A New PCR/LDR-Based Multiplex Functional Molecular Marker for Marker-Assisted Br

Letter

A New PCR/LDR-Based Multiplex Functional Molecular Marker for Marker-Assisted Breeding in Rice

Marker-assisted selection or marker-aided selection (MAS) provides an effective complementary approach for conventional rice breeding with precise and speedy mobilization of target genes into elite genetic backgrounds. The targeted genes, however, may not be selected in the course of MAS due to the occasional recombination between the marker and the target gene/QTL during the many cycles of meiosis involved in breeding programs. This leads to failure in the selection of target traits (Gopalakrishnan et al, 2008).

Insertion-deletions (InDels) and single nucleotide polymorphisms (SNPs) are common genetic polymorphisms in rice genome. Functional InDels and SNPs in genes directly give rise to the phenotypic variation, and such polymorphisms are valuable for the development of functional markers (FMs). The utilization of FMs can effectively circumvent the problem of selection of false positive for the target genes in MAS progress (Andersen and Lübberstedt, 2003).

Polymorphisms of InDels can be easily differentiated as co-dominant markers due to varying length of PCR products. However, it has been a long-term challenge of detecting SNPs with traditional molecular techniques (Mammadov et al, 2012). Several methods including allele-specific PCR (AS-PCR), cleaved amplified polymorphic sequences (CAPS), temperature-switch PCR (TSPCR) and gene re-sequencing, have been developed for discrimination of SNPs in the past few decades (Rasheed et al, 2016). Unfortunately, all these methods have limited potential in rice breeding programs due to their low throughput, laborious and time-consuming assays involved.

Recently, a new method known as Kompetitive allele- specific PCR (KASP) has been developed to discriminate SNP and InDel polymorphisms at specific sites (Yang G L et al, 2019). KASP markers have been successfully deployed to convert several functional SNP and InDel sites into FMs, which greatly increases the speed and efficiency of selection in crop breeding programs (Neelam et al, 2013; Pariasca-Tanaka et al, 2015; Rasheed et al, 2016; Steele et al, 2018). However, KASP assays often have limited multiplexing capabilities and require several assays to be run for the detection of multiple target genes in rice breeding programs, which involves multiple genes pyramiding into a desirable genetic background.

Thus far, over 50 SNP arrays and 15 different types of genotyping- by-sequencing (GBS) platforms have been developed in more than 25 crops and trees (Rasheed et al, 2017; Arbelaez et al, 2019). These high-throughput platforms are powerful tools for SNP discovery, genomic background selection and genome- wide association analysis. However, they may not be the ideal platforms for MAS, which needs quick data yield time, low per-sample cost and high reliability. MAS generally uses a set of specific markers of target functional polymorphisms to pyramid the underpinning economic traits into an elite variety (Bernardo et al, 2015).

Several high-throughput multiplex molecular marker assays have been developed for MAS in rice (Masouleh et al, 2009; Kim et al, 2016), wheat (Bérard et al, 2009; Bernardo et al, 2015), legume (Varshney et al, 2016) and other crops. However, the utilization of these assays in MAS is hampered due to the low efficiency of customization, high cost and costly equipment needed for application (Rasheed et al, 2016).

Polymerase chain reaction/ligase detection reaction (PCR/LDR) is a powerful technique that has been used in detecting single- base mutations (Khanna et al, 1999; Luo et al, 2010), and when coupled with universal array (PCR/LDR/UA), it is a powerful tool for sequencing polymorphism discrimination and detecting of low abundant DNA point mutations (Gerry et al, 1999) and SNPs (Qin et al, 2005) in genetically modified rice (Xu et al, 2006). In this study, we developed a multiplex FM based on PCR/LDR technology. To assess the reliability of this FM, we selected five functional allelic genes,NRT1.1B(Hu et al, 2015),(Ma et al, 2015; Shi and Gong, 2015),(Li et al, 2015),(Bradbury et al, 2005)and(Sato et al, 2002), as targeted polymorphism loci. This newly developed multiplex FM co-segregates with the all functional polymorphisms, and can simultaneously genotype multiple allelic genes in a single experiment. This multiplex FM platform is convenient, rapid, cost-effective and highly-efficient, and hence provides an effective approach for pyramiding of multiple favorable genes in rice improvement breeding.

To check the accuracy and reliability of the multiplex FM, we initially chose five rice varieties Fan 14, 9311, WYJCG14, Fan 24 and Nanjing 9108 to detect polymorphisms using the multiplex FM. The multiplex FM is designed to discriminate the different alleles ofNRT1.1B,COLD1,,andgenes. Each allelic gene is distinguished by differing lengths of LDR products, which are detected by a DNA sequencer. The chromatographs and genotypes of five rice varieties are shown in Fig. 1-A and Table S1. To confirm the results of the multiplex FM, we used Sanger sequencing to detect the functional SNP sites ofNRT1.1B,COLD1,andgenes, and a PCR-based functional InDel marker to detect the polymorphism sites of. The results were agreement with those of multiplex FM (Fig. S1).

