overall sequence assembled is 54 M and 45 M bp. When seed and leaf transcriptome are combined, a total of 106,190 contigs are assembled, in which 69,043 and 56,345 contigs belong to seeds and leaf, respectively. Of all the contigs, 22,857 and 9,889 are exclusively exist in seed and leaf, respectively. After mixed assembling, 27,258 contigs, 25.6% of all assembled contigs, are considered false assembled as the RPKM value is 0 in both seed and leaf transcriptome. The successfully assembled contigs are then blasted with databases and annotated. The gene ontology (GO) is analyzed according to cellular component, biological process and molecular functions. The contigs are also blasted with KEGG (Kyoto Encyclopedia of Genes and Genomes) database and results in 306 metabolic pathways data. As the amount of information is tremendous, at the beginning stage we only inspect the metabolic pathway of flavonoids, flavones and flavonol. In the project, we are interested in seed properties include seed storage protein, LEA protein, cupin family and allergenic protein. All the above sequence are sorted and retrieved, which would be valuable for future breeding.

(8). Development of variety identification technology for grapevine and chrysanthemum.

Molecular marker assisted variety identification technology is the basis of protecting variety right. Variety identification technology has been developed for the aim of protecting breeders’right, strengthening the seedling market mechanism, and encouraging development of new varieties. Seven SSR primer pairs have been screened for grapes, and these primers could generate clear markers on 7 loci. These markers can be utilized to identify 30 grape varieties independently. The copy number of each SSR could be recognized by capillary electrophoresis and sequencing. The allele lengths information could be transformed into allele classes information. This technology could identify violation events. The SOP of grape variety identification has been sent to research team of plant seedling. For developing chrysanthemum variety identification technology, 500 SRAP primer pairs have been tested on 34 varieties. Forty five markers have been selected, these markers could recognize all varieties independently, and the SOP for chrysanthemum variety identification has been sent to research team of plant seedling.

(9). Development and application of pea molecular marker technology on powdery mildew resistance breeding.

Pea is an important legume vegetable crop in Taiwan. For establishing pea variety identification technology and enhancing pea breeding efficiency, molecular marker technology has been developed. The SOP of pea variety identification has been developed and sent to research team of plant seedling. Eighty polymorphic SSR primer pairs have been screened for assisting breeding. The genetic map of 7 linkage groups has been created. The positioning of important trait loci could be proceeded and would be helpful for assisting breeding. The cross by powdery mildew resistance plants has been made. In the F 1 generation, the self-pollenated individuals have been picked out by the assistance of markers. This movement could ensure the correctness of gene positioning in F 2 generation.

(10). Application of ACC synthase gene detection technology on pear breeding.

Pears’ shelf life is the main factor of fruit value. Ethylene plays an important role during ripening, the shelf life of fruit could be prolonged if the efficiency of ethylene synthesis could be low down on the biochemical pathway. The ACC synthase gene detection technology has been adapted from reference of oversea experts. By this technology, the domestic cultivars’genotypes have been identified. The genotypes 358 F 1 plants derived from the cross of Ru-Yi and Heng-Shan also had been detected. Among all F 1 plants, 51 plants with recessive genotypes have been selected. This technology could overcome the restriction of juvenility and evaluate the fruit traits, it could enhance the efficiency of pear breeding.