Evaluating the existing genetic diversity among local maize inbred lines towards developing new hybrids, with superior qualities and increased productivity

A collaborative effort of:
                          1. Institute of Biological Research, Cluj-Napoca (coordinator - CO)
                          2. Agricultural R&D Station, Turda (partner - P1)
                          3. Oncology Institute "Prof. Dr. Ion Chiricuță" (partner - P2)

© Mihai Miclaus


3 October 2016

Mihai Miclaus, Ovidiu Balacescu, Ioan Has, Loredana Balacescu, Voichita Has, Dana Suteu, Samuel Neuenschwander, Irene Keller, Rémy Bruggmann (2016) Maize cytolines unmask key nuclear genes that are under the control of retrograde signaling pathways in plants. Genome Biology and Evolutiondoi: 10.1093/gbe/evw245 (First published online: October 3, 2016)

25 March 2016
Plenary talk at:

Life sciences in the dialogue of generations: connections between universities, academia and business community, Chișinău, Republic of Moldova.
Title: Unveiling the molecular structure of Romanian maize germplasm through genotyping and transcriptome sequencing.  
Presenting author: M. Miclăuș

20 March 2016
Invited talk at:

Maize Genetics Conference (16-20 March 2016)
Title: Maize cytolines unmask key nuclear genes that are under the control of retrograde signaling pathways in plants.
Authors: Miclăuș, M.*, Balacescu, O., Has, I., Balacescu, L., Has, V., Șuteu, D., Neuenschwander, S., Keller, I., Bruggmann, R.  
Presenting author: M. Miclăuș 

8 January 2015
Poster presentation at:

Plant and Animal Genome Conference, San Diego, CA, SUA (10-14 January 2015)
Title: Transcriptome analysis of maize inbred lines reveals important shifts in gene expression between parents, hybrids, and isonuclear lines
Authors: Bălăcescu O, Bălăcescu L, Haș I, Haș V, Șuteu D, Neuenschwander S, Keller I, Bruggmann R, Miclăuș M 
Presenting author: M. Miclăuș 

Poster details

24 November 2014
Two examples of new hybrids that have been generated based on our initial molecular analysis of the 90 inbred lines.
Illustrated above are parental lines flanking their respective hybrids. The crosses that generated the hybrids above were predicted based on the genetic background of the parental lines.

15 November 2014
Following last year's crosses, the first hybrids were produced and tested in the field this summer

The image illustrates this year's harvest from a subset of inbred lines and their respective hybrids.

15 September 2014
Starting date for a six-months collaboration with the Bioinformatics Department at University of Bern, Switzerland.

The collaboration is centred on:
  • Transcriptome analysis of several Romanian maize inbred lines by means of NGS (Next Generation Sequencing). The data will be corroborated with those generated by the Oncology Institute "Prof. Dr. Ion Chiricuță" (Partner 2 in ZEAHYBR), for an in-depth view of gene expression patterns in the germplasm of interest.
  • Training of human resource. 
    • Mihai Miclaus will get trained in NGS library preparation, sequencing with an Illumina HiSeq2500 machine, and analyze the data by means of bioinformatics tools (@ Uni. of Bern).
    • Ovidiu Balacescu, together with Rémy Bruggmann, will both travel for short term visits at the partner institution for knowledge transfer and strengthen the collaboration.
19 June 2014
Public presentation of the intermediate results. Bucharest, Romania

13 March 2014
The intermediate evaluation of the project, by the Romanian National Authority for Scientific Research (February 2014) places us in the "A+" category.
Evaluation sheet in Romanian, here

31 December 2013
Șuteu, D., Băcilă, I., Haș, V., Haș, I., Miclăuș, M. Romanian Maize (Zea mays) Inbred Lines as a Source of Genetic Diversity in SE Europe, and Their Potential in Future Breeding Efforts, PLoS ONE 8(12): e85501. doi:10.1371/journal.pone.0085501, 2013

7 September 2013  
Poster presentation at:
Plant Genome Evolution Conference, Amsterdam, The Netherlands (8-10 September 2013)
Title: Insights into the Romanian maize germplasm
Authors: D. Suteu, I. Bacila, V. Has, I. Has, M. Miclaus
Presenting author: M. Miclaus 

Team members (key persons)

  • CS II Dr. Mihai Miclăuș (team leder)
  • CS III Dr. Dana Șuteu-Mireșan and CS III Dr. Ioan Băcilă

