TRANSCRIPTOME ANALYSIS OF THE PHOTOPERIODIC RESPONSE ON THE FLOWERING GENE NETWORKS IN SOYBEAN.

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 0224438
• Grant No.: 2011-67013-30038
• Cumulative Award Amt.: $499,983.00
• Proposal No.: 2010-04138
• Project Start Date: Apr 1, 2011
• Project End Date: Mar 31, 2015
• Grant Year: 2011
• Program Code: [A1101]- Plant Health and Production and Plant Products: Biology of Agricultural Plants

Recipient Organization
UNIVERSITY OF ILLINOIS
2001 S. Lincoln Ave.
URBANA,IL 61801

Performing Department
Crop Sciences

Non Technical Summary
Flowering response to seasonal photoperiod changes is a critical factor to environmental adaptation of crop plants. Flowering is a key trait that determines plant's survival and productivity, and a major target of the domestication process of crop species by human selection. Flowering gene pathways are one of the best-understood molecular networks in plants; nevertheless, applications of such knowledge for modern agricultural use have been limited, largely because most of our current knowledge has been restricted to model species, and studies in crop species are well behind. Soybean is an ideal crop to address this problem. One approach to increase soybean production is to extend the seed filling period between the flowering time and the beginning of seed maturation. Modification of photoperiod responsiveness would make this possible by advancing the flowering time, allowing cultivation of soybean at wider latitudes while improving yield. To obtain genetic tools and information to allow modification of flowering time independently of seed maturation through molecular breeding, we will elucidate global gene expression patterns on floral transition in soybean using transcriptome sequencing technique by comparing the standard variety and known flowering QTLs, and construct the soybean flowering gene networks. Our approaches will clarify the genetic basis of environmental adaptation of soybean and provide valuable information to breed superior germplasm that is highly adaptive to diverse environments to maximize the yields.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
201	1820	1040	30%
201	1820	1050	10%
201	1820	1080	60%

Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1820 - Soybean;

Field Of Science
1040 - Molecular biology; 1080 - Genetics; 1050 - Developmental biology;

Keywords

soybean

flowering

photoperiod

transcriptome

genomics

genetics

Goals / Objectives
Flowering response to seasonal photoperiod changes is a critical factor to environmental adaptation of crop plants. Flowering is a key trait that determines plant's survival and productivity, and a major target of the domestication process of crop species by human selection. Flowering gene pathways are a complex network that consists of more than 100 genes, yet one of the best-understood molecular networks in plants. Nevertheless, applications of such knowledge for modern agricultural use have been limited, largely because most of our current knowledge has been restricted to model species, and studies in crop species are well behind. Soybean is an ideal crop to address this problem. Soybean flowers in response to a photoperiod change from short day to long day at the summer solstice. One approach to increase soybean production is to extend the seed filling period between the flowering time and the beginning of seed maturation. Modification of photoperiod responsiveness would make this possible by advancing the flowering time, allowing cultivation of soybean at wider latitudes while improving yield. Addressing this requires genetic tools and information to allow modification of flowering time independently of seed maturation. To achieve this, we aim to understand the molecular basis of the photoperiodic response on the flowering gene networks in soybean. We will elucidate global gene expression patterns on floral transition in soybean by comparing the standard variety and known flowering QTLs to obtain genetic tools and information to allow modification of flowering time independently of seed maturation through molecular breeding. We will construct the soybean flowering gene networks using the obtained data. In addition, we will clarify the function of key flowering genes to deeper understand the soybean flowering gene networks. Furthermore, we will explore the genome of the wild soybean for novel genes and polymorphisms that underlie variation in photoperiodic flowering. These approaches will clarify the genetic basis of environmental adaptation of soybean and provide valuable information to breed superior germplasm that is highly adaptive to diverse and changing environments with higher productivity. The primary goal of this project is to increase our understanding of the photoperiodic response on the flowering gene networks in a gene expression level. This work will provide a deep insight into the molecular basis of the evolution of a key trait that underlies adaptation of plants and domestication processes by human selection. Discoveries in this work will contribute to address a central question in plant biology and help to make a significant advance in our knowledge in fundamental plant science. Although genetic tools such as genome sequences are being well set up, resources for functional genomics in soybean are still limited. Global gene expression data and the gene networks obtained in this project will enhance further development of plant functional genomics and stimulate multiple areas of plant biology research and beyond.

