Estimation of phenolic content in yellow, red, blue and red-blue maize by near-infrared reflectance spectroscopy

Red and blue colors in maize kernel are the consequence of anthocyanin pigments, responsible for the increased phenolic content and antioxidant activity in maize. Therefore, interest in pigmented maize as a novel functional food due to its possible health benefits and unique taste has grown. The red color in maize kernel originates from pericarp and is maternally inherited while the blue color originates from aleurone, which is triploid in nature and its mode of inheritance is more complicated. Red and blue maize are usually low-yielding populations, and at the Maize Research Institute Zemun Polje (MRIZP), commercial inbred lines have been converted to red and blue colors to produce high-yielding red and blue maize hybrids. Maize extractable phenolic content (EPC) can be directly determined through wet chemistry and measures phenolic amides, glycerides, free phenolic acids, and some flavonoids. In this study, however, Fourier-transformed near-infrared reflectance spectroscopy (FT-NIRS), as a non-destructive, inexpensive, and rapid technique has been used to scan EPC profiles of 56 MRIZP maize samples (14 genotypes in two replications and two environments) differing in kernel color: yellow, red pericarp, blue aleurone and red pericarp with blue aleurone. Twenty samples with outlying EPC values have been directly analyzed for total phenolics and these values have been used to adjust and improve NIRS calibration. EPC values ranged from 1.69 to 6.82 mg g-1 across genotypes and environments. Two red inbred lines had the highest EPC content in 2017, 6.82 and 5.11 mg g-1, and, overall samples from 2017 had a higher content of EPC compared to the same genotypes grown in 2018. Red genotypes on average had the highest EPC content, followed by red-blue, while yellow and blue kernels had similar and the lowest content within the samples

UGRADNJA GА1-S GENA U RODITELJSKE LINIJE HIBRIDA KUKURUZA BELOG ZRNA POMOĆU MOLEKULARNIH MARKERA

Maize Research Institute Zemun Polje has a marker assisted backcross breeding (MABB) program aimed to incorporate the strong allele of incompatibility gene Gа1 into parental components of the hybrids with specific traits (white kernel). The main objective of this research was marker assisted development of the white maize hybrid´s parental lines with the complete cross-incompatibility to foreign pollens. Ga1-S-tightly-linked molecular markers successfully identified heterozygous plants with the percentage of approximately 50% after all three backcrossing. Regarding the background selection, 69% of progenies had recurrent parent’s genome (RPG) above the theoretical value for BC3 generation. Also, a few individuals even had 99%, the value theoretically achieved in BC6 generation, showing that MABB made a genetic gain in RPG recovery. Foreground selection in this generation aimed to identify homozygous dominant individuals. Out of 264 BC3F2 plants, 70 (26.5%) were dominant homozygotes, which is in accordance with the expected Mendelian ratio. Progenies of the BC3F2 homozygous dominant plants were planted alternatively with yellow-kernel maize of the same maturity to check cross-incompatibility. Unfortunately, none of the dominant progenies had a 100% white kernel, most likely due to the presence of modifier genes that increase/decrease the effectiveness of pollen exclusion or that, alternatively, modifiers were lost during the backcrossing. It could be expected that successfully employed functional markers (developed from the sequence polymorphisms present within functional gene(s) associated with phenotypic trait variations) would outbalance the noted impediments and enhance MABB efficiency to transfer the desired gene(s) controlling simple or complex trait(s) into cultivated varieties.U Institutu za kukuruz “Zemun Polje” postoji program selekcije pomoću molekularnih markera (MAS) čiji je cilj ugradnja gena za inkompatibilnost Gа1-S u linije kukuruza koje su roditeljske komponente hibrida belog zrna. Glavni cilj ovog rada je bio stvaranje linija kukuruza belog zrna sa potpunom unakrsnom inkompatibilnošću sa stranim polenom pomoću molekularnih makera. Molekularnim markerima koji su blisko vezani za Ga1-S gen uspešno su identifikovane heterozigotne biljke (50%) nakon sva tri povratna ukrštanja. Rezultati utvrđivanja procenta genoma rekurentnog roditelja (RPG) u BC3 generaciji pokazali su vrednost iznad očekivane kod 69% potomstva, dok je kod određenog broja RPG iznosio 99%, što predstavlja vrednost koja se teoretski dostiže u BC6 generaciji. Od 264 BC3 F2 pojedinačnih biljaka, 70 (26,5%) je bilo dominantno homozigotno, što je u skladu sa očekivanim, prema pravilima Mendelovog nasleđivanja. Potomstva BC3 F2 dominantno homozigotnih biljaka posejana su sa kukuruzom žutog zrna iste grupe zrenja radi provere unakrsne inkompatibilnosti. Nijedno od dobijenih potomstava nije imalo 100% belo zrno, najverovatnije usled prisustva gena modifikatora koji menjaju efikasnost isključivanja polena, ili je došlo do gubljenja ovih gena prilikom povratnog ukrštanja. Moglo bi se očekivati da će uspešna primena funkcionalnih markera (stvorenih na osnovu polimorfizama sekvenci unutar samog gena vezanog za fenotipsku osobinu) prevazići uočene prepreke i poboljšati efikasnost MAS za prenos željenih gena koji kontrolišu prostu ili složenu osobinu u gajene kulture

