Source: WEST VIRGINIA STATE UNIVERSITY submitted to
MAPPING BIOMASS QTLS FOR ARABIDOPSIS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0209960
Grant No.
(N/A)
Project No.
WVAX-BIOMASS
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2006
Project End Date
Sep 30, 2010
Grant Year
(N/A)
Project Director
Reddy, U.
Recipient Organization
WEST VIRGINIA STATE UNIVERSITY
PO BOX 1000
INSTITUTE,WV 25112
Performing Department
AGRICULTURAL & ENVIRONMENTAL RES STATION (AERS)
Non Technical Summary
Numerous physiological and molecular mechanisms underlying biomass are known to have complex epistatic as well as environmental interactions. In several plants, including Arabidopsis, several studies identified several morphological and physiological traits to have strong genetic correlations to final biomass accumulation. Literature shows these components are more are less common and are important to pin point source and sink relationships in biomass accumulation. If the phenomenon of biomass accumulation is a widespread occurrence, the vast genomic and technological resources available for model plant Arabidopsis could be used to rapidly identify several useful markers, positive and negative QTLs and individual genes that directly or indirectly contribute to biomass and these markers and located nearby sequences of various candidate genes can be used for marker assisted selection of similar loci in several crop plants that are important for biomass traits. . The purpose of this study is to identify QTLs for biomass.
Animal Health Component
30%
Research Effort Categories
Basic
40%
Applied
30%
Developmental
30%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2012499106040%
2022499108030%
2062499104030%
Goals / Objectives
1. Identify QTLs that are linked to various growth parameters from RIL populations that are available at Arabidopsis Biological Resource Center 2.) Validate these QTLs in various F2s that are generated from the crosses of different ecotypes 3) Resolving the identified QTLs on different sizes of populations and 4) Understanding dynamics of the selection coefficient in subsequent generations of segregation.
Project Methods
We will be using recombinant inbred lines of various ecotypes for which marker genotyping information is already documented at www.arabidopsis.org (The Arabidopsis Information Resource). The selected RIL populations are, for which known marker genotypes i.e.1. Ler X Cvi with 26 markers on chromosome 1, 15 markers on chromosome 2, 66 markers on chromosome 3, 14 markers on chromosome 4 and 25 markers on chromosome 5 with a population size of 193; 2. Col X Kas with a total of 26 frame work markers on 129 RILs; 3. Ler X NO with a population size of 137 and mapping data for 46 frame work markers; 4.) Ler X Sha with a population size of 114 RILs with 67 mapped markers; and 5) Ler X Col with a population size of 100 with 67 markers are currently publicly available resources. These populations will be evaluated under diverse growing conditions such as normal conditions and various water stress levels in field as well as green house conditions. Morphological data pertaining to fresh weight, dry weight, leaf number, plant height, silique number, seed size and seeds weight will be observed. Using the available marker data, we propose to map QTLs related to various traits. A set of F2 populations will be generated from newly made crosses of various ecotypes. Apart from simple morphological and physiological traits, photosynthetic efficiency, and chemical parameters such as release and residual fibers will be estimated. The identified markers that are linked to major QTLs will be validated on newly made population. Genetic gains in subsequent generations will be accounted after applying various levels of selection intensities. Over a period of time, we have set up a cutting edge genomic facility that has high throughput genotypers such as ABI 3130XL, microarray platform, CEQ 8800 and two LICOR genotypes. Our lab is currently housed in 1,000 sq. ft. of wet laboratory space, and an additional 500 sq. ft. of office and instrumentation space and is well equipped for modern plant genetic and molecular biological research. Other equipment includes numerous horizontal (agarose) gel electrophoresis rigs with power supplies, large- and small-format vertical (acrylamide) electrophoresis rigs with power supplies, micro-centrifuges, hybridization chambers, thermal cylers (6), refrigerators (7), -20 degrees centigrade (5) and -80 degrees centigrade (3) freezers, and both Macintosh (3) and PC (10) desktop computers and one 12 bit CCD camera digital imaging System. Additional common use equipment available to the project in the Biology Department includes high-speed centrifuges and one 2000 Beckman liquid handling system. The equipment currently available in our program is adequate to support the research proposed here. In addition, we have university's well-equipped research station and two greenhouses and one growth chamber.

