Page 1 Page 2 Page 3 Page 4 Page 5 Page 6 Page 7 Page 8 Page 9 Page 10 Page 11 Page 12 Page 13 Page 14 Page 15 Page 16 Page 17 Page 18 Page 19 Page 20 Page 21 Page 22 Page 23 Page 24 Page 25 Page 26 Page 27 Page 28 Page 29 Page 30 Page 31 Page 32 Page 33 Page 34 Page 35 Page 36 Page 37 Page 38 Page 39 Page 40 Page 41 Page 42 Page 43 Page 44 Page 45 Page 46 Page 47 Page 48 Page 49 Page 50 Page 51 Page 52 Page 53 Page 54 Page 55 Page 56 Page 57 Page 58 Page 59 Page 60 Page 61 Page 62 Page 63 Page 64 Page 65 Page 66 Page 67 Page 68 Page 69 Page 70 Page 71 Page 72 Page 73 Page 74 Page 75 Page 76 Page 77 Page 78 Page 79 Page 80 Page 81 Page 82 Page 83 Page 84JANUARY 2017 SEEDWORLD.COM / 47 Scientists Form a Second Line of Plant Defense SCIENTISTS HAVE DEVELOPED a new improved method for capturing longer DNA fragments, doubling the size up to 7,000 DNA bases that can be analyzed for novel genes, which provide plants with immunity to disease. RenSeq is the method to sequence Resistance (R) genes that confer disease resistance in plants. “R genes can control diverse plant diseases including major threats to global crop production,” says Ingo Hein, principal investigator at the James Hutton Institute and co-author. “The ability to capture and sequence long genomic DNA fragments that contain full-length R genes enables the rapid identification of novel, functional resistance genes from wild species. These genes, if introgressed into new cultivars via breeding or alterna- tive routes, could significantly reduce the dependency on pesti- cides for crop production.” Each plant typically carries hundreds of potential R gene sequences, encoding NB-LRR proteins, identified by the pres- ence of specific sequence motifs. R genes are often part of families of closely related sequences. While shared sequences make it possible to capture the R-genes, it also makes it hard to tell them apart and find the exact gene that enables plants to survive attack. Longer mol- ecules and sequences of DNA allow easier and more accurate genetic analysis to identify variation. Activate Defense Responses The NB-LRR gene family enables plants to withstand infection from a suite of diseases and form a second line of defense. After a pathogen has managed to invade a plant, it uses “effector” molecules to weaken a plant’s defenses — the R gene proteins recognize these “effector” molecules and signal to the plant to activate defense responses — killing cells around the site of infec- tion in an attempt to stop it spreading. This constant evolutionary arms race between plants and patho- gens, whereby the organisms causing disease in plants are mutating to avoid plant defenses, causes the plants to evolve through changes in their own genetic makeup. This is where a huge variety of R genes come into play that are highly similar in structure and DNA sequence. Researchers at the Earlham Institute (EI), The Sainsbury Laboratory (TSL) and the James Hutton Institute, have found a new way to decipher these large stretches of DNA to discover and annotate pathogen resistance in plants. Using the PacBio, which can read longer stretches of DNA in their entirety, along with the developed NB-LRR gene work- flow “RenSeq” (Resistance gene enrichment sequencing), the data not only targets R genes, but also the important regulatory regions of DNA – promoters and terminators that signal when to start making a protein and when to stop. “Wild relatives of crops contain a huge repertoire of novel genes that could be used to breed more resistant varieties that need less pesticide treatments,” says Matt Clark, head of tech- nology development at EI and lead author of the study. “When it comes to identifying key genes it can be very difficult for researchers to find the exact resistance gene due to the sheer similarity of their DNA sequences. “Typical sequencing methods use short reads eg from the Illumina HiSeq, but these are often too short to prise similar genes apart. “RenSeq diverges from normal DNA sequencing on the PacBio by focusing the sequencing effort on a specific gene family, i.e. R-genes. In this study, by optimizing multiple steps in the library construction, we can identify the protein-coding sequences and the neighboring regulatory regions; indeed in many cases, we can reconstruct the entire DNA region even if it contains many similar genes, which normally are too hard to tell apart. This means we can identify the exact gene that confers resistance to a certain infection, and used in breeding programs.” Professor Jonathan Jones, senior scientist at TSL and co- author, adds: “This improvement to the RenSeq method will greatly facilitate building reliable inventories of R genes in mul- tiple plant species, helping us clone additional genes that could protect our crops.” SW