Project Methods
To understand the molecular basis of the short-day flowering response of soybean, surveys of changes in gene expression in response to a photoperiod change will be performed. The NILs of the flowering loci, E1, E2, and E3 (PHYA), and standard varieties Clark and Williams 82 will be used for this study. Seedlings are first grown under long day with 16 hrs light / 8 hrs dark for three weeks in a greenhouse with a controlled lighting system, allowing for a day-length shift to short day with 8 hrs light / 16 hrs dark. To capture the circadian-regulated mRNA expression of some of the flowering genes, samplings are being performed every 4 hours on the day before the photoperiod shift, and 5 days and 10 days after the shift. All samples are collected in triplicate for biological replication and statistical significance. mRNA will be isolated and cDNA library will be prepared using all samples, and expression analysis of key flowering genes will be performed using quantitative reverse transcriptase PCR (qRT-PCR). 26 candidate flowering genes will be targeted in this analysis. Separately, twelve mRNA samples from four time points, time point 2 (mid-day) and time point 4 (end of day) on the day before and 5 days after the photoperiod shift in triplicate, will be used for deep-sequencing of transcriptomes. This will reduce the sequencing cost, while capturing both morning-expressed genes and evening-expressed genes. The cDNA library construction and sequencing of the libraries will be performed by the Keck Center on the University of Illinois campus using their existing 454 sequencing instrument. The variety Clark will be used for this study to compare the gene expression effects with the flowering alleles that are introgressed into the Clark genomic background. Correlations between gene expression levels, photoperiod, and genotypes will be assessed to determine which of the RNAs are differentially expressed between genotypes in association with photoperiod. To better understand gene-to-gene interactions, we will attempt to re-construct soybean flowering gene networks based on our mRNA and small RNA expression data using a co-expression classification approach. To further explore genes and polymorphisms that underlie variation in photoperiodic flowering response, we have developed a new mapping population that consists of 115 RILs from a cross between G. max and the wild soybean G. soja in close collaboration with Professor Randall Nelson in USDA and Department of Crop Sciences at University of Illinois. Several key traits are currently being measured in the fields at four locations, including flowering time, maturation time, height, tilling, and yield. Their segregation will be scored and interaction with flowering time phenotype will be calculated to obtain a genomic map of flowering QTLs. We will select QTLs based on the obtained map and characterize gene expression patterns in these QTLs and the parental lines by deep-sequencing and qRT-PCR. Through these approaches, we expect our results would bring robust and useful information to identify genes and gene networks controlling flowering transition in soybean.

Progress 04/01/11 to 03/31/15

Outputs
Target Audience:The target audience includes plant and animal bioligists, soybean biologists, and soybean breeders. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project provided training opportunities for 3 undergraduate students, 3 graduate students, and 2 postdoctoral researchers. Undergraduate students:Mahmoud Elrakhawy,Thanaporn Chittiphalungsri,and Isaiah Song. Graduate students:Waseem Haider,Eric Sedivy,and Daniel Wickland. Postdoctoral researchers:Valpuri Sovero andFaqiang Wu. How have the results been disseminated to communities of interest?The results obtained have been shared with communities through publications and conference presentations as listed under products. Several additionalpublications are currentlyunder review. In addition, the results have been sharedwith communities throughpresentations at scientific institutions as listed underother products. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The achievements for specific objectives of this project are described below: Objective 1. Analyze changes in gene expression in response to a photoperiod shift. To understand the molecular basis of the short-day flowering response of soybean, surveys of changes in gene expression in response to a photoperiod change will be performed by taking two complementary approaches: 1) transcriptome deep-sequencing, and 2) qRT-PCR of candidate flowering genes. qRT-PCR analyses were carried out for soybean CO homologs and FT homologs using RNA samples from a total of 189 soybean samples from six time points per day under long-day (LD), short-day (SD), and 5/10 days long-day after 3 weeks short-day (Williams 82, Clark, the NILs of E-loci for E1, E2, E3 and E5, and G. soja). Selected CO and FT homologs were subjected to functional analysis via overexpression in Arabidopsis and soybean as well as RNAi in soybean. Statistical analysis of the transcriptome sequencing data indicated notable difference between cultivated soybean and wild soybean in their responses to photoperiods. Flowering genes exhibited stronger response to photoperiod, time point and E loci than non-flowering genes. Objective 2. Identify genes that are expressed differently in the known flowering QTLs. To better understand the effects of QTLs that affect photoperiodic flowering response in soybean, changes in transcriptomes on the photoperiod shift are being analyzed by deep-sequencing using the NILs of the flowering loci in comparison with a reference variety. We have identified specific E loci affecting a total of 5,605 genes responded to Eloci. E3 showed the highest differentially expressed genes (DEG) (3,058), followed by E2 (2,926), E1 (2,202) and E5 (1,862). We observed E loci responded differently to photoperiods.This part of the results is summarized in a manuscript and submitted. Objective 3. Investigate the function of GmCOLs in photoperiodic flowering. RNAi soybean plants of a GmCOL gene showed early flowering, suggesting that this gene acts as a flowering repressor throughdownregulation of GmFT genes under long day. We have also found that one of GmFT homologs mayunderlie soybean domestication and improvement processes. This GmFT gene is non-functional in cultivated soybean but functional in wild soybean G. soja and shows genomic signatures of selection, suggesting that the non-functional form of this gene is favored by human selection. This part of the results has summarized in a manuscript and submitted.

