Marker assisted selection for quality protein maize inbred lines adapted to temperate regions

Hranljiva vrednost kukuruza je niska zbog niskog sadržaja dve esencijalne aminokiseline - lizina i triptofana. Poboljšanje hranljive vrednosti kukuruza predstavlja jedan od glavnih ciljeva savremenih programa oplemenjivanja zbog sve značajnijih klimatskih promena i rasta ljudske populacije, odnosno sve veće potrebe za hranom. Najveći uspeh postignut je 60-ih godina prošlog veka otkrićem opaque2 (o2) mutanta sa superiornim hranljivim svojstvima. Međutim, plejotropni efekti o2 mutacije čine endosperm zrna kukuruza mekim, a samo zrno osetljivim na pucanje, trulež i štetočine. Istraživači Međunarodnog centra za poboljšanje pšenice i kukuruza u Meksiku (eng. International Maize and Wheat Improvement Center - CIMMYT) su metodama konvencionalnog oplemenjivanja stvorili kukuruz visokog kvaliteta proteina (eng. Quality Protein Maize – QPM), koji istovremeno ima visoku hranljivu vrednost proteina endosperma i dobre agronomske performanse. Iako je QPM razvijen klasičnim metodama selekcije, poslednjih godina se sve više primenjuje selekcija upotrebom molekularnih markera (eng. Marker Assisted Selection - MAS) sa ciljem poboljšanja elitnih linija za kvalitet proteina. Unutar sekvence o2 gena identifikovana su tri različita SSR markera – phi057, phi112 i umc1066, koji se koriste za utvrđivanje polimorfizma inbred linija i kao selekcioni markeri za o2 gen (eng. foreground selection). Molekularni markeri se koriste i radi utvrđivanja procenta genoma rekurentnog roditelja u potomstvima povratnih ukrštanja (eng. background selection). Primena foreground i background selekcije smanjuje broj generacija potrebnih za stvaranje željenog genotipa klasičnim metodama selekcije koje se zasnivaju na fenotipskom odabiru biljaka. Osnovni cilj ovog rada bio je dobijanje linija kukuruza poboljšanog kvaliteta proteina adaptiranih na umereno klimatsko područje, korišćenjem specifičnih molekularnih markera za opaque2 gen, kao i da se održe dobre agronomske osobine i kombinaciona sposobnost poboljšanih linija...Maize nutritional quality is poor due to the low levels of two essential aminoacids - lysine and tryptophan. Because of the climatic changes and increased food demands due to the human population growth, improvement of the maize nutritional quality is one of the main goals of many breeding programs. A major breakthrough has been the isolation of the opaque2 (o2) mutant with superior nutritional properties in the 1960s. However, pleiotropic effect of the o2 mutation makes the maize endosperm soft and susceptible to cracking, ear rots, and storage pests. Using conventional breeding methodologies, interdisciplinary research team in the International Maize and Wheat Improvement Center (CIMMYT), Mexico, created the new, agronomically acceptable and nutritionally improved opaque2 types by the name of Quality Protein Maize (QPM). Although QPM was created through conventional breeding, marker assisted selection (MAS) is becoming increasingly used approach for improvement of protein quality in maize. Three simple sequence repeats (phi057, phi112 and umc1066), located as internal repetitive sequences within the o2 gene, are being utilized as foreground selection markers for the o2 gene. Molecular markers are also effectively employed for identification of the genotypes with the highest proportion of recurrent parent genome (background selection). Both foreground and background selection decrease the number of the generations required to create desirable genotype through conventional breeding based on the fenotypic selection. The main objective of this research was to create high quality protein maize lines adapted to temperate regions using opaque2 specific molecular markers, while maintaining their good agronomic performances and combining abilities. Two elite inbred lines - ZPL 3 and ZPL 5 were selected as the recurrent parents, to be the recipients of the o2 allele and to improve the tryptophan content. CML 144, a QPM tropical inbred line, was selected as the donor line of o2. Identification and confirmation of the QPM selection results involved biochemical analysis (determination of protein content, tryptophan content and quality index - tryptophan to protein ratio) and evaluation of the agronomicaly important traits in the field trials..