To test whether the multiplex FM can be used to discriminatehomozygous and heterozygous lines, three F1hybrids, F1-1, F1-2and F1-3, generated from the crosses of Fan 14/9311, Fan 14/ WYJCG14and Nanjing 9108/Fan 24, respectively, were genotyped using the multiplex FM. Based on the genotypes of the parental lines, we speculated that F1-1should be heterozygous in the lociNRT1.1Band COLD1, F1-2should be heterozygous in the locus, and F1-3should be heterozygous in the lociand. As expected, 77 and 79 nt, 81 and 86 nt LDR products were simultaneously detected in the line F1-1, indicating the presence of heterozygosity of theNRT1.1BandCOLD1loci in the line F1-1. Similarly, the results showed that thelocus was heterozygous in the line F1-2, whereas theandloci were heterozygous in the line F1-3(Fig. 1-B and Table S1). These results implied that the multiplex FM developed in this study is robust enough to differentiate allele positive, negative and heterozygotes for each gene locus, thereby confirming the co-dominant inheritance pattern of this multiple FM.

Three F2segregation populations, F2-1, F2-2and F2-3, derived from crosses of Fan 14/9311, Fan 14/WYJCG14and Nanjing 9108/ Fan 24, respectively, were screened with the new multiplex FM. The parents of F2-1, F1-1, is heterozygous inNRT1.1BandCOLD1, while the other three target sites were negative homozygous (Fig. 1-B and Table S1). The genotyping analysis of the F2-1population revealed that, among the 48 individuals, 12 had positive alleles, 7 had negative alleles, and 29 were heterozygous in theNRT1.1Blocus; while 10 had positive alleles, 16 had negative alleles, and 22 were heterozygous in theCOLD1locus (Tables S2 and S3, Fig. S2-A and -C). Like their parental line of F1-1, all the individuals of F2-1showed negative alleles in the,andloci (Tables S1 and S3). In order to confirm the co-segregation of the newly developed multiple FM with the functional SNPs ofNRT1.1BandCOLD1, the same samples were screened with the functional AS-PCR marker ofNRT1.1B(Tian et al, 2016) and functional dCAPS marker ofCOLD1(Yang J et al, 2019). These genotyping results are concordant with those from our multiplex FM (Table S3 and Fig. S2), suggesting the multiplex FM co-segregates with the functional SNPs ofNRT1.1BandCOLD1.

Fig. 1. Genotype analysis of five elite rice varieties using multiplex functional marker (FM).

A, Genotype analysis of five elite rice varieties using the multiplex FM. Ligase detection reaction (LDR) products differing in length are generated based on variant lengths of probes (Table S4). ForNRT1.1B(+/-), the LDR products are 77 nt (+) and 79 nt (-). ForCOLD1(+/-), the LDR products are 86 nt (+) and 81 nt (-). For(+/-), the LDR products are 91 nt (+) and 89 nt (-). For(+/-), the LDR products are 93 nt (+) and 95 nt (-). For(+/-), the LDR products are 97 nt (+) and 99 nt (-).B, Genotype analysis of three independent F1lines using the multiplex FM.F1-1is heterozygous inNRT1.1BandCOLD1loci, F1-2is heterozygous inlocus, and F1-3is heterozygous inandloci.

Genotyping of 24 individuals of F2-2population using the multiplex FM revealed that, 5 had positive alleles, 6 had negative alleles, and 13 were heterozygous in thelocus (Tables S2 and S3), and all individuals had positive alleles inCOLD1and negative alleles inNRT1.1B,andas their parental line F1-2(Tables S1 and S3). Sequencing thegene in the F2-2population gained consistent results (Table S3 and Fig. S3), suggesting the co-segregation of the multiplex FM with the functional SNPs of

In the 66 individuals of F2-3, 12 had positive alleles, 19 had negative alleles and 35 were heterozygous in thelocus; while 18 had positive alleles,18 had negative alleles and 30 were heterozygous in thelocus (Tables S2 and S3), whereas all the individuals had positive alleles inCOLD1and negative alleles inNRT1.1Bandas their parental line F1-3(Tables S1 and S3). To confirm the co-segregation of multiple FM with the functional polymorphisms ofandloci, the F2-3samples were screened with the reported functional InDel marker of(Cheng et al, 2018) and CAPS marker(Wang et al, 2009), and the results perfectly matched with those of the multiple FM (Table S3, Fig. S2-B and -D).

In marker-assisted breeding, MAS should usually be performed before flowering, to allow breeders have sufficient time to make crosses or backcrosses between different gene resources for gene pyramiding. Therefore, the development of practical, high-efficiency and cost-effective multiplex FMs of allelic genes associated with important agronomic traits is very helpful in crop marker-assisted breeding (Kim et al, 2016).