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: Wickland, D.P. and Hanzawa, Y. 2015. The FLOWERING LOCUS T / TERMINAL FLOWER 1 Gene Family: Functional Evolution and Molecular Mechanisms. Molecular Plant. 8(7):983-997.
• Type: Journal Articles Status: Under Review Year Published: 2015 Citation: Baumann, K., Venail, J., Berbel, A., Domenech, A.J., Money, T., Conti1, L., Hanzawa, Y., Madueno, F. and Bradley, D. 2015. The level, timing and spatial pattern of TFL1 expression affects Arabidopsis flowering architecture. Journal of Experimental Botany. 66(15):4769-4780.
• Type: Journal Articles Status: Under Review Year Published: 2015 Citation: Serrata, A., Haider, W., Mei, Y., Emad, A., Gregis, V., Kamater, M., Bradley, D., Alon, U., Milenkovic, O., Madue�o, F. and Hanzawa, Y. 2015. Paradoxical feedback circuits fine-tune the flowering gene network.
• Type: Journal Articles Status: Under Review Year Published: 2015 Citation: Sedivy, E.J., Mei, Y., Donahue, J., Nakamura, Y., Teo, N.Z.W., Wenk, M.R., Ito, T., Bradley, D., Gillaspy, G. and Hanzawa, Y. 2015. Phospholipid signaling modulates flowering time controlled by TFL1 in Arabidopsis.
• Type: Journal Articles Status: Submitted Year Published: 2015 Citation: Sedivy, E., Akpertey, A., Khan, A., Nelson, R. and Hanzawa, Y. 2015. Identification of novel QTLs controlling flowering time and seed maturation in soybean.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Wang, M. and Hanzawa, Y. 2014. Comparative transcriptome analysis of photoperiodic flowering response of maturity loci (E loci) in soybean. The Molecular & Cellular Biology of the Soybean Conference, Minneapolis, USA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Sedivy, E. and Hanzawa, Y. 2014. Association mapping of temperature response in wild soybean Glycine soja. The Molecular & Cellular Biology of the Soybean Conference, Minneapolis, USA.
• Type: Journal Articles Status: Submitted Year Published: 2015 Citation: Wang, M., Haider, W., Price, W.B., Nelson, R. and Hanzawa, Y. 2015. Transcriptome analysis of photoperiodic flowering of soybean.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Wu, F., Price, W.B., Haider, W., Nelson, R. and Hanzawa, Y. 2014. Functional and evolutionary characterization of the CONSTANS gene family in short-day photoperiodic flowering in soybean. The Molecular & Cellular Biology of the Soybean Conference, Minneapolis, USA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Wickland, D., Faqiang, W. and Hanzawa, Y. 2014. Evolution of FT11c and its function in flowering control in soybean. The Molecular & Cellular Biology of the Soybean Conference, Minneapolis, USA.

