Recombinase polymerase amplification (RPA) has been emerged as an alternative to PCR due to its high specificity and sensitivity in diagnostics of animal and plant pathogens.In this study,an RPA protocol was developed and validated for detection of aromagene in rice.We considered the functional nucleotide polymorphism (FNP) with 8 bp deletion and three single nucleotide polymorphisms (SNPs) in exon 7 of the geneBetaine Aldehyde Dehydrogenase 2(BADH2) on chromosome 8,the key factor of fragrance in rice.Out of four designed markers,one dominant marker (Aro-RPA F1/R1) in coupling phase was identified by the RPA assay for successful differentiation between aromatic and non-aromatic rice varieties.The developed assay was highly specific and sensitive in detecting the functional mutation both from diverse aromatic rice germplasm as well as from the hybrid plants of an aromatic and non-aromatic cross (Pusa Basmati 1 × Sahbhagi Dhan).Additionally,the RPA assay was also able to detect the 8 bp-FNP from the crude leaf extracts at a dilution up to 10-7.The time and cost involved in performing RPA were considerably lower than the conventional PCR based method.RPA can be easily done within 1 h with a minimum laboratory set up.Therefore,the developed assay can be utilized for a rapid screening of rice germplasm for the aroma gene to select parents in aromatic rice breeding programme and rice varieties by the seed industries.

Aroma in rice is controlled by a recessive geneBADH2on chromosome 8 (Lorieux et al,1996;Bradbury et al,2005a;Chen et al,2006).The FNP is well characterized by three SNPs and an 8-bp deletion in the 7th exon ofBADH2resulting in a premature stop codon and partially truncated BADH2 protein (Bradbury et al,2005a;Amarawathi et al,2008;Bourgis et al,2008).The 8 bp FNP is the key factor in most aromatic varieties,while other functional mutations are rare and limited to small sets of varieties (Bourgis et al,2008;Fitzgerald et al,2008;Kovach et al,2009).Sequence-specific markers to detect FNPs within theBADH2gene have been developed to facilitate marker-assisted breeding (Bradbury et al,2005b;Amarawathi et al,2008;Shi et al,2008;Myint et al,2012).However,such marker-assisted selections rely on detection of the FNP using PCR,and thus require considerable laboratory resources which are still limitations in developing countries.Isothermal DNA amplification strategies have been extensively used as promising alternative to PCR facilitating nucleic acid replication at constant temperatures (Li et al,2018).Among these isothermal techniques,RPA is remarkable due to its simplicity,efficiency,high sensitivity and selectivity,and compatibility with multiplexing (Piepenburg et al,2006).The fundamental reaction mechanism of RPA relies on a synthetically engineered adaptation of a natural cellular process called homologous recombination,a key process in DNA metabolism.The RPA relies on three key proteins (recombinase,recombinase loading factor and single-stranded binding protein),which subsequently coordinate with ancillary components such as DNA polymerase,crowding agents (a high molecular polyethylene glycol),energy components (ATP) and salt molecules to perform the RPA reaction (Piepenburg et al,2006).

The present study reported the development and evaluation of an RPA-based selection of aroma in rice.A panel of 27 rice genotypes,including 17 rice genotypes from the Basmati/Sadri group of 3 000 rice genome panel,8 aromatic varieties and 2 non-aromatic varieties,was initially categorized for their aroma traits based on phenotyping by KOH test and PCR analysis using aroma trait linked functional markers (Table S1).The PCR analysis of 27 rice genotypes with allele specific amplification(ASA) markers (Bradbury et al,2005b) revealed the presence of 8-bp deletion in the exon 7 ofBADH2in 15 rice genotypes (Fig.S1).All these genotypes were characterized as aromatic with an aroma score of ‘3’ or ‘2’ based on sensory evaluation of cut leaves and grains.A few of the moderately to strongly aromatic varieties such as Hamsraj,Kataribhog and Napnang Hangmei were null for the 8-bp FNP (Table S1 and Fig.S1).The aromatic variety Pusa Basmati 1 having 8-bp FNP showed aroma score of‘3’,while the non-aromatic variety Sahbhagi Dhan recorded aroma score of ‘1’ with null allele (Table S1 and Fig.S1).These two genotypes were further used as the aromatic and nonaromatic checks for standardization of RPA.Initially,four pairs of primers were designed from theBADH2exon 7 spanning the 8-bp FNP (Table S2 and Fig.S2).In the PCR evaluation,the primer pair Aro-RPA F1/R1 (Fig.1-A) successfully differentiated aromatic and non-aromatic genotypes by producing the specific amplification of desired size (197 bp)only in aromatic genotype (Fig.S3-A).The remaining three sets of primers (Aro-RPA F1/R2,Aro-RPA F2/R3 and Aro-RPA F3/R3) failed to differentiate aromatic and non-aromatic checks(Fig.S3-A).Further,primer pair Aro-RPA F1/R1 amplified desired amplicon of 197 bp in all the 15 aromatic rice genotypes carrying the 8-bp FNP (Fig.S3-B).

Fig.1.Development and validation of recombinase polymerase amplification (RPA) assay for detection of aroma gene in rice.