    2. Agricultural R&D Station (P1)
  • Prof. univ. Dr. Ioan Haș (group leader)
    Foto: Camelia Terec (www.turdalive.ro)
  • CS II Dr. Voichița Haș

  • Dr. Ovidiu Bălăcescu (group leader)
  • Dr. Loredana Bălăcescu

Expected impact

Our project offers a viable alternative to using seed from multinational companies, who also pressure towards the large-scale use of transgenic maize in agriculture. So, instead of using foreign germplasm, the Romanian growers will have the choice of using hybrids generated from inbred lines that have been developed in our country over the years. But due to the lack of knowledge in terms of their genetic diversity, and therefore potential for amelioration, they were not used for that purpose. We do not plan to use any transgenic plants, but instead harness the power of the existing pool of maize diversity in our country. We think that this is a sustainable approach towards conserving the local maize genetic diversity and avoiding sensitive issues, like the use of transgenics.
The project end products will be a set of hybrids, well characterized at molecular, biochemical, and morphological levels, and that will be mapped to different regions of Romania. The molecular characterization will be of uttermost importance. By using modern molecular biology techniques, like gene expression profiling through microarray platform, we’ll get new insights into the mechanism of heterosis.   

Project Objectives

  1. Evaluate the genetic diversity of the Romanian maize inbred lines, using SSR (Simple Sequence Repeat) and SNP (Single Nucleotide Polymorphism) markers. 
  2. Bioinformatic analysis of SSR and SNP data in order to define the heterotic groups present in the Romanian germplasm.
  3. Map those heterotic groups to regions of the country and correlate their location to gene diversity. 
  4. Classify them according to the international reference standards, by including representatives of those groups in our molecular analysis. 
  5. Predict crosses among inbreds analysed that will lead to hybrids characterized by a high amount of heterosis. 
  6. Validate the presence of heterosis in the field. 
  7. Correlate the genetic diversity with heterosis, oil content, resistance to pests and physical damage, and starch content. 
  8. Measure the amount of heterosis in terms of: 
    1. Gene expression
    2. Phenotype
    3. Biochemical properties (especially zein content, the main storage proteins in the maize kernel)
    4. Productivity 
  9. Compare gene expression between progenies and parental lines by microarray analysis and identify the genes that are up regulated in the hybrids, as possible key players in explaining heterosis. 
  10. Study gene expression of those copies in more detail by qRT-PCR (quantitative Real-Time Polymerase Chain Reaction) in order to identify patterns that can be associated to heterosis. 
  11. If several of these genes form QTLs (Quantitative Trait Locus), start introgressing them into a common background and create elite lines.
  12. Cross opaque7(o7) (a high-lysine mutant of maize, recently cloned – Miclaus et al., 2011; Wang et al., 2011) to the hybrids generated in order to further improve their amino acid composition. 

Executive summary

Maize is the most important crop of the world in terms of production, serving as staple food in many countries around the globe. According to FAO statistics, maize production reached 817 million tons last year, while rice and wheat were trailing behind with 678 and 682 million tons, respectively. This tremendous productivity is explained mainly by the large-scale use of hybrids, worldwide. They owe their superior characteristics to heterosis, a phenomenon that Darwin hinted to and that is currently being used by breeders in their constant efforts of ameliorating crops. They have also slowly replaced the local races and inbred lines, but the latter two have always been a constant reservoir of genetic biodiversity that one has to turn back to when trying to create new hybrids. The tools for evaluating genetic diversity are various nowadays but the most important ones come from the filed of molecular biology. Whereas studies with isoenzymes and RFLP markers are a thing of the past, they have the merit of opening the way to marker-assisted breeding. With the advent of new technologies, like high-throughput sequencing and microarray analysis, one can take this to a whole new level. In this context, we propose here the use of SSR and SNP markers in evaluating the genetic diversity among local maize inbred lines and races nationwide, defining the so-called heterotic groups, and then use these knowledge in designing cross-pollinating schemes that would render us with new hybrids, having superior qualities and increased productivity. We’ll analyse the effect of heterosis on these newly developed hybrids at three main levels: molecular, biochemical, and morphological. Besides the immediate applied results of our research, i.e. development of superior crop material, our observations will potentially offer a paradigm to understanding the underlying mechanisms of heterosis in plants.