DNA and biochemical analysis of a potential opaque2 maize population

Maize has low nutritional value because it is poor in essential amino acids lysine and tryptophan, but different mutations have been identified that increase their content. Two high lysine/tryptophan populations from Maize Research Institute genebank (IP1 and IP2) were identified in a previous research. In both populations, analysis with umc1066 opaque2 specific marker detected a recessive (o2), a dominant (O2) and an unknown allele (UA). However, IP2 lacked homozygous recessive o2o2 genotypes. The aim of the present research was to determine by DNA and biochemical analysis if UA allele was a recessive allele and/or if high tryptophan content was due to the o2 or some other mutation. Tree more opaque accessions with different mutations - IP3o5, IP4o14 and IP5floury (no data on type of mutation) were used in biochemical analysis for comparison with IP1 and IP2. Kernels were divided into two samples - with hard and with soft kernels. The UA allele sequencing revealed that it was a dominant allele with four GCCAGA repeats. SSR analysis showed presence of o2 in IP1 in both hard and soft kernels. Decrease in 22 kDa, 19 kDa and 27 kDa zeins in soft kernels was observed only in IP1 and IP2. Tryptophan content was high in soft kernels of IP1 (0.081) and IP2 (0.085), and in both hard and soft kernels of -IP3o5 (0.083 and 0.085, respectively). It can be concluded that IP1 is an o2 mutant and that IP2 carries a high tryptophan mutation other than o2, o5, o14 or floury

Poređenje sitnozrnih žitarica prema sadržaju dijetalnih vlakana i proteina

The content of dietary fibres (cellulose, hemicellulose, lignin, NDF, ADF), tryptophan and proteins, as well as their quality index were determined in whole grains of bread and durum wheat, rye, hull-less barley and hull-less oat, each represented with four genotypes. In addition, content of β-glucans in hull-less barley were determined. In average, hull-less barley and oat had the lowest content of hemicellulose (22.54 and 13.11% d.m., respectively), cellulose (1.36 and 1.41% d.m., respectively), lignin (0.98 and 0.49% d.m., respectively), as well as NDF (24.84 and 15.16% d.m., respectively) and ADF (2.30 and 2.04% d.m., respectively). In average, the highest content of hemicellulose was in durum wheat (33.47% d.m.), followed by rye (29.63% d.m.), and bread wheat (23.24% d.m.). Among tested hull-less barley genotypes the the content of β-glucans ranged from 4.1% d.m. (IWHBON 97-18) to 5.6% d.m. (Apolon). The highest content of proteins (on average 15.65% d.m.) and tryptophan (on average 0.206% d.m.) was in hull-less oat. Hull-less barley had the highest protein quality index (1.48%) followed by bread and durum wheat and hull-less oat (IQ 1.35, 1.34 and 1.31%, respectively), and rye (IQ 0.93%). The results indicate that there is genetic diversity in content of dietary fibres and proteins among tested genotypes and that it should be possible to selectively breed for lines with high nutrition capacities, as well as, to improved diet requirements.U zrnu genotipova hlebne i durum pšenice, raži, golozrnog ječma i golozrnog ovsa određen je sadžaj dijetalnih vlakana (celuloze, hemiceluloze, lignina, NDF-a, ADF- a), triptofana i proteina, kao i njihov indeks kvaliteta. Pored toga određen je i sadržaj β-glukana u zrnu četiri reprezentativna genotipa golozrnog ječma. U proseku, golozrni ječam i ovas imali su najniži sadržaj hemiceluloze (22.54 i 13.11% s.m.), celuloze (1.36 i 1.41% s.m.), lignina (0.98 i 0.49% s.m.), kao i NDF (24.84 i 15.16% s.m.) i ADF (2.30 i 2.04% s.m.). Najviši sadržaj hemiceluloze bio je u zrnu durum šenice (u proseku 33.47% s.m.), sledi raž (u proseku 29.63% s.m.) i hlebna pšenica (u proseku 23.24% s.m.). U zrnu ispitivanih genotipovima golozrnog ječma sadržaj β-glukana se kretao od 4.1% d.m. (IWHBON 97-18) do 5.6% d.m. (Apolon). Najviši sadržaj proteina (u proseku 15.65% d.m.) i triptofana (u proseku 0.206% d.m.) bio je u zrnu golozrnog ovsa. Najviši indeks kvaliteta proteina bio je u zrnu golozrnog ječma (u proseku 1.48%), sledi hlebna i durum pšenica i golozrni ovas (1.35, 1.34 i 1.31%), a zatim raž (0.93%). Rezultati ukazuju na genetičku divergentnost u sadržaju dijetalnih vlakana i proteina između ispitivanih genotipova i mogućnost odabira genotipova za selekcione linija visokog nutritivnog kapaciteta, kao za i poboljšane zahteva ishrane