To determine the effectiveness of selected primer pair Aro-RPA F1/R1 in RPA,standard procedure was followed using TwistAmp?Basic Kit (TwistDx,Cambridge,UK) by taking purified DNA and crude leaf extract of aromatic and non-aromatic checks.Desired amplicon of 197 bp was observed only in Pusa Basmati 1 with both genomic DNA and crude leaf extract after incubation of the reaction product at 65 °C for 10 min (Fig.1-B).However,a smear-like pattern was observed when the incubation step was not included.Further,the sensitivity of RPA in comparison to PCR was assessed using serial dilutions of genomic DNA (starting with initial concentration of 100 ng/μL) and crude leaf extract up to 10-7-fold of Pusa Basmati 1.The RPA showed higher sensitivity than the PCR in detecting the 8-bp FNP at different dilution levels.In case of PCR with genomic DNA as template,amplicons were visible only up to 10-3dilution,whereas PCR with crude leaf extract was unable to generate desired amplicon at all the dilution levels (Fig.1-C).However,the RPA could generate amplicons at all the dilutions of DNA as well as crude extract (Fig.1-C).The results clearly indicated a higher efficiency and sensitivity of RPA over PCR in detecting a plant gene.Earlier studies on RPA based detection of plant pathogens also reported a greater sensitivity of RPA over PCR (Londo?o et al,2016;Kapoor et al,2017;Lu et al,2021;Priti et al,2021).

Next,the RPA assay was applied in the whole germplasm set(Table S1).RPA using both genomic DNA and crude leaf extract showed desired amplifications of 197 bp (Fig.1-D) in all the 15 aromatic rice genotypes earlier identified as carrying the 8-bp FNP based on ASA marker.The RPA assay was also applied in detecting the 8-bp FNP in F1hybrid plants of Pusa Basmati 1 and Sahbhagi Dhan,where it resulted in similar pattern of amplification in both hybrids and homozygous aromatic parent (Fig.1-E).

Aromatic rice is a highly valued rice varietal group because of its pleasant and highly desirable aroma,and other grain quality(Roy et al,2020;Luo et al,2022).This group boasts the iconic basmati rice from India and Pakistan,Jasmine rice from Thailand,and the Sadri rice from Iran,in addition to many traditional cultivars.A number of sensory methods are available for rice breeders and seed industries to select desired aromatic varieties (Sood and Siddiq,1975;Reinke et al,1991;Hien et al,2006).However,evaluating a large sample using these objective estimations is cumbersome,time consuming and less reliable due to the fact that aroma is subjected to human perception.Hence,marker-assisted selection of the trait offers a rapid and reliable way of screening aroma in rice samples (Bradbury et al,2005b;Amarawathi et al,2008).In this study,the RPA assay using the newly designed dominant marker Aro-RPA F1/R1 successfully detected 8-bp FNP ofBADH2gene from both purified DNA and crude leaf extract of aromatic rice plants.This assay could detect the FNP in diverse aromatic genotypes as well as in the F1hybrid plants of an aromatic × non-aromatic cross.Although it could not differentiate the homo-and hetero-zygotes in the segregating generations,this assay can be useful for a rapid survey of large germplasm collections based on the presence of 8-bp FNP for selecting desired lines.The time and cost involved to perform RPA was considerably lower than the conventional PCR.RPA amplifications can be completed within 1 h with a minimum laboratory set up.Although the reagents required for RPA are costlier than PCR reagents,the overall cost of the assay is greatly reduced considering that PCR requires prior purification of genomic DNA and availability of thermal cyclers.RPA also has several advantages over other available isothermal techniques.It has been successfully utilized in diagnostics of animal and plant pathogens (Li et al,2018;Ghosh et al,2020;Lu et al,2021).

In this study,we successfully demonstrated the detection of key factor of aroma in rice through RPA assay using both purified DNA and crude leaf extract of aromatic rice plants.The RPA assay was more sensitive than PCR based detection both in case of purified DNA and crude leaf extract.RPA can detect a plant gene up to 10-7dilution of both purified DNA and leaf extract.Therefore,the developed assay will be highly useful for a rapid and sensitive selection of aromatic lines under resource-limited setup.This study offers scope for developing lateral flow strip-based RPA assay for rapid on-site detection of aroma gene by rice breeders and seed industry directly from crude extracts.The outcome of the study can also encourage the development of RPA-based detection protocol for other important plant genes,such as disease resistance genes for selection of donors,and transgenes as a part of biosafety measures during exchange of plant materials.

ACKNOWLEDGEMENTS

The authors thank Director,Indian Council of Agricultural Research,National Rice Research Institute (ICAR-NRRI),Cuttack,Odisha,India for providing necessary infrastructure and support.The study was conducted under the project‘Development of Resilient Production Technologies for Rice under Rainfed Drought-Prone Agro-Ecosystems’ of the ICARNRRI,Cuttack,India.

SUPPLEMENTAL DATA

The following materials are available in the online version of this article at http://www.sciencedirect.com/journal/rice-science;http://ricescience.org.

File S1.Methods.

Fig.S1.PCR survey of 27 rice germplasm using allele specific amplification for detecting 8 bp-FNP inBADH2gene.

Fig.S2.Location of recombinase polymerase amplification primers inBADH2gene sequences of aromatic cultivar Basmati 385 and non-aromatic cultivar Nipponbare.

Fig.S3.Validation of four sets of designed recombinase polymerase amplification primers.

Table S1.Rice genotypes used in this study and their aroma characteristics.

Table S2.Primers used in this study.