Development and validation of SNP-based functional codominant markers for two major disease resistance genes in rice (O. sativa L.)

Abstract

Blast and bacterial leaf blight are major diseases of rice that limit grain yield significantly. These two devastating biotic stresses have to be controlled to meet the demand for 23 % more rice production by 2035 to feed the increasing number of rice consumers. Incorporating appropriate disease resistance genes into elite varieties is considered as the best method to enhance crop resistance. Molecular markers play an important role in multiple gene pyramiding programs to select desirable genotypes with targeted genes. Two major resistance genes, Pita and xa5, for blast and bacterial leaf blight races, respectively, have been used in many gene pyramiding programs. However, simple PCR-based functional codominant markers have not been reported for these genes. Hence, in the present study, time-and costeffective codominant markers for Pita and xa5 have been developed and validated in segregating populations. High-throughput screening has been demonstrated using parallel capillary electrophoresis to replace laborious gel-based electrophoresis. Additionally, the presence of Pita and xa5 alleles was evaluated with 260 diverse rice varieties that were collected from different parts of the world. Of the 260 cultivars tested, 55 were identified with the Pita resistance allele while all the tested cultivars had the susceptible Xa5 allele. The identified Pita allele-derived cultivars can be used as an alternative resistance source for blast disease. The newly developed Pita and xa5 functional markers will help toward tracking the two target genes for blast and bacterial leaf blight resistance in breeding programs.

Allele-specific marker-based assessment revealed that the rice blast resistance genes Pi2 and Pi9 have not been widely deployed in Chinese indica rice cultivars

“…However, due to the relatively low density of SSR markers, most of these SSR markers did not completely co-segregate with the mapped blast R -genes, which can lead to false positive selections. SNP and InDel polymorphisms are very abundant in the rice genome, providing potential targets for designing allele-specific markers directly targeting distinct blast- R gene alleles (Costanzo and Jia 2010 ; Hayashi et al 2010 ; Ramkumar et al 2011 ; Hua et al 2015 ; Ramkumar et al 2015 ). Because of their co-segregation with target blast R -genes (Ramkumar et al 2011 ; Ramkumar et al 2015 ), allele-specific markers are more effective than the random genomic SSR marker, allowing for selection of target blast R -genes with less labor and cost in MAS programs.…”

“…However, owing to genetic recombination, the genetically linked markers may give rise to false positives (Frisch et al 1999 ). With the progress in cloning of blast R -genes and the availability of sequences of more functional R -genes, functional allele-specific markers have been developed for several blast R -genes, such as Pi-km (Costanzo and Jia 2010 ), Pit (Hayashi et al 2010 ), Pi54 (Ramkumar et al 2011 ), Pi2 / Piz-t / Pi9 (Hua et al 2015 ), and Pita (Jia et al 2002 ; Ramkumar et al 2015 ) by using the strategy of allele mining. These allele-specific markers provide more efficient selection of desired genotypes compared to DNA markers at nearby, but functionally irrelevant site (Andersen and Lubberstedt 2003 ).…”

Pyramiding of Pi46 and Pita to improve blast resistance and to evaluate the resistance effect of the two R genes

“…Because the gene provides broad-spectrum resistance against different blast isolates, it is used to control blast disease worldwide. Moreover, it is one of the most common genes used in blast gene pyramiding programs and allele mining studies (Hittalmani et al 2000;Jia et al 2003;Ramkumar et al 2015). In this study, it was introgressed into HH179 using MABB.…”

“…Thus far, more than 90 blast R genes that confer resistance to different races of M. oryzae have been reported. However, only a few of these genes have been cloned (Huang et al 2011;Wang et al 2012;Koide et al 2013;Xu et al 2014;Ramkumar et al 2015). Molecular markers that are tightly linked to these R genes have been developed and are widely used in marker-assisted selection (MAS) for transferring R genes in rice breeding programs and for improving resistance to blast disease.…”

Development and validation of a PCR-based functional marker system for identifying the low amylose content-associated gene <i>Wx</i><i>^hp</i> in rice

“…Tetra-primer ARMS-PCR is a novel method for detecting single nucleotide polymorphisms (SNPs) and was widely used in rice molecular breeding (Chen et al 2013, Ramkumar et al 2015. Because Taq DNA polymerase lacks of 3′→5′ exonuclease activity, allele-specific primers containing a mismatch at their 3′-terminus can only be extended at low speed, and the reaction stops when a large number of mismatched bases are present and thus the formation of 3′-terminus nucleotide phosphodiester bonds is less likely to occur.…”