Genetics of resistance to Fusarium crown rot in barley and pan-genome sequence anchors of this species

Fusarium crown rot (FCR), caused primarily by Fusarium pseudograminearum, is a devastating disease for cereal production in semi-arid regions worldwide. Apart from severe yield loss, this disease can also lead to accumulation of mycotoxins in foods or feeds, which is harmful to health of human and livestock. It has long been recognized that breeding and growing resistant varieties is an important and indispensable component in FCR management. Three large-effect quantitative trait loci (QTL) conferring FCR resistance had been identified in barley when I first started my PhD research. They were located on the chromosome arms 1HL, 3HL and 4HL, respectively. Pyramiding these QTL was proved to be effective in further enhancing the resistance to FCR. I started my PhD research by characterizing a new source of resistance. It (AWCS799) is a landrace identified from a systematic screening of more than 1,000 genotypes. Genetic control of its resistance was investigated by generating and analysing two populations of recombinant inbred lines with AWCS799 as the common parent. One of the populations was used for QTL detection and the other for validation. A novel QTL, located on the long arm of chromosome 6H (designated as Qcrs.caf-6H), was consistently detected in each of the four tests conducted against the mapping population. The QTL explained up to 28.3% of the phenotypic variance and its effect was confirmed in the validation population. Significant interaction between this resistance locus and either plant height or heading date was not detected in the populations used, further facilitating its manipulation in breeding programs. The interactions between FCR severity and other characteristics indicate that QTL detected through mapping can only be treated as putative. Benefiting from the uniform genetic backgrounds for untargeted genomic region, near isogenic lines (NILs) could be used to validate the effectiveness of QTL for various characteristics. Validating an existing locus on chromosome arm 1HL thus become another part of my PhD research program. This QTL is named as Qcrs.cpi-1H. Five pairs of NILs targeting this locus were generated. Analysing the NILs found that the resistant allele at Qcrs.cpi-1H significantly reduced FCR severity. Transcriptomic analysis was then conducted against three of the NIL pairs, which placed the 1HL locus in an interval spanning about 11 Mbp. A total of 56 expressed genes bearing single nucleotide polymorphisms (SNPs) were detected in this interval. Five of them contain non-synonymous SNPs. NILs developed in this study and the transcriptomic sequences obtained from them would be valuable for identifying genes and generating diagnostic markers targeting this locus. As the limited resolution caused by the heterogeneous genetic backgrounds in mapping populations, markers obtained from QTL mapping is usually not tightly linked with a given locus. To develop markers that are able to reliably trace 1HL locus in breeding programs, we developed and assessed a fine mapping population consisting of 1,180 recombinant inbred lines derived from one of the above NIL pairs. Using this population, we delineated Qcrs.cpi-1H into an interval of 0.4 cM covering a physical length of about 487 kb. Six markers co-segregating with this locus were generated. Three of the five genes with non-synonymous variations identified from the multiple pairs of NILs were further confirmed to be located within the interval. In addition to generate extra markers for breeding programs, the refined location of Qcrs.cpi-1H should also facilitate the cloning of the causal gene(s) underlying this locus. It has become clear in recent years that many genes in a given species cannot be found in any given genotype, thus a comprehensive pan-genome can be highly valuable for genetic research and breeding. Clearly, obtaining a comprehensive pan-genome requires deep-sequencing large numbers of genotypes which is still not practical for species like barley which has a huge and highly repetitive genome. However, large quantity of genotype-by-sequencing (GBS) data has been made available for barley. We thus attempted to identifying barley pan-genome sequence anchors using these data based on an approach combining genetic mapping and machine learning. Based on the GBS sequences from 11,166 domesticated and 1,140 wild barley genotypes, we identified 1.844 million reliable tags. Of them, 532,253 were identified as presence/absence variation (PAV) tags. Based on those present in the genome of the hulless barley genotype Zangqing320, positions for 83.6% of the Morex-absent tags from the domesticated genotypes and 88.6% from the wild barley genotypes should be correct. Association analyses against flowering time, plant height and kernel size showed that the relative importance of the PAV and Non-PAV tags varied for different traits. This project provided a series of genetic resources, including a novel locus and the detailed genetic profile of Qcrs.cpi-1H, for breeding FCR resistant barley varieties. Moreover, the high-resolution physical map based on pan-genome sequences should not only facilitate the construction of a comprehensive barley pan-genome, but also assist various genetic studies including identification of structural variation, genetic mapping and breeding in barley.