Molecular and biochemical characterisation of rust and Fusarium head blight resistant wheat lines

Abstract

English: Wheat is one of the most important food crops and consumption in the past year was higher than production worldwide, which indicates improved wheat cultivars need to be developed to maintain the demand for wheat. One of the biggest threats of wheat is ever-evolving pathogens that overcome resistance. To overcome threatening diseases, breeders need to incorporate new resistant genes/QTL into improved cultivars. Incorporating different resistant genes/QTL into a single line using gene-pyramiding and MAS can enhance the breeding process. However, pyramiding of genes for resistance can lead to a decrease in baking quality characteristics, which are important for the milling and baking industry as well as for consumers. To overcome this problem, lines should also be tested for bread-making quality characteristics such as HMW-GS and the LUPP% which are directly linked to protein quality. The aim of the study was to identify rust and FHB resistant lines with good protein content. Rust and FHB resistant wheat lines were developed during previous studies. The best rust and FHB resistant lines were planted and self-pollinated. Lines were evaluated for the presence of five rust resistant genes/QTL (Lr19, Lr34/Yr18/Sr57, QYr.sgi.2B-1, Sr2 and Sr26) and four FHB resistant genes/QTL (Fhb1, Qfhs.ifa-5A-1, Qfhs.ifa-5A-2 and Fhb2) for two consecutive years. These selected rust and FHB resistant lines were also subjected to three biochemical tests namely SDS-PAGE, SEHPLC and RP-HPLC. PCR-based markers linked to HMW-GS alleles and the IBL.1RS translocation, associated with weak dough strength, were also tested. Since the breeding programme was still in an early stage, only few seeds per line were available and therefore biochemical tests that can be performed using single seeds were selected. Results from SDS-PAGE and molecular markers linked to HMW-GS were similar and therefore lines were further evaluated using only molecular markers. No correlations were detected for the RP-HPLC data of lines therefore it was excluded during further analysis. The rust and FHB resistant genes/QTL co-segregated based on their related parental lines. An additional round of self-pollination either led to higher levels of homozygosity for the selected traits or to the loss of traits due to recombination. MAS enabled the selection and enhancement of homozygous lines. Only a few offspring of one of the rust resistant lines contained the Sr26 gene, while the Lr34/Yr18/Sr57 gene was present in all tested lines. None of the rust resistant lines contained the Sr2 gene which is a major gene for resistance against stem rust and especially effective against the threatening Ug99 race. The top ten rust resistant lines all had the same rust resistant gene/QTL present (Lr19, Lr34/Yr18/Sr57 and QYr.sgi.2B-1) as well as the same protein quality alleles (Ax2*, Bx7+By8 and Dx5+Dy10). The LUPP% of these lines showed high levels of variation and was optimal (40% to 50%) for only one line. High levels of variation were detected for the FHB resistant markers in the FHB resistant populations. The top ten lines contained all six markers although the level of homozygosity varied. Four of these lines expressed both the Bx7+By8 and Bx17+By18 alleles and only one line did not express the Bx7OE allele. None of these lines expressed the 1BL.1RS translocation. Two of these lines showed a desirable LUPP%. Results indicated the preference towards rust or FHB resistance selection followed by selection for protein quality alleles and lastly the LUPP%. The top ten rust and FHB lines can serve as resistance sources in further breeding programmes.