Progress 04/01/13 to 03/31/14

Outputs
Target Audience: The target audience includes plant and animal bioligists, soybean biologists, and soybean breeders. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? The project provided training opportunities for 3 undergraduate students, 3 graduate students, and 2 postdoctoral researchers. Undergraduate students: Mahmoud Elrakhawy (Spring 2013–current): Soybean planting and management. Sampling for the gene expression analyses and phenotyping. Thanaporn Chittiphalungsri (Fall 2013–Spring 2014): Soybean planting and management. Sampling for the gene expression analyses and phenotyping. Isaiah Song (Spring 2012–Fall 2013): Soybean planting and management. Sampling for the gene expression analyses and phenotyping. Graduate students: Waseem Haider (PhD in summer 2014): Bioinformatics analyses of the transcriptome sequencing data. Eric Sedivy (MS in summer 2013, currently a PhD student): QTL mapping and association mapping of flowering loci. Daniel Wickland (MS in summer 2014, currently a PhD student): Functional characterization of flowering genes in Arabidopsis and soybean. Postdoctoral researchers: Faqiang Wu (June 2012-current): Gene expression analyses using qRT-PCR and transcriptome sequencing. Gene cloning and gene function testing by transgenic approach. Minglei Wang (March 2014-current): Bioinformatics analyses of the transcriptome sequencing data. How have the results been disseminated to communities of interest? The results obtained have been shared with communities through publications as listed under products. Several additional publications are currently being prepared and some are accepted or under review. In addition, the results have been shared with communities through presentations at scientific institutions and scientific meetings/conferences as listed under products/other products. What do you plan to do during the next reporting period to accomplish the goals? Our major goal for the next reporting period is producing additional publications and public presentations at scientific meetings to disseminate the obtained results through this project.

Impacts
What was accomplished under these goals? The achievements for specific objectives of this project are described below: Objective 1. Analyze changes in gene expression in response to a photoperiod shift. To understand the molecular basis of the short-day flowering response of soybean, surveys of changes in gene expression in response to a photoperiod change will be performed by taking two complementary approaches: 1) transcriptome deep-sequencing, and 2) qRT-PCR of candidate flowering genes. Expression analysis of candidate flowering genes. A total of 504 soybean samples from six time points per day under long-day (LD), short-day (SD), and 5/10 days long-day after 3 weeks short-day (Williams 82, Clark, the NILs of E-loci for E1, E2, E3 and E5, and G. soja) were used to extract RNAs, and selected 189 RNA samples from three time points per day were used for transcriptome sequencing. qRT-PCR analysis were carried out using RNA samples from six time points for verification for the important flowering gene families GmCOLs and GmFTs. The obtained expression data indicated that GmCOL1a and GmCOL1b are inducers of flowering under short day. This part of the results was published in Wu et al., 2014. Transcriptome analysis. Statistical analysis of the RNA sequencing data indicated more genes are rhythmically expressed in SD than LD, with an intermediate number of genes under the shift condition, in G. max genotypes. However, in G. soja as well as the E5 NIL, the numbers of rhythmically expressed genes are approximately the same in SD and LD, unlike other G. max genotypes. Notably, G. soja showed significantly less genes that are rhythmically expressed under the shift condition than other genotypes. The candidate flowering genes exhibited stronger response to photoperiod (P<0.01), time point (P<0.05) and E loci (P<0.01) than non-flowering genes. Objective 2. Identify genes that are expressed differently in the known flowering QTLs. To better understand the effects of QTLs that affect photoperiodic flowering response in soybean, changes in transcriptomes on the photoperiod shift are being analyzed by deep-sequencing using the NILs of the flowering loci in comparison with a reference variety. Among 315 pair-wise comparisons of our RNA sequencing data, 189 comparisons allowed us to investigate the response of different E loci to photoperiod. The results indicated different effects of E loci on gene expression at different time points and light-length treatments. While E loci exhibited effects in all conditions, we observed specific E loci affected gene expression width (gene numbers) and depth (gene expression levels) under specific conditions, as well as opposite directions of gene regulation (up/down), involving more than 2,000 differentially expressed genes in total. Total of 5,605 genes responded to E loci. E3 showed the highest differentially expressed genes (DEG) (3,058), followed by E2 (2,926), E1 (2,202) and E5 (1,862). E loci responded differently to photoperiods. E2 showed highest DEG in SD, E3 showed highest DEG in LD, and E5 in the shift condition. This part of the results is currently being summarized in a manuscript. Objective 3. Investigate the function of GmCOLs in photoperiodic flowering. Characterization of GmCOLs. To understand the function of CO in soybean, comprehensive characterization of the soybean CO gene family was carried out. The obtained results were published in Wu et al., 2014. Testing the CO-FT model in soybean. Based on our expression study, we selected GmCOL1b, GmCOL2a and GmCOL5a for further functional study. Their full-length cDNAs and partial fragments for gene silencing were cloned in a binary vector that contained the CaMV 35S promoter and transgenic soybean plants are created. T2 seeds are currently being collected. The overexpression constructs were also transformed into Arabidopsis. T3 plants currying homozygous single insertion are currently being identified. The full-length cDNAs and partial fragments were also subcloned into a VIGS vector for ectopic transient expression and gene silencing. The resulted VIGS vectors are currently being created. The full-length cDNAs and partial fragments were also subcloned into a VIGS vector for ectopic transient expression and gene silencing. The resulted VIGS vectors are currently being used for soybean infection and the effects in flowering phenotype and gene expression are being monitored.