Progress 10/01/06 to 09/30/10

Outputs
OUTPUTS: We used classical QTL mapping and association mapping approaches to identify SNPs located on transposon (SGCSNP299) of chromosome 2 that controls sepal length, petal length and anther length variation to the extent of 16.3, 18.3 and 19 percents respectively. Interestingly, out of the QTLs identified in this study, none of them are from classical floral genes. Among the SNPs (SGCSSNP 299, 38, 298, 149 and 96) were found to be significant QTLs affecting natural variation of floral traits. The SNP, which is called as `er' of chromosome 2, a CAPs marker located in receptor kinase (AT2G26330), previously known to be linked to the transformation efficiency and resistance to nectrotrophic fungus, in the current study has shown to be a strong QTL controlling biomass traits such as plant height, fruit size, number of branches and total plant weight. This QTL explains biomass variation to the extent of 15 to 37.7% for various growth related traits. We also conducted experiments to resolve epigenetic differences to understand genomic complexity of growth. DNA methylation changes across various plant tissues throughout the Arabidopsis growth elucidated DNA methylation differences related to phenotypic plasticity and alteration of growth. The Arabidopsis ecotypes Columbia (COL), Landsberg erecta (LER), Shakdara (SHA), and Cape Verde Islands (CVI) were used in the current study. Methylation Specific AFLP (MSA)was applied using primer combinations were EAAG-JS5 (EcoRI+ACT/ HpaII/MspI+CGG), EAAC-JS11 (EcoRI+AAG/ HpaII/MspI+CCT), EACG-JS12 (EcoRI+AGG/ HpaII/MspI+CAC), EAGG-JS13 (EcoRI+ACG/ HpaII/MspI+CAT), EACG-HS1 (EcoRI+ACG/ HpaII/MspI+CG), EACA-HS2 (EcoRI+ACA/ HpaII/MspI+AC), EACG-HS3 (EcoRI+ACA/ HpaII/MspI+CC), and EACA-HS5 (EcoRI+AAG/ HpaII/MspI+GG) to resolve methylations. The primer combinations EACT-JS5 and EACG-HS1 were selected to elute differentially methylated bands. The sequences were analyzed and annotated on the Arabidopsis genebank (www.tair.org). The annotated sequences from the eluted methylated bands are the transposon genes distributed across the Arabidopsis genome. Transcripts from transposons and repetitive sequences are known to form non-coding RNA and dsRNA, which are then processed to siRNA to maintain posttranscriptional gene silencing through methylation and subsequent histone modifications. The silique formation stage accumulated the highest number of methylations as compared to the other stages. A trend can be noted that, as development progresses, the methylation events increase. The information pertaining to methylations in individual ecotypes showed that all the ecotypes underwent a similar pattern of methylations. The number of methylations across the stages for all the ecotypes is also comparable, with minor deviations. To exemplify our findings, we also subjected the total methylations to similarity analysis and constructed a UPGMA (Unwieghted pair group method with arithmetic mean) tree. This tree depicts the extent of methylations that occurred across the stages. Methylations that occurred at each stage resolved into different clusters. The silique which underwent a lot of methylations has shown 45% of diverse methylations, whereas the rest of the stages had ≤ 40% diverse methylations. To corroborate resolutions of the tree, a Principal component analysis was constructed using the first three eigen vectors, which cumulatively absorbed 55.16% of the effect. This explains that our MSA captured 55.16% of causative factors of differential methylation across the development. The timing of transition from juvenile vegetative to adult vegetative to reproductive stages can differ considerably, even between closely related taxa. In current study, we have identified similar methylated profiles across a set of Arabidopsis ecotypes throughout their development. Our results clearly indicated a trend in pattern of methylations across various stages of growth; in other words, as development progressed, methylation events increased. Another important finding in our study is that methylations in all ecotypes underwent a similar pattern and amount of methylations throughout their development. PARTICIPANTS: GRADUATE and UNDERGRADUATE STUDENTS TARGET AUDIENCES: Biologists, graduate and undergraduate students PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
We validated some of the QTLs identified through classical mapping approach and also association mapping approach. The QTLs identified in the current study will be of immense use to further allele mine growth related QTLs in conventional biomass crops. We also generated tetraploids from the RIL progeny for ongoing research to identify if gene doubling increases the impact of the QTLs to enhance natural variation in biomass related traits. Two graduate students and two undergraduate students are trained in QTL mapping and association mapping using the software TASSEL.

Publications

  • Reddy UK, Rahman MA and P.Nimmakayala. 2009. QTLs for floral and leaf morphology and natural variation in Arabidopsis thaliana. 20th International Conference on Arabidopsis Research held at Edinburgh, Scotland, UK on 30th June to 4th July: 113.
  • Umesh K. Reddy and R. Gist 2011. Role of DNA methylation in regulation of growth and development in Arabidopsis ecotypes. Journal of Heredity (in review)


Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: One hundred recombinant inbred lines of the cross Col x Ler were evaluated in the multiple environments for various floral (length and width of petal, sepal, pistil and anther as well as rosette and cauline leaf characters (LAI, length and width), and identified several QTL (quantitative trait loci) locations throughout the genome. We noted many QTLs that are linked to various traits from the noncoding part of the genome, which do not possess known floral/leaf genes. Thirty ecotypes belonging to the diverse geographical regions were grown in replications for collecting the data pertaining to natural variation among the observed QTLs. We subjected the data to association mapping and obtained interesting results pertaining to causative SNPs underlying the natural variation. PARTICIPANTS: GRADUATE and UNDERGRADUATE STUDENTS TARGET AUDIENCES: Biologists graduate and undergraduate students PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
QTLs pertaining to non coding regions produced effects ranging from 2 to 10% to the variation of various floral traits. We validated some of the QTLs identified through classical mapping approach and also association mapping approach. We also generated tetraploids from the RIL progeny known to have candidate QTLs. Research and further analysis is in progress. Two graduate students and two undergraduate students are trained in QTL mapping and association mapping using the software TASSEL.

Publications

  • Reddy UK, Rahman MA and P.Nimmakayala. 2009. QTLs for floral and leaf morphology and natural variation in Arabidopsis thaliana. 20th International Conference on Arabidopsis Research held at Edinburgh, Scotland, UK on 30th June to 4th July: 113.