Individual and combined (Plus-hybrid) effect of cytoplasmic male sterility and xenia on maize grain yield

Plus-hybrid effect refers to a combined effect of cytoplasmic male sterility (CMS) and xenia in maize (Zea mays L.) It could be used in commercial production by growing a mixture of 80% CMS hybrid and 20% of another fertile hybrid. The aim of this research was to examine individual and combined CMS and xenia effects on two hybrids widely grown in Serbia. Sterile and fertile versions of ZP 1 and ZP 2 hybrids (three-way; Iodent x Lancaster dents) were used as females, while ZP 1, ZP 2, ZP 3, ZP 4, and ZP 5 (three-way or single cross; Iodent (BSSS) x Lancaster dents) were used as pollinators. All of them belong to medium maturity group. The trial was set up at one location in Serbia (Zemun Polje) in 2009, 2010, and 2011. Molecular analysis of the five genotypes was done using simple sequence repeat (SSR) primers. Plus-hybrid effect on grain yield ranged from -6.2% to 6.2%; on thousand kernel weight from -1.7% to 5.2%; on number of kernels per area from -1.0% to 8.0%. The poor response could be due to a use of three-way instead of single cross hybrids in S type of sterility. Modified Rogers' distance between hybrids was in the range 0.211 to 0.378 and was not relevant for the effect, which depended mostly on the sterile hybrid genotype and the fertile hybrid pollinator ability. This approach should be more suitable for female hybrids with slightly poorer performance, already being produced on a sterile base

DNA and biochemical analysis of a potential opaque2 maize population

Maize has low nutritional value because it is poor in essential amino acids lysine and tryptophan, but different mutations have been identified that increase their content. Two high lysine/tryptophan populations from Maize Research Institute genebank (IP1 and IP2) were identified in a previous research. In both populations, analysis with umc1066 opaque2 specific marker detected a recessive (o2), a dominant (O2) and an unknown allele (UA). However, IP2 lacked homozygous recessive o2o2 genotypes. The aim of the present research was to determine by DNA and biochemical analysis if UA allele was a recessive allele and/or if high tryptophan content was due to the o2 or some other mutation. Tree more opaque accessions with different mutations - IP3o5, IP4o14 and IP5floury (no data on type of mutation) were used in biochemical analysis for comparison with IP1 and IP2. Kernels were divided into two samples - with hard and with soft kernels. The UA allele sequencing revealed that it was a dominant allele with four GCCAGA repeats. SSR analysis showed presence of o2 in IP1 in both hard and soft kernels. Decrease in 22 kDa, 19 kDa and 27 kDa zeins in soft kernels was observed only in IP1 and IP2. Tryptophan content was high in soft kernels of IP1 (0.081) and IP2 (0.085), and in both hard and soft kernels of -IP3o5 (0.083 and 0.085, respectively). It can be concluded that IP1 is an o2 mutant and that IP2 carries a high tryptophan mutation other than o2, o5, o14 or floury

Effect of preceding crops nitrogen fertilization and cobalt and molybdenum application on yield and quality of soybean grain