Publications

• Type: Book Chapters Status: Published Year Published: 2014 Citation: Wu, F. and Hanzawa, Y. Photoperiodic Control of Flowering in Plants. Handbook of Plant and Crop Physiology, the 3rd edition, CRC Press, Taylor & Francis Group, LLC.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Wu, F., Price, B., Haider, W., Seufferheld, G., Nelson, R. and Hanzawa, Y. Functional and evolutionary characterization of the CONSTANS gene family in short-day photoperiodic flowering in soybean. PLOS ONE 9: e85754.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Rosas, U., Mei, Y., Xie, Q., Banta, J., Seufferheld, G., Gerald-Martinez, S., Chou, L., Bhambra, N., Parks, J.D., Flowers, J., McClung, C.R., Hanzawa, Y. and Purugganan, M.D. 2014. Variation in Arabidopsis flowering time associated with cis-regulatory variation in CONSTANS. Nature Commun. 5:3651-3658.
• Type: Journal Articles Status: Submitted Year Published: 2014 Citation: Sedivy, E., A. Akpertey, A. Khan, R. Nelson and Y. Hanzawa. Identification of novel QTLs controlling flowering time and seed maturation in soybean.
• Type: Journal Articles Status: Other Year Published: 2014 Citation: Wang, M., Haider, W., Price, W.B., Nelson, R. and Hanzawa, Y. Transcriptome analysis of photoperiodic flowering of soybean.
• Type: Theses/Dissertations Status: Published Year Published: 2013 Citation: Sedivy, E. Molecular characterization of signaling mechanisms in flowering transition in Arabidopsis and identification of novel flowering QTL in soybean.
• Type: Theses/Dissertations Status: Published Year Published: 2014 Citation: Haider, W. Exploring flowering gene networks in soybean and Arabidopsis through transcriptome analysis.
• Type: Theses/Dissertations Status: Published Year Published: 2014 Citation: Wickland, D. Functional characterization of the FT/TFL1 gene family in photoperiodic flowering pathway in Arabidopsis and soybean.
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Hanzawa, Y. Genomic approaches to photoperiodic flowering in soybean. The Plant and Animal Genome Conference XXI, San Diego, USA.

Progress 04/01/12 to 03/31/13

Outputs
Target Audience: Members of the target audience include plant and animal biologists, soybean biologists, and soybean breeders. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This project provided training opportunities for 2 undergraduate student, 3 graduate students, and 2 postdoctoral researchers. Undergraduate students: Mahmoud Elrakhawy (current): Soybean planting and management. Sampling for the gene expression analyses and phenotyping. Isaiah Song (current): Soybean planting and management. Sampling for the gene expression analyses and phenotyping. Graduate students: Waseem Haider (current): Bioinformatics analyses of the transcriptome sequencing data.Eric Sedivy (current): QTL mapping of flowering loci. Brian William Price (MS, Aug 2012): Gene expression analyses of CONSTANS homologs using qRT-PCR and transcriptome sequencing. Gene cloning and gene function testing by transgenic approach. Postdoctoral researchers: Faqiang Wu (June 2012 - current): Gene expression analyses using qRT-PCR and transcriptome sequencing. Gene cloning and gene function testing by transgenic approach. Valpuri Sovero (May 2012): Gene expression analyses using qRT-PCR and transcriptome sequencing. Gene cloning and gene function testing by transgenic approach. How have the results been disseminated to communities of interest? The results obtained have been shared with communities through presentations at scientific institutions and scientific meetings / conferences as listed under other products. In addition, several publications are currently being prepared and some are accepted or under review. What do you plan to do during the next reporting period to accomplish the goals? Our major goal during the next reporting period is to reconstruct the photoperiod-regulated flowering gene networks of soybean based on the obtained transcriptome data. Currently we are testing multiple algorithms for gene clustering and gene interaction prediction for this purpose. Our additional goal for the next reporting period is publication of obtained results through this project.

