Association analysis of agronomical traits and SSR loci of wheat

Asocijativno mapiranje predstavlja relativno nov i perspektivan metod za rasvetljavanje genetičke osnove kompleksnih osobina. Zasniva se na korišćenju prirodnog genetičkog diverziteta i rekombinacija nastalih tokom evolucije određene biljne vrste, kako bi se detektovao broj i položaj gena koji determinišu osobinu od agronomskog značaja. Asocijativna analiza je izgrađena na konceptu gametske neravnoteže, odnosno neslučajnog vezanog nasleđivanja alela dva lokusa. Prednost ovog metoda u odnosu na tradicionalno mapiranje, bazirano na primeni mapirajućih populacija nastalih ukrštanjem dva genetički divergentna roditelja, jeste veća rezolucija mapiranja jer se pri analizi koristi materijal koji karakteriše veća polimorfnost alela i veći broj rekombinacija. Pored toga, ova metoda je i ekonomičnija jer nije potrebno dodatno vreme za stvaranje mapirajuće populacije. U radu je primenom seta od trideset jednog mikrosatelitskog markera izvršena molekularna evaluacija 282 genotipa pšenice, koji predstavljaju deo genetičke kolekcije Instituta za ratarstvo i povrtarstvo u Novom Sadu i poreklom su iz dvadeset šest zemalja sveta. Pored toga, analizirani su i podaci fenotipske evaluacije za navedeni materijal, koji su prikupljeni tokom pet vegetacionih sezona, od 1995. do 1999. godine, na lokalitetu Rimski Šančevi i to za sledećih jedanaest agronomski značajnih osobina: visina stabljike, dužina klasa, vreme klasanja i cvetanja, masa klasa, broj klasića po klasu, broj sterilnih klasića po klasu, masa zrna po klasu, broj zrna po klasu, masa 1000 zrna i prinos. Na osnovu podataka molekularne i fenotipske evaluacije odabranog materijala, izvršena je asocijativna analiza između navedenih agronomski važnih osobina i ispitivanih mikrosatelitskih lokusa. Ustanovljeno je ukupno 112 statistički značajnih veza marker-osobina, koje su bile zajedničke za opšti i mešoviti linearni model. Dva lokusa sa hromozoma 2AS, Xbarc212 i Xwmc177 bila su u vezi sa najvećim brojem agronomski značajnih osobina. Xbarc212 bio je u vezi sa vremenom klasanja, vremenom cvetanja i brojem klasića po klasu. U okviru ovog lokusa alel od 215 bp determinisao je kasnije sazrevanje i veći broj klasića po klasu, dok je alel od 219 bp imao suprotan efekat na navedene osobine. Za mikrosatelitski marker WMC177 ustanovljena je asocijacija sa dužinom klasa, brojem klasića po klasu, brojem sterilnih klasića po klasu, masom klasa i masom zrna po klasu. Alel od 177 bp, detektovan u ovom lokusu, imao je pozitivan efekat na navedene osobine. Pored toga, markeri WMC24 (1A), GWM261 (2D), BARC12 (3A), WMC238 (4B), WMC553 (6A) i GWM297 (7B), su takođe bili u asocijaciji sa određenim osobinama ispitivanim u ovom radu, te se mogu koristiti pri marker asistiranoj selekciji u našem agroklimatskom regionu.Association mapping is relatively new and perspective method for genetic dissection of complex traits. Here, detection of number and location of genes for some agronomically important traits relies on occurring variation in diverse germplasm and on historical recombination events within some plant species. Association analysis is built on the basis of linkage disequilibrium concept, which is defined as the non-random co-segregation of alleles at two loci. The advantage of association mapping, compared with traditional quantitative trait mapping, based on mapping populations, is a relatively detailed mapping resolution. Besides that, this method is far less time consuming since no mapping populations need to be generated. In this paper, thirty one microsatellite loci were used for molecular evaluation of 282 wheat genotypes originated from twenty six countries across five continents. These genotypes are part of core collection of Institute of Field and Vegetable Crops Novi Sad. Phenotypic data for eleven agronomically important traits (height, spike length, heading, flowering, spike weight, number of spikelets per spike, number of sterile spikelets per spike, grain weight per spike, number of grains per spike, thousand kernel weight and grain yield) of this germplasm were collected during five growing seasons, from 1995 to 1999 at locality Rimski Sancevi. According to association analysis based on data of molecular and phenotipic evaluation, 112 statistically significant associations were detected. These associations were common for general and mixed linear model. Two microsatellite loci from 2AS chromosome, Xbarc212 and Xwmc177 were associated with the greatest number of agronomically important traits. Xbarc212 was associated with heading, flowering and number of spikelets per spike. Within this locus allele of 215 bp contributed to later maturity and more spikelets per spike than allele of 219 bp. SSR marker WMC177 was associated with spike length, number of spikelets per spike, number of sterile spikelets per spike, spike weight and grain weight per spike, where allele of 177 bp had positive effect on all listed traits. Besides that, markers WMC24 (1A), GWM261 (2D), BARC12 (3A), WMC238 (4B), WMC553 (6A) and GWM297 (7B) were also associated with some of traits analysed in this paper and could be used for marker assisted selection in our agro-climate region.Doktorska disertacija je odbranjena 2015. godine na Poljoprivrednom fakultetu Univerziteta u Beogradu