The three-year study was conducted on a calcareous chernozem experimental plot of the Institute of Field and Vegetable Crops. The experimental field trial was designed as corn soybean wheat crop rotation and performed in four replications. The aim of this study was to determine the effects of soybean seed inoculation with microbiological fertilizer, seed treatment with cobalt and molybdenum, as well as the effect of preceding maize crop fertilization with different dosages of nitrogen, on soybean yield and its protein and oil contents. This would allow defining optimal seed treatment that would enable production of high and stable yield of quality soybeans with the rationalization of the use of mineral nitrogen fertilizers. The application of the largest dose of 250 kg N ha(-1) was responsible for significantly (by 12.11%) higher yields compared with the control. Grain yield was not affected by the application of cobalt and molybdenum. No significant yield increase was found when seed that was treated only with Nitragin. The contents of protein and oil were statistically very significantly different between the three experimental years because the investigated qualitative properties of grain are highly dependent on hydrothermal conditions. The protein content in the soybean increased very significantly following the increasing amounts of nitrogen. Very significant effect of seed treatment on protein and oil content was found. The two-field system of maize and wheat cultivation should be upgraded to the three-field system: maize - soybean - wheat. The use of cobalt and molybdenum in slightly alkaline and alkaline soils did not contribute to the increase of grain yield or protein content in the grain. However, it caused very significant increase in soybean oil content on slightly alkaline soils. The effect of the increase was 1.77%

Effects of adapted quality protein maize on broiler performance

Quality protein maize (QPM) has high contents of essential amino acids lysine and tryptophan. The substitution of standard maize (SM) with QPM in feeds can be profitable due to improved livestock performance and decreased costly dietary lysine supplementation and protein ingredients. Herein, effects of QPM adapted to temperate climate on broiler performance were tested. Feeding experiment was performed on control (fed with SM) and treatment (fed with QPM) broilers. Each feeding trial of 42 days duration was carried out in three phases - starter (1-14), grower (15-35) and finisher (36-42). Diets for each growth phase was formulated based on the biochemical analysis of maize kernels. At the end of each growth phase, feed intake (FI), body weight gain (BWG), average daily gain (ADG) and feed conversion ratio (FCR) were calculated. In the first experiment, SM was completely replaced with QPM in treatment group diets. The results indicated better FCR in the treatment group for grower (1.51:1.56) and finisher (1.56:1.61) phases. In the second experiment QPM was increased and soybean component decreased by 3%. This time, FCR improvement was even better but in all three phases – 1.16:1.33 for starters, 1.28:1.36 for growers and 1.30:1.46 for finishers. These results indicate significant financial reduction in feed as the price of soybean is usually two to four times higher than the price of maize. Overall, it can be concluded that using adapted QPM in broiler diets can be profitable for both feed industry and broiler producers