Impacts
What was accomplished under these goals? The specific objectives of this project and current achievements are: Objective 1. Analyze changes in gene expression in response to a photoperiod shift. To understand the molecular basis of the short-day flowering response of soybean, surveys of changes in gene expression in response to a photoperiod change will be performed by taking two complementary approaches: 1) transcriptome deep-sequencing, and 2) qRT-PCR of candidate flowering genes. Sampling and expression analysis. Plants were grown under long-day (LD), short-day (SD), and 5 / 10 days long-day after 3 weeks short-day, and sampled every 4 hours with four biological replicas. Williams 82, Clark, the NILs of E-loci for E1, E2, E3 and E5, and G. soja were used. 504 samples (with three biological replicas) were used to extract RNAs and qRT-PCR analysis was carried out for GmCOs and GmFTs (for Objective 3). Selected RNA samples total 189 were used for transcriptome sequencing. The sequencing data was cleaned and statistical analysis was performed. G. soja shows more rhythmic genes. More rhythmically expressed genes were observed in SD than LD, with an intermediate number of genes in the shift condition. The pattern, however, was different in the E5 NIL and G. soja. In the E5 NIL, approximately 3,000 more genes than other genotypes were rhythmically expressed in the shift condition, suggesting that E5 may have an important role in responsiveness to a photoperiod change. In G. soja, approximately 5,000 more genes than other genotypes were rhythmically expressed in LD, indicating that G. soja maintains the ability to respond to different photoperiods, whereas cultivated soybean may have lost such ability during the process of domestication. GO-term analysis showed that signaling and transcription related functions were overrepresented G. soja-specific rhythmic genes in LD. In particular, we identified one of GmFT homolog showing a rhythmic pattern in G. soja, but not in other genotypes. Currently, we are testing the function of the GmFT homolog. Reconstruction of the soybean flowering gene networks. Gene clustering and prediction of regulatory interactions of flowering genes are currently being performed to reconstruct the soybean flowering gene networks. We are currently testing two methods: a co-expression based clustering method that predict nondirected edges and a novel state-of-art algorithm of causal compressive sensing developed by Dr. Milenkovic (UIUC). This analysis will yield a small set of novel candidate flowering genes and their predicted genetic interactions that are to be tested experimentally in the future. Objective 2. Identify genes that are expressed differently in the known flowering QTLs. To better understand the effects of QTLs that affect photoperiodic flowering response in soybean, changes in transcriptomes on the photoperiod shift are being analyzed by deep-sequencing using the NILs of the flowering loci in comparison with a reference variety. Key flowering genes show differential expression patterns in flowering QTLs. We used a combination of the NILs that contain the previously identified flowering time QTLs: E1, E2, E3, and E5. The samples for the expression analyses described in Objective 1 include the NILs. Our qRT-PCR and transcriptome analyses using LD and SD samples suggest significant difference among the genotypes in the expression levels of GmFTs, while very little or no difference was observed in GmCOs that were known regulators of FT in model plants, suggesting a potential post-transcriptional regulation of GmCOs function. Genes under control of E loci were identified. 50,474 representative gene models were expressed in our datasets. Total 5,605 genes responded to E loci. E3 showed the highest differentially expressed genes (DEG) (3,058), followed by E2 (2,926), E1 (2,202) and E5 (1,862). E loci responded differently to photoperiods. E2 showed highest DEG in SD, E3 showed highest DEG in LD, and E5 in the shift condition. We are currently reconstructing gene networks of E loci-regulated genes in addition to candidate flowering genes (approximately 500 genes were identified based on sequence homology) as described above, aiming at identification of novel flowering genes that are connected with candidate flowering genes. Objective 3. Investigate the function of GmCO in photoperiodic flowering. CO is the central gene in photoperiodic flowering that is widely conserved among flowering plants. In particular, CO is known to activate a key flowering inducer gene FT, often referred as the CO-FT module. However, it is also suggested that the function of CO varies depending on environments and plant species. We aim to clarify the function of CO in soybean flowering in this project. To understand the function of CO in soybean, comprehensive characterization of the soybean CO gene family was carried out. Our BLAST search identified a total 26 soybean CO homologs, including many previously unreported genes. Phylogenetic analysis showed that the 26 GmCO homologs were classified into three clades that were