Table_5_Sequence Composition of Bacterial Chromosome Clones in a Transgressive Root-Knot Nematode Resistance Chromosome Region in Tetraploid Cotton.XLSX

Plants evolve innate immunity including resistance genes to defend against pest and pathogen attack. Our previous studies in cotton (Gossypium spp.) revealed that one telomeric segment on chromosome (Chr) 11 in G. hirsutum cv. Acala NemX (rkn1 locus) contributed to transgressive resistance to the plant parasitic nematode Meloidogyne incognita, but the highly homologous segment on homoeologous Chr 21 had no resistance contribution. To better understand the resistance mechanism, a bacterial chromosome (BAC) library of Acala N901 (Acala NemX resistance source) was used to select, sequence, and analyze BAC clones associated with SSR markers in the complex rkn1 resistance region. Sequence alignment with the susceptible G. hirsutum cv. TM-1 genome indicated that 23 BACs mapped to TM-1-Chr11 and 18 BACs mapped to TM-1-Chr 21. Genetic and physical mapping confirmed less BAC sequence (53–84%) mapped with the TM-1 genome in the rkn1 region on Chr 11 than to the homologous region (>89%) on Chr 21. A 3.1-cM genetic distance between the rkn1 flanking markers CIR316 and CIR069 was mapped in a Pima S-7 × Acala NemX RIL population with a physical distance ∼1 Mbp in TM-1. NCBI Blast and Gene annotation indicated that both Chr 11 and Chr 21 harbor resistance gene-rich cluster regions, but more multiple homologous copies of Resistance (R) proteins and of adjacent transposable elements (TE) are present within Chr 11 than within Chr 21. (CC)-NB-LRR type R proteins were found in the rkn1 region close to CIR316, and (TIR)-NB-LRR type R proteins were identified in another resistance rich region 10 cM from CIR 316 (∼3.1 Mbp in the TM-1 genome). The identified unique insertion/deletion in NB-ARC domain, different copies of LRR domain, multiple copies or duplication of R proteins, adjacent protein kinases, or TE in the rkn1 region on Chr 11 might be major factors contributing to complex recombination and transgressive resistance.

植物演化出包含抗病基因在内的先天免疫系统，以抵御害虫与病原物的侵染。本团队此前在棉属（Gossypium spp.）植物中的研究显示，陆地棉（G. hirsutum）品种Acala NemX的11号染色体（Chr 11）上的一段端粒区段（rkn1位点），对植物寄生线虫南方根结线虫（Meloidogyne incognita）表现出超亲抗性；而其部分同源染色体21号（Chr 21）上的高度同源区段则无任何抗性贡献。为进一步解析该抗性的分子机制，研究人员以Acala N901（Acala NemX的抗性供体材料）的细菌人工染色体（bacterial artificial chromosome, BAC）文库为基础，筛选、测序并分析了与复杂rkn1抗性区域内简单序列重复（simple sequence repeat, SSR）标记相关的BAC克隆。将测序序列与感病陆地棉品种TM-1的基因组进行序列比对后发现，共有23个BAC克隆定位到TM-1的11号染色体，18个BAC克隆定位到TM-1的21号染色体。遗传与物理图谱验证显示，相较于21号染色体上的同源区域（比对覆盖率>89%），11号染色体上rkn1区域内与TM-1基因组匹配的BAC序列占比更低（53%~84%）。在Pima S-7 × Acala NemX的重组自交系（recombinant inbred line, RIL）群体中，rkn1侧翼标记CIR316与CIR069之间的遗传距离为3.1 厘摩（cM），对应TM-1基因组中的物理距离约为1 百万碱基对（Mbp）。NCBI Blast与基因注释结果表明，11号与21号染色体均富集抗病基因簇区域，但11号染色体上的抗病（R）蛋白同源拷贝数以及相邻转座元件（transposable elements, TE）的数量均多于21号染色体。在靠近CIR316的rkn1区域中发现了CC-NB-LRR型R蛋白，而在距离CIR316约10 cM（对应TM-1基因组中约3.1 Mbp）的另一抗病富集区域中鉴定到了TIR-NB-LRR型R蛋白。本次研究中鉴定到的11号染色体rkn1区域内NB-ARC结构域的独特插入/缺失、不同拷贝数的LRR结构域、R蛋白的多拷贝或复制事件、相邻蛋白激酶以及转座元件，可能是导致复杂重组与超亲抗性的关键因素。