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Wheat is an important food crop, but adversity stresses are becoming more common, resulting in significant yield losses. Accelerating molecularly assisted resistance breeding is critical. Through statistical analysis of published loci in wheat over the last two decades, we selected 60 loci with main breeding objectives, high heritability, and reliable genotyping, such as stress resistance, yield, plant height, and resistance to spike germination. Then, using genotyping by target sequencing(GBTS) technology, we developed a liquid phase chip based on 101 functional or closely linked markers. The genotyping of 42 loci was confirmed in an extensive collection of Chinese wheat cultivars, indicating that the chip can be used in molecular-assisted selection (MAS) for target breeding goals. Besides, we can perform the preliminary parentage analysis with the genotype data. The most significant contribution of this paper is to evaluate the target traits of breeding materials without conducting field experiments. Breeders can quickly screen germplasm resources, parental breeding materials, and intermediate materials for the presence of excellent allelic variants using the genotyping data by this chip, which is high throughput, convenient, reliable, and cost-efficient.

YrFDC12 and PbcFDC, co-segregated in chromosome 4BL, and significantly interacted with Yr30/Pbc1 to enhance stripe rust resistance and to promote pseudo-black chaff development. Cultivars with durable resistance are the most popular means to control wheat stripe rust. Durable resistance can be achieved by stacking multiple adult plant resistance (APR) genes that individually have relatively small effect. Chinese wheat cultivars Ruihua 520 (RH520) and Fengdecun 12 (FDC12) confer partial APR to stripe rust across environments. One hundred and seventy recombinant inbred lines from the cross RH520 × FDC12 were used to determine the genetic basis of resistance and identify genomic regions associated with stripe rust resistance. Genotyping was carried out using 55 K SNP array, and eight quantitative trait loci (QTL) were detected on chromosome arms 2AL, 2DS, 3BS, 4BL, 5BL (2), and 7BL (2) by inclusive composite interval mapping. Only QYr.nwafu-3BS from RH520 and QYr.nwafu-4BL.2 (named YrFDC12 for convenience) from FDC12 were consistent across the four testing environments. QYr.nwafu-3BS is likely the pleiotropic resistance gene Sr2/Yr30. YrFDC12 was mapped in a 2.1-cM interval corresponding to 12 Mb and flanked by SNP markers AX-111121224 and AX-89518393. Lines harboring both Yr30 and YrFDC12 displayed higher resistance than the parents and expressed pseudo-black chaff (PBC) controlled by loci Pbc1 and PbcFDC12, which co-segregated with Yr30 and YrFDC12, respectively. Both marker-based and pedigree-based kinship analyses revealed that YrFDC12 was inherited from founder parent Zhou 8425B. Fifty-four other wheat cultivars shared the YrFDC12 haplotype. These results suggest an effective pyramiding strategy to acquire highly effective, durable stripe rust resistance in breeding.

Cultivars with durable resistance are the most popular means to control wheat stripe rust. Durable resistance can be achieved by stacking multiple adult-plant resistance (APR) genes that individually have relatively smaller effect. Chinese wheat cultivars Ruihua 520 (RH520) and Fengdecun 12 (FDC12) confer partial APR to stripe rust across environments. One hundred and seventy recombinant inbred lines from the cross RH520 × FDC12 were used to determine the genetic basis of resistance and identify genomic regions associated with stripe rust resistance. Genotyping was carried out using 55K SNP array and eight quantitative trait loci (QTL) were detected on chromosomes 2AL, 2DS, 3BS, 4BL, 5BL (2) and 7BL (2) by inclusive composite interval mapping. Only QYr.nwafu-3BS from RH520 and QYr.nwafu-4BL.2 (named YrFDC12 for convenience) from FDC12 were stable across the four testing environments. QYr.nwafu-3BS is likely the pleiotropic resistance gene Sr2/Yr30. YrFDC12 was mapped in a 2.1 cM interval corresponding to 12 Mb and flanked by SNP markers AX-111121224 and AX-89518393. Lines harboring both Yr30 and YrFDC12 displayed higher resistance than the parents and expressed pseudo-black chaff (PBC) controlled by two loci Pbc1 and PbcFDC12, which were co-located with Yr30 and YrFDC12, respectively. Both marker-based and pedigree-based kinship analyses revealed that YrFDC12 was inherited from founder parent Zhou 8425B. Fifty-four other wheat cultivars shared the same haplotype of YrFDC12 region. These results suggest an effective pyramiding strategy to acquire highly effective, durable stripe rust resistance in breeding.

Genetic information of polymerase chain reaction (PCR)-based markers, one of the main tools of genetics and genomics research in wheat, have been well documented in wheat. However, the physical position in relation to these markers has not yet been systematically characterized. Aim of this study was to characterize the physical information of thousands of widely used molecular markers.We first assigned 2705 molecular markers to wheat physical map, of which 86.1% and 84.7% were the best hits to chromosome survey sequencing (CSS) project (CSS-contigs) and International Wheat Genome Sequencing Consortium Reference Sequence v1.0 (IWGSC RefSeq v1.0), respectively. Physical position of 96.2% markers were predicated based on BLAST analysis, were in accordance with that of the previous nullisomic/aneuploidy/linkage analysis. A suggestive high-density physical map with 4643 loci was constructed, spanning 14.01 Gb (82.4%) of the wheat genome, with 3.02 Mb between adjacent markers. Both forward and reverse primer sequences of 1166 markers had consistent best hits to IWGSC RefSeq v1.0 based on BLAST analysis, and the corresponding allele sizes were characterized. A detailed physical map with 1532 loci was released, spanning 13.93 Gb (81.9%) of the wheat genome, with 9.09 Mb between adjacent markers. Characteristic of recombination rates in different chromosomal regions was discussed. In addition, markers with multiple sites were aligned to homoeologous sites with a consistent order, confirming that a collinearity existed among A, B and D subgenomes. This study facilitates the integration of physical and genetical information of molecular markers, which could be of value for use in genetics and genomics research such as gene/QTL map-based cloning and marker-assisted selection.

SummaryMutations affecting splice sites comprise approximately 7.5 % of the known F8 gene mutations but only a few were verified at mRNA level. In the present study, 10 putative splice site mutations were characterised by mRNA analysis using reverse transcription PCR (RT-PCR). Quantitative real-time RT-PCR (RT-qPCR) and co-amplification fluorescent PCR were used in combination to quantify the amount of each of multiple F8 transcripts. All of the mutations resulted in aberrant splicing. One of them (c.6187+1del1) generated one form of F8 transcript with exon skipping, and the remaining nine mutations (c.602-6T>C, c.1752+5_1752+6insGTTAG, c.1903+5G>A, c.5219+3A>G, c.5586+3A>T, c.969A>T, c.265+4A>G, c.601+1_601+5del5 and c.1444-8_1444del9) produced multiple F8 transcripts with exon skipping, activation of cryptic splice site and/or normal splicing. Residual wild-type F8 transcripts were produced by the first six of the nine mutations with amounts of 3.9 %>, 14.2 %>, 5.2 %>, 19.2 %>, 1.8 °% and 2.5 %> of normal levels, respectively, which were basically consistent with coagulation phenotypes in the related patients. In comparison with the mRNA findings, software Alamut v2.3 had values in the prediction of pathogenic effects on native splice sites but was not reliable in the prediction of activation of cryptic splice sites. Our quantification of F8 transcripts may provide an alternative way to evaluate the low expression levels of residue wild-type F8 transcripts and help to explain the severity of haemophilia A caused by splicing site mutations.