widely conserved among flowering plants. We gathered evidences that strongly indicate that a homeologous pair in Clade I, GmCO1a and GmCO1b are major inducers of flowering, likely through the induction of GmFTs in morning under SD. A key evidence is that mRNA expression of GmCO1a and GmCO1b peaked at dawn in SD, which overlaps with GmFT2a and GmFT5a. In addition, GmCO1a and GmCO1b were expressed high under flowering-inductive SD but low under LD, corresponding well with the high expression of GmFT2a and GmFT5a in SD. Moreover, our expression results also pointed out that mRNA accumulation of GmCO5a in Clade II resembled that of GmCO1b, indicating a possible role of GmCO5a in flowering control despite its sequence divergence from Arabidopsis CO, as well as a potential co-regulation between GmCO1b and GmCO5a. Based of these results, we selected the 5 GmCOs homologs for further functional characterization. The full-length cDNAs of the selected GmCOs were subcloned into a binary vector that contained the CaMV 35S promoter. The resulted constructs were transformed into Arabidopsis and the transgenic plants were identified. In parallel, transgenic soybean plants are currently being created. The full-length cDNAs were also subcloned into a VIGS vector for ectopic transient expression, as well as the partial cDNAs for RNAi. The resulted VIGS vectors are currently being used for soybean infection and the effects in flowering phenotype and gene expression are being monitored. Objective 4. Characterize the novel flowering QTLs by profiling gene expression patterns. To further explore genes and polymorphisms that underlie variation in photoperiodic flowering response, our objective is to identify novel flowering time QTLs using wild soybean G. soja and characterize them in global gene expression patterns aiming at understanding the function of the QTLs. Using the RIL mapping population created by a cross between G. max and its ancestor G. soja, we identified several potential QTLs, including many previously uncharacterized loci. We chose two novel QTLs that were among statistically strongest QTLs and selected 5 plant lines for each QTL. Plant material was sampled and RNA was prepared. Gene expression studies will be performed in summer 2013.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Sedivy, E., Akpertey, A., Khan, A., Nelson, R. and Hanzawa, Y. Identification of novel QTLs controlling flowering time and seed maturation in soybean. The Molecular & Cellular Biology of the Soybean Conference, Des Moines, Iowa.
• Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Haider, W., Price, W.B., Hudson, M., Nelson, R. and Hanzawa, Y. Transcriptome analysis of photoperiodic flowering of Soybean. The ASPB annual meeting, Austin, Texas.
• Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Hanzawa, Y., Price, W.B., Sovero, V. and Nelson, R. Understanding the function of CONSTANS homologs in photoperiodic flowering in soybean. The ASPB annual meeting, Austin, Texas.
• Type: Conference Papers and Presentations Status: Published Year Published: 2011 Citation: Sovero, V., Price, W.B., Haider, W., Hudson, M., Nelson, R. and Hanzawa, Y. Understanding photoperiod regulation of soybean flowering via transcriptome analysis. The ASBP annual meeting, Minneapolis, Minnesota.
• Type: Journal Articles Status: Under Review Year Published: 2013 Citation: Wu, F., Price, B., Haider, W., Nelson, R. and Hanzawa, Y. Functional and evolutionary characterization of the CONSTANS gene family in short-day photoperiodic flowering in soybean.
• Type: Journal Articles Status: Under Review Year Published: 2013 Citation: Sedivy, E., Akpertey, A., Khan, A., Nelson, R. and Hanzawa, Y. Identification of novel QTLs controlling flowering time and seed maturation in soybean.
• Type: Journal Articles Status: Other Year Published: 2013 Citation: Haider, W., Price, B., Hudson, M. and Hanzawa, Y. Global gene expression patterns revealed the photoperiodic flowering gene networks in soybean.
• Type: Book Chapters Status: Awaiting Publication Year Published: 2013 Citation: Wu, F. and Hanzawa, Y. Photoperiodic Control of Flowering in Plants. Handbook of Plant and Crop Physiology, the 3rd edition, CRC press.
• Type: Theses/Dissertations Status: Published Year Published: 2012 Citation: William Brian Price, Understanding the mechanisms of the photoperiod flowering pathway in soybean.
• Type: Theses/Dissertations Status: Awaiting Publication Year Published: 2013 Citation: Eric James Sedivy. Molecular characterization of signaling mechanisms in flowering transition in Arabidopsis and identification of novel flowering QTL in soybean.
• Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Haider, W., Price, W.B., Hudson, M., Nelson, R. and Hanzawa, Y. Transcriptome analysis of photoperiodic flowering of Soybean. IGB Fellows Symposium, Urbana, Illinois.
• Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Price, B., Haider, W., Nelson, R. and Hanzawa, Y. Characterization of the CONSTANS gene family in Glycine max. The Molecular & Cellular Biology of the Soybean Conference, Des Moines, Iowa.