Biohemijska analiza F2 zrna genotipova kukuruza visokog kvaliteta proteina

Protein quality of standard maize kernels is poor, due to the low levels of two essential aminoacids - lysine and tryptophan. The opaque2 mutation which increases lysine and tryptophan content also confers an undesirable phenotype leading to low yields, soft and chalky kernels that renders seeds susceptible to storage pest and ear rots. Quality protein maize (QPM) is the maize with increased levels of these amino acids and good agronomic performances. At the Maize Research Institute, a program on creating QPM germplasm developed for conditions of temperate climatic zone is in progress. The aim of this study was to test quality traits of F2 kernels derived from the crosses between 24 QPM inbred lines with three standard inbred lines and two opaque2 lines. Most of the analyzed 108 genotypes had tryptophan content under the threshold of 0.075 set for QPM (83.5%). However, 50% had elevated tryptophan content in the range from 0.065 to 0.074. High tryptophan content was found in 16.5% of the analyzed genotypes, the highest being 0.088. Only one genotype had quality index over 0.80 (threshold for QPM). Biochemical analysis identified genotypes with good protein quality which will be further tested for agronomic performances in order to potentially be considered as QPM hybrids.Proteini kukuruza su siromašni u sadržaju dve esencijalne aminokiseline - lizina i triptofana. Prirodna opaque2 mutacija udvostručava nivo ovih aminokiselina, ali istovremeno smanjuje prinos, čini endosperm kukuruza mekim i brašnjavim, a seme osetljivim na trulež klipa i štetočine skladištenog zrna. Kukuruz visokog kvaliteta proteina (Quality Protein Maize - QPM) se može definisati kao kukuruz sa visokom nutritivnom vrednošću proteina endosperma i istovremeno dobrim agronomskim performansama. U Institutu za kukuruz već nekoliko godina postoji program stvaranja genotipova kukuruza visokog kvaliteta proteina, adaptiranih na umereno klimatsko područje. Cilj ovog rada je bio da se ispita kvalitet proteina F2 zrna kukuruza dobijenog ukrštanjem 24 QPM inbred linija sa tri standardne inbred linije i dve opaque2 linije. Većina od 108 analiziranih genotipova (83,5%) je imala sadržaj triptofana ispod praga od 0,075, od čega je poboljšani nivo triptofana (0,065-0,074) imalo 50% genotipova. Visok sadržaj je imalo 16,5% analiziranih genotipova, od kojih je najviša vrednost bila 0,088. Samo jedan genotip je imao indeks kvaliteta proteina iznad praga od 0,80. Biohemijskom analizom utvrđeni su genotipovi poboljšanog kvaliteta čija će agronomska svojstva biti ispitana kako bi se utvrdilo da li se mogu smatrati potencijalnim QPM hibridima

High grain quality accessions within a maize drought tolerant core collection

Maize (Zea mays L.) landraces are an important source of genes for improving commercial germplasm. Today, drought tolerance and grain quality are major challenges in maize cultivation due to climatic changes and population growth. The Maize Research Institute gene-bank has a drought tolerant collection, which includes 13 landraces (from the former Yugoslavia) and 12 introduced populations (from different countries). These accessions were analyzed for protein, oil, starch and tryptophan contents, in order to identify drought tolerant accessions with high grain quality. Also, simple sequence repeat (SSR) analysis with specific primers for opaque2 recessive allele (o2) was carried out. All analyzed accessions showed high levels of protein. Oil content ranged from 3.75 % to 5.40 % and starch content from 67.5 % to 71.30%. Average protein content was not different (p lt 0.01) between landraces and introduced populations. Starch and oil contents were higher in introduced populations at 0.84 % and 0.39 %, respectively (p lt 0.01). Twenty-three accessions had high levels of tryptophan content. A high percentage of kernel type 1 and 2 indicated the presence of endosperm hardness modifier genes. Recessive o2 allele was found in most of the accessions. Absence of o2 in some high tryptophan accessions indicated action of another mutation. In two high tryptophan accessions an unknown band was detected. Absence of negative correlations between proteins, tryptophan and oil makes certain accessions suitable for use in the simultaneous improvement of target genotypes for these traits. Identified drought tolerant, high quality accessions can be used in breeding programs aimed at nutritional improvement of maize grown under drought conditions

Dental health care for children with autism spectrum disorders

Introduction: Autism spectrum disorders (ASD) are pervasive disorders beginning in early childhood. The prevalence of these disorders has been on the rise over the last few decades. A disorder of this kind does not have any direct impact on the oral health, but the behavior of the affected children can markedly deteriorate it. Due to a poor compliance to oral hygiene and maintenance of oral health, dental film accumulates in the oral cavity, leading to both caries and its complications, and gingival-periodontal diseases. A high percentage of children with ASD are not sufficiently compliant during dental interventions, being hyperactive, tense, and often highly agitated. Any dental intervention in such children is therefore very complicated or it cannot be performed at all, consequentially broadening the spectrum of indications for dental extractions. In order to preserve the oral health in children with ASD, an individualized approach to each patient is thus necessary. Conclusion: The journey to a healthy oral cavity and teeth in children with ASD is full of twists and turns, but the desired goals can be realized, although requiring considerable patience and perseverance from both the parents and pediatric dentists