Map-based cloning and validation of a gall midge resistance gene, Gm8, encoding a proline-rich protein in the rice...

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 F10 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.