Results

Year 2020

  • A 1 - Establishing the maize inbred panel and DNA preparation: - we establish the working protocol and the lists with the image of the plates containing the maize lines taken in to analysis; we have prepared an optimal dilution for the DNA stocks held for the subsequent amplification processes (30%).


  • A 2. -Growth of plant material in populations required for gDNA extraction: 10 seeds were grown in the growth chamber for 400 inbred lines; the vegetable material was harvested and placed on silica gel for optimal drying in order to extract genomic DNA.


Year 2021

  • A 1. Extraction of DNA from plant material: the genomic DNA was extracted from 400 inbred lines provided in the previous step using specific extraction kits, quantified and diluted accordingly.

Fig. 1: DNA extraction and verification on 1% agarose gel.

  • A 2. PCR amplification of the crtRB1 and lcyE genes: 2746 inbred lines were analyzed - 584 lines with alleles favorable for the crtRB1 gene (21,26%); - 55 lines with alleles favorable for the lcyE gene (2%); 5 lines with alleles favorable for the both genes (0.18%).

Fig. 2: multiplex crtRB1 and lcyE gel profiling of selected 29 genotypes: 1-5: homozygous favorable for both genes; 6-15: homozygous favorable for lcyE gene; 16-25: homozygous favorable for crtRB1 gene; 26-29: heterozygous unfavorable for both genes; M - 100 bp molecular marker.

Table 1: Results obtained after processing the lines from 7 research centers from Romania (5), Rep. Moldova and Serbia.

Fig. 3: Graph with the results obtained for each gene analyzed (percentage dates).

Fig. 4: Graph with the results obtained for each research center.

Fig. 5: Map with the geographical location of the research centers from where the analyzed lines were chosen.

  • A 3. Sequencing and identification of SNPs / InDels:

Fig. 6: Polymorphisms detected following lcyE gene sequencing.

Fig. 7: Polymorphisms detected following crtRb1 gene sequencing.

  • A 4. -Estimating the level of provitamin A:

Fig. 8: HPLC results obtained in the case of 34 Zea mays lines belonging to the following categories: lines with both favorable alleles; lines with one of the two favorable alleles; lines with the two unfavorable alleles. (The name of the obtained genotype will be available after the publication of the obtained data).

Year 2022

  • A1: Analysis of the obtained data

  • A1.1: Genetic diversity analysis using Neghbour joining method

A)

B) cont. of A

C) cont. of B

D) cont. of C

E) cont. of D

Fig. 1 (A,B,C,D, E): Neighbor joining analysis for inbred lines, belonging to ARDS Turda, containing one or both favorable alleles for the crtRB1 and lcyE genes, data obtained previously by Genotyped-By-Sequence method. (Purple - inbreed lines with favourable alelle for ctrRB1 gene; yellow - inbreed lines with favourable alelle for lcyE gene; green - inbreed lines with favourable alelle for both genes)

  • A1.2: Population structure analysis using STRUCTURE software v.2.3.4

A)

B) cont. of A

Fig. 2 (A,B): STRUCTURE analysis for inbred lines, belonging to ARDS Turda, containing one or both favorable alleles for the crtRB1 and lcyE genes, data obtained previously by Genotyped-By-Sequence method. (Purple - inbreed lines with favourable alelle for ctrRB1 gene; yellow - inbreed lines with favourable alelle for lcyE gene; green - inbreed lines with favourable alelle for both genes)

  • A2: Dissemination of results

  • Article published: "Ioan Băcilă, Voichița Haș, Dana Șuteu, Mihai Miclăuș, Ana Coste, Edward Muntean, Carmen D. Vana, Andrei Varga, Roxana Călugăr, Ana Copândean, 2022, Screening of the Romanian maize (Zea mays L.) germplasm for crtRB1 and lcyE alleles enhancing the provitamin A concentration in endosperm. Notulae Botanicae Horti Agrobotanici Cluj-Napoca Volume 50, Issue 3, Article number 12621 DOI:10.15835/nbha50312621

  • Registration and participation at the 21st International Conference "Life sciences for sustainable development", which took place at the University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Romania, 15-17 September 2022 - poster presentation.