Afrikaans: Koring is een van die belangrikste voedselgewasse en die afgelope jaar was die verbruik van koring wêreldwyd hoër as produksie wat daarop dui dat verbeterde koring kultivars ontwikkel moet word om aan die behoefte vir koringte kan voorsien. Een van die grootste bedreigings vir koring is patogene wat steeds bly verander om weerstand te kan oorkom. Ten einde hierdie bedreiging te kan oorkom moet planttelers nuwe weerstandsgene en kwantitatiewe eienskap lokusse (QTL) in verbeterde kultivars inbou. Die teelproses kan aansienlik versterk word deur van geenstapeling en merker-ondersteunde seleksie gebruik te maak om verskillende weerstandsgene en QTL in ‘n enkele lyn in te bou. Die stapeling van gene kan egter tot ‘n afname in bakkwaliteitseienskappe, wat belangrik vir die meulenaars en die bakindustrie asook verbruikers is, lei. Om hierdie probleem te oorkom behoort lyne ook vir broodkwaliteits-eienskappe soos hoë molekulêre gewig gluten sub-eenhede (HMW-GS) en groot onekstraheerbare polimeriese proteïen persentasie (LUPP%) wat direk aan proteïenkwaliteit gekoppel is, getoets te word. Die doel van die studie was om roes en fusariumaarskroei (FHB) weerstandbiedende lyne met goeie proteïeninhoud te identifiseer. Roes en FHB weerstandbiedende lyne is gedurende vorige studies ontwikkel. Die beste roes en FHB weerstandbiedende lyne is geplant en selfbestuif. Lyne is vir die teenwoordigheid van vyf roesweerstandsgene of QTL (Lr19, Lr34/Yr18/Sr57, QYr.sgi.2B-1, Sr2 en Sr26) en vier FHB weerstandsgene of QTL (Fhb1, Qfhs.ifa-5A-1, Qfhs.ifa-5A-2 en Fhb2) gedurende twee opeenvolgende jare, geëvalueer. Hierdie geselekteerde roes en FHB weerstandbiedende lyne was ook aan drie biochemiese toetse, naamlik natrium dodesielsulfaatpoliakrielamiedgelelektroforese (SDS-PAGE), grootte uitsluitings-hoë prestasie vloeistof chromatografie (SE-HPLC) en omgekeerde-fase-hoë prestasie vloeistof chromatografie (RP-HPLC) onderwerp. Polimerase kettingreaksie (PCR) gebaseerde merkers gekoppel aan HMW-GS allele en die 1BL.1RS translokasie, wat met swak deegsterkte geassosieer is, is ook getoets. Aangesien die teelprogram nog in ‘n vroeë stadium van ontwikkeling was, was slegs ‘n klein aantal saad van elke lyn beskikbaar en daarom is biochemiese toetse wat op enkel sade gedoen kan word, geselekteer. Dieselfde resultate is verkry deur van SDS-PAGE en molekulêre merkers gekoppel aan die HMW-GS gebruik te maak en daarom is die res van die lyne slegs getoets deur van die molekulêre merkers gebruik te maak. Geen korrelasies is vir die RP-HPLC data van die verskillende lyne verkry nie en daarom is die tegniek nie verder gebruik nie. Die roes en FHB weerstandsgene of QTL het ko-segregasie getoon gebaseer op hul onderskeie ouers. ‘n Addisionele rondte van selfbestuiwing het tot of hoër vlakke homosigositeit van die geselekteerdeeienskappe, of tot ‘n verlies van van die eienskappe weens rekombinasie gelei. MAS het die seleksie en verhoging van homosigotiese lyne moontlik gemaak. Slegs ‘n klein aantal lyne afkomstig van een van die roesweerstandbiedende lyne het die Sr26 geen bevatterwyl die Lr34/Yr18/Sr57 geen in al die getoetste lyne teenwoordig was. Geen van die roesweerstandbiedende lyne het die Sr2 geen wat ‘n belangrike weerstandsgeen teen stamroes en veral die Ug99 patogeen is, bevat nie. Die top 10 roesweerstandbiedende lyne het almal dieselfde roesweerstandsgene of QTL (Lr19, Lr34/Yr18/Sr57 en QYr.sgi.2B-1) asook dieselfde proteïen kwaliteitsallele(Ax2*, Bx7+By8 en Dx5+Dy10) bevat. Die LUPP% van hierdie lyne het hoë vlakke van variasie getoon en was slegs optimaal (40% tot 50%) in een van die lyne. Hoë vlakke van variasie is vir die FHB weerstandsmerkers in die FHB weerstandbiedende populasies opgespoor. Die top tien lyne het almal ses merkers bevat alhoewel die vlak van homosigositeit gevarieer het. Vier van hierdie lyne het beide die Bx7+By8 en Bx17+By18 allele uitgedruk en slegs een van hierdie lyne het nie die Bx7OE alleel bevat nie. Geen een van hierdie lyne het die 1BL.1RS translokasie uitgedruk nie. Twee van die lyne het aanvaarbare LUPP% gehad. Resultate het aangetoon dat daar tydens seleksie voorkeur verleen is aan roes en FHB weerstand gevolg deur seleksie vir proteïenkwaliteitallele en laastens vir LUPP%. Die top tien roes en FHB lyne kan in die toekoms as weerstandbronne in ander teelprogramme gebruik word.