Analysis Of Genetic Background And Marker-assisted Selection Of Powdery Mildew Resistant Wheat Germplasm

Wheat(Triticum aestivam) is an important grain crop worldwide.Powdery mildew(PM,caused by Blumeria graminis f.sp.tritici) is a major epidemic disease of wheat,causing serious reduction of yield and quality.Breeding and extension of powdery mildew resistant varieties is an efficient and safe strategy to control this disease,lntrogression of powdery mildew resistance genes from relative species into common wheat genetic background is a crucial for disease resistance breeding in wheat. At same time,identification of molecular markers linked with resistance genes and application of them in the selection of cultivars and stocks,will promote the pyramiding efficiency and comprehensive elite traits and the disease resistance breeding steps.1.The 6V/6B alien substitution line 010714 of wheat was bred and identifiedGuinong 21 is an elite wheat line with multiple resistances especially with to powdery mildew.It is a 6V/6A substitution line in which the 6V chromosome was introgressed from Haynadia villosa.Hexaploid of Ae.ventricosa-T,durum is an offspring of T.durum and Aegilops ventricosa.Both of them have good resistance to powdery mildew.Bridge-cross between Guinong 21 and hexaploid of Ae.ventricosa-T. durum was made and a PM-resistant intermediate line 010714 was selected from the F₇ generation.It is relatively stable,highly resistant to PM,and has comprehensive agronomic traits near general common wheat lines.Cytological identifications of 010714 were carried by Genome in Situ Hybridization(GISH),karyotype investigation and C-band analysis.Using Digoxigenin-11-dUTP labeled total genomic DNA of H.villosa and A.ventricosa as the probes.The results revealed that line 010714 has a pair of alien chromosomes from H.villosa and no chromosome or mega fragment from A.ventricosa.Karyotype analysis suggested that the arm ratio of the chromosome from H.villosa is 1.20, simility to the 1.12 of 6V.In 010714,there is only a pair of 1B-like satellite chromosomes with an arm ratio of 1.85.But it lacks another satellite chromosome,6B, whose arm ration is 2.10.C-band analysis showed the substitution chromosome is the 6V chromosome.Is definite that 010714 is a 6V/6B substitution line which is a new material,and its resistance to PM is correlated to the alien chromosome.2.The RAPD marker RM874 was identified,with linkage to the PM-resistance in 010714C050S has the thermo-photo-sensitive genic male sterility(TGMS) and good agronomic traits but is highly susceptible to many diseases especially PM.This study used 010714 as the male parent and C050S as the female parent to make a cross,and identified the molecular markers with linkage to the PM-resisntance.The F₁ plants are resistant to PM,indicating that the PM-resistance of 010714 is a dominant trait.The F₂ plants segregated for a ratio of 139 resistant over 96 susceptible ones.The PM resistance is a dominant trait determined by a single gene.This segregation ratio did not follow the typical Mendelian ratio 3:1,because in 6V substitution line,the transmission rate of the 6V chromosome through gametes was significantly lower than that of common wheat chromosomes.RAPD primer S2018 amplified a specific band of about 874 bp,named RM874 that is a specific RAPD marker for powdery mildew resistance in GN21 and 010714.3.Based on batched sequencing within single PCR bands and multi-alignments, the repeat-containing RM874 marker was successfully converted into SCAR marker SM142RM874 showed a little instability and needed to be converted into a more stable and specific SCAR marker.Sequencing results indicated that RM874 single band contained a family of homologous repetitive sequences with equal lengths.Among the sequenced clones,sequence F2R874-7 from the F₂ resistant pool was completely identical to sequence PR874-910 from 010714 and sequence Hv874-7 from H.villosa. Then the specific F2R874-7/PR874-910/Hv874-7 fragment was named as RS874. RS874 could be traced together with the PM-resistance from H.villosa to Guinong 21, then to 010714,and finally to the F₂ plants of cross C050S×010714.NCBI BLASTn revealed that,in local alignments,RS874 had 58.1%to 72.1%identities to LTR-retrotransposon like repetitive regions from T.monococcum,T.turgidum, common wheat,Hordeum vulgare and Oryza sativa.RM874 and its homologous sequences were in the GAG-POL coding regions of Ty3-gypsy retrotransposons.The family sequences from RM874 were dispersive in 010714's chromosomes.Based on multi-alignments of RS874 with other 43 RM874 sequences and homologous sequences from T.monococcum,T.turgidum and common wheat,a pair of SCAR primers 05-116/05-117were designed.This SCAR primer pair specifically amplified a 142-bp fragment from 010714 and F₂ resistant pool,but not from C050S and F₂ susceptible pool.This SCAR marker was named SM142.SM142 is linked with the PM resistance gene but did not show cosegrating/complete linkage with the PM resistance gene.4.Based on SM142-assited selection,the PM resistance and the TGMS were successfully combinedF₂ and F₅ individual plants from the C050S×010714 were examed for the presentation of RAPD marker RM874 and SCAR marker SM142.The results showed that the markers are linked to the PM resistance,and SM142 is more stablie than RM874.Both of them can be applicated as the markers of PM resistance in MAS.Two TGMS lines with elite PM resistance and other agronomic traits were selected from F₅ population by SM142-assisted selection and traditional breeding.It proved the value of SM142.Because of translocation between 6V chromosome and common wheat's chromosome,the linkage between the marker and the PM-resistance has been disrupted in several progenies of cross C050S×010714.The results of GISH show that the individuals having SM142 but no PM resistance are 6VL translocation lines,while the individuals that having both SM142 and resistance are 6VS translocation line. These suggested that the PM resistance gene is located at the short arm of 6V chromosome.For 010714,its hybrid offsprings,and other wheat materials with 6V chromosome from H.villosa,RM874 and SM142 can act as 6V-specific PCR marker. For wheat stocks harboring other chromosome from H.villosa,RM874 might be capable to certain extent for tracing the alien genetic substances as well.