Seed quality of reciprocal maize single crosses and their parental inbred lines

Five direct and reciprocal early hybrids, five direct and reciprocal late hybrids and a total of their 12 parental inbred lines were used in order to test reciprocal effects among hybrids in early germination phase and seed quality of their parental inbred lines. Five hybrids were of early maturity FAO 300-400 group, and five of late maturity FAO 500- 600 group. In the early group one line of Lancaster origin (L1) was in common, and in direct crosses used as male parent, while in reciprocal crosses as female parent. The same case was with late hybrids where another Lancaster line (L2) was in common. In 2015 and 2016 all the material was multiplied in hand pollination and after six months of storage three tests according to ISTA standards were performed: standard germination test (SGT), cold-test (CT) and vigor radicle test (RE). According to the Analysis of variance, a strong reciprocal effect was found among early hybrids for four out of five parameters in RE and SGT, while it was found only for RE in late hybrids. However, in CT none of the parameters had significant reciprocal effect. Two tested Lancaster inbreds were superior in the hybrids over non-Lancaster lines when used as maternal components, while as lines per se they did not display such superiority. Mid-parent heterosis (MPH) ranged from -62.32% to 211.11% in RE test, with an average of 44.26%; from -10.15% to 17.93% in SGT test, with an average of 6.27%, and from -5.31% to 4.49% in CT, with an average of 0.64%. Significant differences of MPH were found for nine pairs of reciprocal hybrids for RE, three pairs for SGT and seven pairs for CT. This shows that reciprocal differences in MPH were higher as the applied level of stress during early seed growth raised. All the results showed very complicated mode of inheritance of tested traits in the early developmental stages of young plants, including the phenomenon of heterosis which is expressed already at the germination stage

Optimization of the doubled haploid technology for temperate maize breeding programs: a case study from Maize Research Institute Zemun Polje

Doubled haploid (DH) technology is one of the several technological advances greatly impacting modern maize breeding. The main benefit for breeders is the development of parental inbred lines in one year comparing to four using conventional breeding. Maize Research Institute Zemun Polje (MRIZP) initiated doubled haploid program in maize in 2014. In six years up to 8.000 DH lines have been developed from close to 160 donor germplasm sources of different genetic backgrounds. During this period the research group engaged in the DH program identified several issues that need to be addressed in order to optimize the method for use in temperate regions including poor agronomic performance of haploid inducing lines, donor germplasm effect on the overall efficiency of DH method in maize breeding and selection strategies for improving test-cross performance of DH lines. To meet the challenges, MRIZP DH research group started a breeding program aiming at developing inducers with high and stable HIR adapted to temperate climate conditions. Two main goals were defined: development of haploid inducers with HIR up to 20% in a completely inducer genetic background, and second, to develop haploid inducers in 75 and 50% inducer genetic background with moderate HIR but better agronomic performance. Also, nine populations were used to assess the suitability of our most elite breeding materials for use as donors with respect to genetic background, specifically heterotic group origin, and give guidance to breeders on donor germplasm selection. Furthermore, we are studying different selection strategies for DH line breeding with respect to donor size and prior cycles of selection, i.e. recombination (none (F1), one (F2), or two selection cycles (F3)). We hope that our still ongoing research will contribute to already extensive knowledge on doubled haploid methodology

Oplemenjivanje kukuruza uz pomoć metode dupliciranih haploida u Institutu za kukuruz “Zemun Polje”