Estimation of genetic diversity and population structure of IFVCNS wheat collection using molecular markers and pedigrees

Within its 80-year long tradition in crop improvement, the Institute of Field and Vegetable Crops has been focusing on incorporation of the latest scientific innovations and technological achievements into conventional breeding programs. The utilization of molecular markers in the Small Grains Department began two decades ago, with their routine use in screening for major genes, especially Rht, Vrn and Ppd. One of the greatest IFVCNS assets, crucial for its breeding success, is a tremendous wheat collection consisting of breeding material, and more that 3000 genotypes diligently collected for decades from the all over the world, evaluated and classified by the important agronomic traits. Classification exclusively based on geographic origin has proved not to be sufficiently precise to explain all relationships among genotypes as a result of constant exchange of genetic material between breeding centres. For this reason, a subset of the IFVNCS collection, containing 283 representative wheat genotypes from 5 continents, were selected to determine its genetic diversity with 30 microsatellite markers and compare it with available pedigree data. Molecular data was analysed in the program STRUCTURE, applying algorithm model to discriminate 6 assumed clusters. A high level of molecular diversity was detected in the first cluster with genotypes originating from all continents

Detection of QTLs for Important Agronomical Traits in Hexaploid Wheat Using Association Analysis

BRBAKLIC L., TRKULJA D., KONDIC-SPIKA A., TRESKIC S., KOBILJSKI B. (2013): Detection of QTLs for important agronomical traits in hexaploid wheat using association analysis. Czech J. Genet. Plant Breed., 49: 1-8. One of the main wheat breeder's goals is determining specific genomic regions which control important agronomical traits. Association analysis is a new strategy with high resolution in plant molecular breeding that could be used to improve the efficiency of marker assisted selection (MAS) for finding important QTLs (quantitative trait loci) or genes. A set of 96 diverse wheat genotypes was phenotypically measured during three growing seasons (2006/07, 2007/08, 2008/09). Microsatellite markers located near important QTLs were carefully chosen in accordance with existing literature data to validate marker trait associations (MTA). Genomic DNA was extracted using the CTAB method and PCR products were separated by capillary electrophoresis. The population structure was assigned based on molecular data in Structure v. 2.0 software, while association analysis was done by the Tassel program using the Q matrix. Nine significant associations were stable in all years investigated and eight MTA were detected to be significant in two growing seasons. Microsatellite markers which showed significant associations and stability in different seasons can be useful and suitable for marker assisted selection (MAS) in Serbian wheat breeding programs

Microsatellites in the analysis of wheat genetic diversity

Microsatellite markers (SSR) were used to study wheat genetic diversity. A set of 87 wheat genotypes was analysed with four SSR markers. Primers used for the amplification of adequate microsatellite loci (Xgwm) are according to RoDER et al. (2002). Results were obtained using Applied Biosystems 3130 genetic analyser. Total of 28 alleles were determined, i.e. average of 7 alleles per marker. Number of alleles for individual markers ranged from six (Xgwm3) to eight (Xgwm18). The presence of two null alleles for Xgwm18 and Xgwm155 was found. There were five rare alleles (frequency lt 2%). Polymorphism information content (PIC) values ranged from 0.52 for Xgwm408 to 0.80 for Xgwm18. Mean PIC value was 0.69 for all markers, which signifies a high level of the detected polymorphism. According to the data collected through the analysis of four markers, most genotypes can be grouped in clusters. The results show usefulness of microsatellite markers in detecting polymorphism, identifying genotypes and assessing genetic diversity

Marker-trait associations for spike-related characters in a doubled haploid population of wheat

