2012 Annual Report
1a.Objectives (from AD-416):
We will genotype approximately 400 accessions of the USDA cabbage germplasm collection for SSR markers. This data will be used to evaluate diversity in the cabbage collection to avoid difficulties of environmental influences on phenotypic traits and these markers also offer the additional benefits of being ubiquitous and distributed throughout the plant genome. Co-dominant markers such as microsatellite or simple sequence repeats (SSRs) are often preferred for evaluating genetic diversity due to their increased polymorphic information content (PIC).
1b.Approach (from AD-416):
This proposal will include multiple types of cabbage (e.g. green, red, and Savoy). We hope this study will also lead to the eventual development of a core collection of cabbage. We will collaborate with the current curator to select 400 accessions that best represent diversity within and among types. We will include historical accessions as well as more recently developed elite material. We will also take into consideration availability, type, usage, geographical origin, agronomic factors and other available passport information when compiling the list of requested material.
One hundred and twenty one SSR markers will be used to screen accessions at the seedling stage. These SSR markers have amplified at least one band when screening the parents of our F2:3 broccoli population. An initial screening set of 16 accessions will be utilized to detect amplification and polymorphisms on agarose gels using unlabeled primers. A set of 25 to 30 markers will be chosen on the basis of this initial screen for analysis of the 400 accessions.
Primers (25-30) displaying the greatest degree of polymorphisms during initial screening will be used to genotype all 400 lines. A similarity matrix will be generated with Jaccards coefficient, (Sij) using the software package NTsys. A dendogram will be generated from the similarity matrix using the average unweighted group mean (UGMA) and principle component analysis (PCA) estimates will also be generated from the same program. Population genetic substructure will be estimated using the program STUCTURE 2.1 that utilizes a Bayesian algorithm to assign accessions to putative sub-populations (k) in Hardy-Weinberg equilibrium and can provide an estimate of the degree of admixture of the accessions that can be utilized as a matrix of co-factors in structured association programs.
During this year, seeds from 350 cabbage accessions obtained from the USDA were grown in the NCRC’s greenhouse at Kannapolis, NC to obtain enough leaf tissues for DNA extraction. For each accession, leaves from 12 seedlings were harvested and tissues were lyophilized. Leaf tissues were fine ground using mortars and pestles with liquid nitrogen and kept at -80 ºC freezer. Extraction of DNA has been completed from the 350 cabbage accessions and DNA samples are stored at -20 ºC freezer for further analysis. Gels for extracted DNA samples have been run to verify quality and DNA has been quantified using a nanometer. The extraction procedure using CTAB protocol has resulted in ideal DNA concentrations with no noticeable degradation. Concentrations of DNA samples were adjusted to suit PCR reactions. Initial screening of 50 PCR markers with a subset of the lines (16) reveal that significant genetic variation exists among the USDA accessions to successfully complete the project. However, the genotyping of the 350 lines using the 25 most polymorphic SSR markers has been delayed due to maintenance issues regarding our lab's ABI 3730 xl sequencer. These issues have been resolved and completion of the genotyping is expected by the end of Nov 2012. Clustering analysis and Principal Component Analysis will be applies to DNA SSR marker data to classify cabbage accessions based on their genetic relatedness.
In addition to genotyping cabbage accessions using SSR markers, we had the opportunity to transplant a subset of the accessions (100 accessions) in the field of Piedmont Research Station located at Salisbury, NC. This allowed us to take some phenotypic observations on the plant growth and head formation and color. Some of the accessions showed to flower with no head formation (PI 176440). With accessions that formed heads, head size varied from 2661g ( PI 214148) to 238g (PI 343520). The head shape and compactness also varied ranging from very loose (PI 1518837) to mostly compact (most of the accessions tested). Some of the accessions had formed good flat head (1800g) such as PI 419104, while others had cone-shaped heads (290g) such as PI 343610. Most of other tested accessions showed a normal cabbage head shape. Though this was not included in the agreement, samples were collected from cabbage heads and lyophilized for further analyses including phytochemical contents such as glucosinolates, carotenoids, and flavonoids. This phenotypic evaluation will be conducted after all SSR genotyping is complete, and will provide the USDA with this data to aid in selection for certain genotypes with specific phytonutrient profile later. We are glad to have the opportunity to genotype and evaluate such large number of cabbage accessions provided by the USDA at Geneva, NY, and hope information generated from this project will aid in selecting accessions with the potential to be used successfully in a breeding program that can result in improved cabbage lines.