Progress 04/01/11 to 03/31/12

Outputs
OUTPUTS: The outputs for the specific objectives are: 1. Analyze changes in gene expression in response to a photoperiod shift. Surveys of changes in gene expression in response to a photoperiod change are being performed by taking two complementary approaches: 1) transcriptome deep-sequencing, and 2) qRT-PCR of candidate flowering genes. We grew plants under long-day, short-day, and 5 / 10 days long-day after 3 weeks short-day, and sampled every 4 hours with four biological replicas. 504 samples (contain three biological reps) were used to extract RNAs and qRT-PCR analyses were carried out for 9 GmCOs (see Objective 3) and 4 GmFTs. 189 selected RNA samples were used for transcriptome sequencing. The sequencing data has been cleaned and is currently being analyzed. 2. Identify genes that are expressed differently in the known flowering QTLs. Changes in transcriptomes on the photoperiod shift are being analyzed by deep-sequencing using the NILs of the flowering loci in comparison with a reference variety. We used a combination of the NILs that contain the previously identified flowering time QTLs: E1, E2, E3, and E5. The samples for the expression analyses described in Objective 1 include the NILs. Our preliminary qRT-PCR and transcriptome analyses suggest significant differences across the genotypes in the expression levels of GmFTs, while very little or no difference was observed in GmCOs. We are carefully replicating this result, as well as looking into genome-wide gene expression patterns for genes under control of the flowering QTLs. 3. Investigate the function of GmCO in photoperiodic flowering. Two major approaches are being conducted: The expression analysis using qRT-PCR and the transgenic approaches. We chose to focus on 9 CO homologs in soybean. The expression patterns of GmCOs suggested 2 of them may be involved in photoperiodic flowering control through the regulation of GmFTs. Based of this result, we selected the 2 GmCOs and other 2 close homologs for further functional characterization. The full-length cDNAs of the selected GmCOs were subcloned into a binary vector that contained the CaMV 35S promoter. The resulted constructs are being transformed into Arabidopsis and soybean. In parallel, the full-length cDNAs were subcloned into a VIGS vector for ectopic transient expression, as well as the partial cDNAs for RNAi. Currently, the resulted VIGS vectors are being used for soybean infection and the effects in flowering phenotype and gene expression are being monitored. 4. Characterize the novel flowering QTLs by profiling gene expression patterns. Our objective is to identify novel flowering time QTLs using wild soybean G. soja and characterize them in global gene expression patterns aimed at understanding the function of the QTLs. Using the mapping population created by a cross between G. max and its ancestor G. soja, we identified several potential QTLs, including two previously uncharacterized loci. We have chosen these novel QTLs and selected five plant lines for each QTL for this approach. Sampling for expression studies will be performed in summer 2012. PARTICIPANTS: Training opportunities for one undergraduate student, two graduate students, and one postdoctoral researcher were provided directly or indirectly through this research. Collaborations / Interactions were made possible from this research with Randall Nelson (USDA / University of Illinois) and Steven Whitham (Iowa State University). TARGET AUDIENCES: Outcomes of this research will reach a broad range of audiences, including plant and animal biologists, soybean biologists, soybean breeders. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Invited lecture, The molecular mechanisms for the photoperiodic flowering control in soybean, was presented at Nara Institute of Science and Technology, Japan (2012). Poster presentation based on the results obtained through this research was done by Sovero, V., Price, W.B., Haider, W., Hudson, M., Nelson, R. and Hanzawa, Y. Understanding photoperiod regulation of soybean flowering via transcriptome analysis. The ASBP annual meeting, USA (2011). This research produced resources listed below that will become available to the public upon publication. 1. Full-length cDNAs of GmCOs and GmFTs, 2. Transcriptome sequence data, 3. A population of RILs created from a cross between G. max and G. soja, 4. Phenotype data of the RIL population, and 5. Information of the QTLs obtained from the RIL population. Training opportunities for one undergraduate student, two graduate students, and one postdoctoral researcher were provided through this research. Collaborations / Interactions were made possible from this research with Randall Nelson (USDA / University of Illinois) and Steven Whitham (Iowa State University).

Publications

• No publications reported this period