Phenotypic assessment of yield and its component traits needs to be performed in replicated trials under multiple environments. Mapping populations such as doubled haploids (DHs) are suitable material for studying marker-trait associations, because they can be evaluated repeatedly in different years or in variable environments, thus having considerable potential for the identification of reliable quantitative trait loci. In the present study a DH mapping population, generated from the crossing of two hexaploid high yield potential wheat varieties, Savannah (UK) and Renesansa (SRB), was used to identify associations between five microsatellite markers and seven spike-related traits. Statistically significant associations were observed between marker GWM261 on 2DS chromosome and six spike-related traits: spike length, spikelet number per spike, sterile spikelet number per spike, grain weight, spike weight and spike index. The phenotypic variation explained by this marker was the highest for the trait spike length (from 8.3 to 24.7%)

Changes in leaf appearance and developmental phases associated with breeding progress in six-rowed barley in the Pannonian Plain

The aim of this study was to analyze traits of leaf appearance and phenological development related to grain yield gain in winter barley cultivars released over the past 50 years. Field trials with 15 six-rowed winter barley cultivars were conducted during two growing seasons. The main leaf appearance and developmental traits were studied. The duration of the emergence-anthesis and emergence-physiological maturity phases decreased by 2.35 and 2.16 GDD yr(1), respectively. The duration of the stem elongation-anthesis phase was 10% longer in modern cultivars. The results showed no clear trend of improvement in final leaf number and phyllochron. The duration of the stem elongation-anthesis phase was positively related with grain number and yield. Constant improvements in grain number and grain weight by fine manipulation of the crop developmental phases could represent an essential strategy for further increases in barley grain yield potential

Induced mutations in cereals breeding

Induced mutations are used for the production of mutant varieties with altered plant characteristics, which result in a significant increase in production and / or improvement in quality. The resulting mutants have contributed to overcoming numerous obstacles in plant breeding and creating new and valuable forms, such as e.g. semi-dwarf wheat varieties, early maturing, disease resistance, etc. The application of induced mutations was widely spread between the 1950s and 1980s, and great successes have been made in creating mutated varieties. The early years of the 21st century have witnessed a return of induced mutation technologies due to a better understanding of mutagenesis and related disciplines, leading to a number of different applications. The economic value of this technology has been proven by the fact that more than 3,300 mutant varieties of different plant species have been created in the world so far, including 291 varieties of bread wheat and 316 varieties of barley. Using this technology, only one variety of paprika (Capsicum annuum L.) was registered in Serbia - Horgoška slatka -X-3 in 1974. Since that time, there have been no examples of successful mutation use in crop varieties creation in Serbia

Mogućnosti primene marker-asistirane selekcije u oplemenjivanju pšenice tolerantne na bor

The aim of this study was to investigate the possibilities of applying molecular markers-microsatellites in breeding boron tolerant wheat. The study comprised the investigation of allelic variability of sixty bread wheat accessions in two microsatellite loci (Xgwm46-7B and Xgwm577-7B) for which was assumed that are placed near the 7B chromosome locus involved in the expression of boron tolerance in wheat. Phenotypic variability concerning boron tolerance was assessed via root length reduction of wheat seedlings grown in the presence of high external boron, applied as boric acid solution (concentrations 50, 100 and 150 mg/l, boron treatments B50, B100 and B150). The indication of marker-trait associations was determined by comparing the allelic variability in the two microsatellite loci with the phenotypic variability in boron tolerance. Nonparametric Kruskal-Wallis test was used for the comparisons. The indication of marker-trait association was found for both Xgwm46-7B and Xgwm577-7B; on B150 and B50 treatments, respectively. Allelic forms identified in Xgwm577-7B locus may be related to tolerance, medium tolerance and sensitivity to high boron. This was not the case for Xgwm46-7B, where the identified alleles were related only to boron tolerance and sensitivity. Therefore, Xgwm577-7B may be preferred over Xgwm46-7B when studying boron tolerance in wheat. However, a considerable portion of boron tolerant accessions carried different alleles in the investigated loci, implying boron tolerance as a quantitative trait with more than one chromosomal region involved in its expression. Therefore, the allelic variability of more than the analyzed two loci should be investigated.Cilj ovog rada je bilo ispitivanje mogućnosti primene molekularnih markera-mikrosatelita u oplemenjivanju pšenice tolerantne na bor. Ispitana je alelna varijabilnost kod šezdeset genotipova hlebne pšenice u dva mikrosatelitna lokusa (Xgwm46-7B i Xgwm577-7B) za koje se pretpostavlja da su pozicionirani u blizini lokusa na 7B hromozomu koji utiče na ekspresiju ovog svojstva. Fenotipska varijabilnost u pogledu tolerantnosti na bor je utvrđena na osnovu redukcije dužine korenčića klijanaca pšenice gajenih u uslovima suviška bora (rastvor borne kiseline koncentracija 50, 100 i 150 mg/l, tretmani B50, B100, B150). Indikacija veze marker-svojstvo je utvrđivana na osnovu poređenja alelne varijabilnosti mikrosatelitnih lokusa i fenotipske varijabilnosti u pogledu tolerantnosti na bor. Za poređenja je korišćen neparametarski Kruskal-Wallis test. Indikacija veze marker-svojstvo je konstatovana za Xgwm46-7B na tretmanu B150, a za Xgwm577-7B na tretmanu B50. Alelne forme identifikovane u lokusu Xgwm577-7B se mogu dovesti u vezu sa tolerantnošću, srednjom tolerantnošću i osetljivošću na bor. Ovo nije bio slučaj kod Xgwm46-7B, gde su identifikovani aleli dovedeni u vezu samo sa tolerantnošću i osetljivošću na bor. Prema tome, za dalji rad se preporučuje Xgwm577-7B. Međutim, kod značajnog dela tolerantnih genotipova su identifikovani različiti aleli u proučavanim lokusima, što upućuje na zaključak da je u pitanju kvantitativno svojstvo u čiju ekspresiju je uključeno više od jednog hromozomskog regiona. Potrebno je ispitati alelnu varijabilnost više od dva mikrosatelitna lokusa

