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Genetic Map Construction Willow ( Salix integra )

Genetic Map Construction Willow ( Salix integra ). Calvin Means. Introduction 介绍 The Salix Integra is a species of willow native to northeastern China, but cultivated for many of years in other places.

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Genetic Map Construction Willow ( Salix integra )

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  1. Genetic Map Construction Willow (Salix integra) Calvin Means

  2. Introduction介绍 • The Salix Integra is a species of willow native to northeastern China, but cultivated for many of years in other places. • It is a deciduous shrub growing to 2–6 m tall with greyish-green bark and reddish to yellowish shoots. • Salix Integra can blossom in 1~2 years, it also grow very fast, and more shorter than Poplar. • It is dioecious, with male and female catkins on separate plants. • Because Salix Integra and Poplar share the family Salicaceae, their genomes are similar enough for gene comparison and future introduction application.

  3. What is Genetic Mapping • Genetic Mapping is the creation of a genetic map assigning DNA fragments to chromosomes. • When a genome is first investigated, this map is nonexistent. • The map improves with the scientific progress and is perfect when the DNA sequencing of the species has been completed.

  4. My Research研究 • I participated on a research project under Miss Wang Tiantian ( a MS student under the direction of Dr. Tongming Yin) • The experiment was focused on using Molecular techniques: • Restriction Enzyme Fragment Length Polymorphism (RFLP) and • Amplified Fragment Length Polymorphism (AFLP) to construct a genetic linkage map for this important species

  5. Research Cont.: AFLP • In the last four weeks, I was involved in conducting research mainly focused on AFLP. • Amplified Fragment Length Polymorphisms (AFLPs) are differences in restriction fragment lengths caused by SNPs or INDELs that create or abolish restriction end nuclease recognition sites. • The AFLP technique is based on the selective PCR amplification of restriction fragments from a total digest of genomic DNA.

  6. These steps make up AFLP, collectively they have many uses in the world of genetics such as, genetic mapping, determination of relation among cultivators, establishment of linkage groups in species crossing, and genetic diversity studies just to name a few

  7. Research Objective • Constructing a genetic linkage map of Salix integra to determine the location and position of fragments generated by AFLP as described earlier.

  8. Research Methods • Several Experiments were conducted: • Sample Collection • DNA Isolation • DNA Purification and Quantification • Primer Design • Amplification and Ligation • Gel Electrophoresis

  9. Collection and DNA Extraction • DNA was isolated from 92 F2 seedlings from a cross of two genetically distant parent trees, using CTAB Protocol. • DNA was purified using Qiagen Dneasy Kit • DNA quality and Quantity was determined using the NanoDrop Method.

  10. Msel ad EcoR1 were used to generate different Restriction fragments of the DNA that were utilizited for downstream application. • Msel is a four base cutter that generates more fragments than EcoR1-a six base cutter. Restriction限制

  11. Adapters are specific for either EcoR1 or Msel sites • On majority of the sites we worked with we used EcoR1 • Ligation of the adapter to the restricted DNA alters the restriction sites in order to prevent a second restriction from happening after ligation • The adapter consists of a known DNA sequence of nucleotides which is later • used in PCR. Ligation of Adapters结扎

  12. Research研究 Methods • This experiment includes the testing of 105 primers with high polymorphism. • The primers were composed with 16 EcoRI and 12 MseI selective amplification primers respectively. • During the duration of my time here, we tested about 30 primers.

  13. Amplification was carried out as described below: • After adding the samples and the master mix which consists of • 0.3 ul HiDi • 1.5 ul 1O X Buffer • 0.15 ul BSA • 0.2 ul Taq • 1.2 ul Mg 2+ (25 mM) • 0.3 ul DNTP (10mM) • 0.3 ul E-NN • 7.05 ul H2O • 3 ul of DNA to the above master mix followed by PCR. • Fragments generated were resolved using 1.2% agarose gel electrophoresis. Amplification products were detected by ABI － 3730 electrophoresis and the clear and repeatable gel profiles were obtained． Amplification放大

  14. Summary of Research • Fragments from AFLP will be outsourced for sequencing before a complete gene map can be made. • This research has many possible downstream applications including new poplar varieties through gene transfer. • Techniques Learned: PCR, Gel Electrophoresis, AFLP, DNA extraction and Designing Primers.

  15. My Chinese Experience

  16. 谢谢! Acknowledgments • National Science Foundation (NSF) • 国家科学基金 • Alabama A&M University • Nanjing Forestry University • 南京林业大学 • Professors • Dr. Tongming Yin • Dr. Soliman • Dr. Wong • Ms. Lisa Gardner and Dr. Elica Moss • Miss Wang and her colleagues • 王仲伟和他的同事

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