In vivo metoda dupliciranih haploida u Institutu za kukuruz „Zemun Polje“ koristi se u oplemenjivanju kukuruza od 2014. godine. Pristup omogućava dobijanje potpuno homozigotnih inbred linija tzv. duplohaploidnih (DH) linija za samo dve godine (dve do tri generacije), za razliku od konvencionalnog oplemenjivanja gde je potrebno bar sedam do osam generacija kako bi se postigla neophodna homozigotnost. Metoda se sastoji iz nekoliko koraka: 1) indukcija haploida iz odabranih populacija ukrštanjem sa linijom induktorom haploida (prva godina/letnja generacija); 2) selekcija haploidnih zrna (prva godina/letnja generacija); 3) duplikacija hromozoma (druga godina/zimska generacija) i 4) umnožavanje dobijenih DH linija (druga godina/letnja generacija). Do sada je dobijeno oko 13000 DH linija iz oko 350 populacija koje pripadaju različitim heterotičnim grupama. U okviru programa sprovode se i aktivnosti sa ciljem razvoja novih induktora haploida sa povišenim procentom indukcije haploida i poboljšanim agronomskim svojstvima. Osam induktora-kandidata je specifičnom procesom selekcije odabrano i u 2023. godini ocenjeno u mini ogledu sa komercijalno dostupnim induktorima, a mereni su visina biljke, produkcija polena, ozrnjenost u ukrštanju, kao i procenat indukcije haploida. U okviru rada na optimizaciji metode, ispitivan je i uticaj populacije (heterotične grupe) i spoljašnje sredine na uspešnost metode. Utvrđen je značajan uticaj spoljašnjih uslova, kao i heterotične grupe odnosno genotipa na indukciju i duplikaciju, kao i konačnu uspešnost metode. Prvi hibridi sa DH linijom kao roditeljskom komponentom već su registrovani od strane Sortne komisije Republike Srbije za komercijalno gajenje u Srbiji

Marker assisted Gа1-S incorporation into white maize hybrid´s parental lines

U Institutu za kukuruz “Zemun Polje” postoji program selekcije pomoću molekularnih markera (MAS) čiji je cilj ugradnja gena za inkompatibilnost Gа1-S u linije kukuruza koje su roditeljske komponente hibrida belog zrna. Glavni cilj ovog rada je bio stvaranje linija kukuruza belog zrna sa potpunom cross-inkompatibilnošću sa stranim polenom pomoću molekularnih makera. Molekularnim markerima koji su blisko vezani za Ga1-S gen uspešno su identifikovane heterozigotne biljke (50%) nakon sva tri povratna ukrštanja. Rezultati utvrđivanja procenta genoma rekurentnog roditelja (RPG) u BC3 generaciji pokazali su vrednost iznad očekivane kod 69% potomstva, dok je kod određenog broja RPG iznosio 99% što predstavlja vrednost koja se teoretski dostiže u BC6 generaciji. Od 264 pojedinačne biljke BC3F2 generacije, 70 (26,5%) su bili dominantni homozigoti, što je u skladu sa očekivanim prema pravilima Mendelovog nasleđivanja. Potomstva BC3F2 dominantno homozigotnih biljaka posejana su sa kukuruzom žutog zrna iste grupe zrenja radi provere cross-inkompatibilnosti. Nijedno od dobijenih potomstava nije imalo 100% belo zrno, najverovatnije usled prisustva gena modifikatora koji menjaju efikasnost isključivanja polena, ili je došlo do gubljenja ovih gena prilikom povratnog ukrštanja. Moglo bi se očekivati da bi se primenom funkcionalnih markera (stvorenih na osnovu polimorfizama sekvenci unutar samog gena vezanog za fenotipsku osobinu) uspešno prevazišle uočene prepreke i poboljšala efikasnost MAS za prenos željenih gena koji kontrolišu prostu ili složenu osobinu u gajene kulture.Maize Research Institute Zemun Polje has a marker assisted backcross breeding (MABB) program aimed to incorporate the strong allele of incompatibility gene Gа1 into parental components of the hybrids with specific traits (white kernel). The main objective of this research was marker assisted development of the white maize hybrid´s parental lines with the complete cross-incompatibility to foreign pollens. Ga1-S-tightly-linked molecular markers successfully identified heterozygous plants with the percentage of approximately 50% after all three backcrossing. Regarding the background selection, 69% of progenies had recurrent parent’s genome (RPG) above the theoretical value for BC3 generation. Also, a few individuals even had 99%, the value theoretically achieved in BC6 generation, showing that MABB made a genetic gain in RPG recovery. Foreground selection in this generation aimed to identify homozygous dominant individuals. Out of 264 BC3F2 plants, 70 (26.5%) were dominant homozygotes, which is in accordance with the expected Mendelian ratio. Progenies of the BC3F2 homozygous dominant plants were planted alternatively with yellow-kernel maize of the same maturity to check cross-incompatibility. Unfortunately, none of the dominant progenies had a 100% white kernel, most likely due to the presence of modifier genes that affect the effectiveness of pollen exclusion or that, alternatively, modifiers were lost during the backcrossing. It could be expected that successfully employed functional markers (developed from the sequence polymorphisms present within functional gene(s) associated with phenotypic trait variations) would outbalance the noted impediments and enhance MABB efficiency to transfer the desired gene(s) controlling simple or complex trait(s) into cultivated varieties