Divya D, Sahu Nihar, Nair Suresh, Bentur J S
ICAR-Indian Institute of Rice Research, Rajendranagar, Hyderabad, 500030, India.
Agri Biotech Foundation, Rajendranagar, Hyderabad, 500030, India.
Mol Biol Rep. 2018 Dec;45(6):2075-2086. doi: 10.1007/s11033-018-4364-8. Epub 2018 Sep 12.
The Asian rice gall midge (ARGM), Orseolia oryzae is an important insect pest causing an annual yield loss of about US$ 80 million in India. Till now 11 R genes and seven biotypes of the pest have been characterized and reported. The indica rice variety Aganni, a landrace from the state of Kerala, is known to carry the gall midge resistance gene Gm8 with HR-type of resistance. This gene has been fine mapped within 0.43 Mb region with the flanking markers RM22685 and RM22709. We identified 63 possible candidate genes through in silico analysis in the reference Nipponbare rice genome between 7.5 and 9.5 Mb region. One of the markers targeting the proline rich protein (PRP) gene (LOC_Os08g15080) showed polymorphism between the parents and also exhibited complete co-segregation with the trait in 426 F RIL populations. Functional validation of this gene through RT-PCR in contrasting parents and Pre-NILs (near isogenic lines) revealed that this is an early responsive gene with rapid induction at 24 h after gall midge infestation (hai) followed by subsequent reduction in the expression levels at late hours. Validation of this gene in five gall midge resistant rice varieties carrying different resistance genes revealed that the induction was unique to Aganni rice carrying Gm8 gene. Further, cloning and sequencing of the alleles of this gene including promoter region from TN1 (susceptible parent) and Aganni (resistant parent) revealed 153 nucleotide substitution, four amino acid substitutions and three mutations at putative cis-acting elements in TN1 when compared to Aganni. In addition, we also developed a functional marker (PRP-del) for detection of the gene for use in marker-assisted introgression of Gm8.
亚洲稻瘿蚊（ARGM），即稻瘿蚊（Orseolia oryzae），是一种重要的害虫，在印度每年造成约8000万美元的产量损失。到目前为止，已经鉴定并报道了该害虫的11个抗性基因（R基因）和7种生物型。印度喀拉拉邦的地方品种籼稻阿加尼已知携带抗稻瘿蚊基因Gm8，具有过敏性坏死反应（HR）型抗性。该基因已被精细定位在0.43兆碱基区域内，侧翼标记为RM22685和RM22709。我们通过在参考日本晴水稻基因组7.5至9.5兆碱基区域进行电子分析，鉴定出63个可能的候选基因。其中一个靶向富含脯氨酸蛋白（PRP）基因（LOC_Os08g15080）的标记在亲本之间表现出多态性，并且在426个F重组自交系群体中也与该性状完全共分离。通过对对比亲本和近等基因系（Pre-NILs）进行逆转录聚合酶链反应（RT-PCR）对该基因进行功能验证，结果表明这是一个早期响应基因，在稻瘿蚊侵染后24小时迅速诱导表达，随后在后期表达水平下降。在五个携带不同抗性基因的抗稻瘿蚊水稻品种中对该基因进行验证，结果表明这种诱导作用是携带Gm8基因的阿加尼水稻所特有的。此外，对该基因的等位基因进行克隆和测序，包括来自感虫亲本TN1和阿加尼（抗性亲本）的启动子区域，结果显示与阿加尼相比，TN1中有153个核苷酸替换、4个氨基酸替换以及在假定的顺式作用元件处有3个突变。此外，我们还开发了一个功能标记（PRP-del）用于检测该基因，以用于Gm8的标记辅助导入。