Modern plant phenotyping in breeding programs of the Institute of Field and Vegetable Crops Novi Sad (IFVCNS)

The Institute of Field and Vegetable Crops Novi Sad, National Institute of the Republic of Serbia has more than 85 years long tradition of plant phenotyping work, but first projects related to use of modern phenotyping platforms were established a decade ago. From these first steps till now, the IFVCNS become one of the leading institutions in this research area in Serbia. So far, the Institute’s researchers finalised four Transnational Access to European Plant Phenotyping Network projects. In these studies, some wheat and sunflower genotypes were identified as potential sources for biotic and abiotic stress tolerance. Thus, it was found that wheat cvs. NS 40S, Subotičanka and NS Avangarda have relatively high levels of drought and salt tolerance. Use of Flower Colour Image Analysis (FloCIA) software enhanced ray florets colour determination and paved the way to more objective and accurate sunflower phenotyping, while thermal imaging was used for screening of sunflower for tolerance to Sclerotinia head and stem rot. Also, a collaborative work with the Department of Agri-Food Sciences and Technologies, University of Bologna, Italy have shown a good tolerance of camelina to drought at the germination stage. The Institute has a strategy for more intensive use of modern phenotyping technologies in its future research and practice. Recently approved projects: SmartSun (7732457), CROPINNO (101059784), EMPHASIS-GO (101079772) and AgroServ (101058020), funded by the National Science Foundation of Serbia and EU Commission, are aimed at institutional capacity building in the application of phenomics in crop improvement

Potentials of gene editing application in wheat breeding

Constant increase in human population causes an increased demand for wheat, which production should rise at a rate of 1.6% annually until 2050. To achieve this goal, scientists and plant breeders must have access to all possible breeding tools. The most recent is precision breeding with CRISPR/Cas9 system, which allows creation of desired crop varieties in a fast, simple and much mote direct way compared to previous breeding techniques. Many agriculturally important traits of wheat have been targeted by genome editing, resulting in resistance to powdery mildew and Fusarium graminearum, i~aproved drought tolerance, water use efficiency and herbicide tolerance, enhanced gtain size and yield, as well as reduced amount of alfa-gliadins and immunoreactivity for consumers with coeliac disease. Also, mutant wheat plants which abort pollen development were produced using this technology, resulting in male sterility. Production of male-sterile and doubled haploid plants can facilitate development of hybrid seed production in wheat. However, all these promising results obtained by advanced methods will have only scientific significance if their wide application in wheat breeding is not allowed. In the European Union plants obtained by precision breeding techniques like CRISPR are considered as genetically modified organisms (GMOs) which are not exempt from the GMO legislation. Even crops with the smallest CRISPR-mediated alteration, which can also arise spontaneously in nature, are subjected to these provisions. Some part of the scientific community thinks that there are no scientific reasons to consider genome-edited crops differently than conventionally bred varieties. They stated that plants obtained by simple and targeted genome editing and which do not contain foreign genes are at least as safe as varieties derived from conventional breeding techniques. Recently, many European research institutions have taken the initiative to change the EU legislation by signing an open statement which says that gene editing with CRISPR should be used as a faster and mote efficient way of producing food sustainably. If this initiative is accepted, it will allow new methods to be applied more widely in breeding and their results can be translated to the field through the precise introgression of desired traits into conventional wheat varieties