Thermostable ligase has been cloned for use in the PCR/LDR assay to detect single base mutations (Barany, 1991). The PCR/LDR assay procedure involves a primary PCR amplification followed by LDR detection. For each bi-allele polymorphism site, LDR probes consist of three different species: one fluorescently labeled, gene-specific probe and two allele-specific probes with different lengths, so that the LDR products will migrate to different positions in the DNA sequence analysis. PCR/LDR assay is ideal for multiplexing, since several probe sets can anneal to PCR products without interference faced in polymerase-based assays (Khanna et al, 1999). Furthermore, PCR/LDR assay is known as an accurate reliability assay for polymorphism genotyping due to the high thermostability and fidelity of the DNA ligase (Luo et al, 2010). This can effectively reduce false positives and it is procedurally simple, as it eliminates the need to purify PCR and LDR products for polymorphisms analysis.

The PCR/LDR assay has been successfully deployed to detect SNPs in many genes and varied species including(Khanna et al, 1999),,and(Favis and Barany, 2000), human mitochondrial DNA (Luo et al, 2010) and human allele polymorphisms (Belgrader et al, 1996). It is recognized as a simple, high-reliability and cost-effective assay for the detection of DNA sequence variations, and has enormous potential to be applied in development multiple FMs of SNPs and/or InDels underpinning important agronomic traits in MAS of crop breeding. In this study, we developed a 5-plex FM from four SNP and one InDel polymorphism sites that represent five genes of agronomic importance in rice, and the FAM (carboxyfluorescein) labeled LDR resultant products were analyzed using an ABI 3730 DNA sequencer (Applied Biosystems, USA). The newly developed FM can successfully discriminate the positive, negative and heterozygous alleles of the five selected polymorphism sites (Fig. 1 and Table S1). Validations using three independent F2populations prove that the multiple FM is in co-segregation with all target allelic genes (Table S3, Figs. S2 and S3). Our results confirmed that the PCR/LDR-based assay is suitable for development of molecular markers for both SNP and InDel loci. More than two alleles may exist in some gene loci, e.g.COLD1(Ma et al, 2015),(Kovach et al, 2009) and(Zhang et al, 2019). Allele specific PCR/LDR-based molecular markers can be developed using distinct MAS breeding programs with alternative alleles. This procedure can also be applied to design markers for other polymorphism sites associated with other agronomic traits, and hence is a very promising assay in the development of multiple FMs for crop improvement breeding program.

The utilization of high-efficient, reliable and cost-effective molecular markers plays a crucial role in MAS breeding during pyramiding of several valuable genes. SNPs and InDels have become the most promising polymorphism loci owing to their wide distribution within genomes. A massive number of unique SNPs and InDels have been identified via next generation sequencing in rice, and the gap between the identification of SNP and InDel markers and the application in breeding is filled by KASP. KASP is a cost-effective single-step method, cheaper than conventional PCR and gel electrophoresis based molecular markers (Rasheed et al, 2016; Yang G L et al, 2019). However, the disadvantage of the KASP approach is disable multiplexing. As a result, five assays have to be used to detect the genotypes of five loci using KASP, whereas only one assay is needed using the PCR/LDR method. Therefore, the cost of PCR/LDR method is about half as much as KASP. We hold the opinion thatPCR/LDR is a more high-efficient and cost-effective method in pyramiding several useable genes in MAS breeding, even though it requires performing both PCR and LDR.

The analysis of PCR/LDR-based multiplex FM is performed using the ABI DNA sequencer, which is a popular equipment used for analysis such as microsatellites, SNP analysis, mutation detection and Sanger sequencing of DNA fragment. ABI 3730 and 3730XL DNA Sequencer can analyze 48 and 96 samples in parallel in 2 h, providing the capacity for analyzing hundreds of samples per day. DNA fragment size differences of as small as two nucleotides can be detected accurately using the DNA sequencer. The use of PCR/LDR-based multiplex FMs will provide greater convenience and costs savings for breeders in MAS breeding.

ACKNOWLEDGEMENTs

This research was supported by the National Key Research and Development Project of Ministry of Science and Technology, China (Grant No. 2016YFD0101106), Program of Shanghai Technology Research Leader (Grant No. 18XD1424300) and Agriculture Research System of Shanghai, China (Grant No. 201903). We acknowledge Dr. Hong Jinghan for valuable critiques and comments on the manuscript.

Supplemental DatA

The following materials are available in the online version of this article at http://www.sciencedirect.com/science/journal/ 16726308; http://www.ricescience.org.

File S1. Methods.

Fig. S1. Validations of multiplex functional markers.

Fig. S2. Validations of genotypes ofNRT1.1B(A),(B),COLD1(C) and(D)in F2segregation populations.

Fig. S3. Sanger sequencing ofgene in F2-2segregation population.

Table S1. Genotypes of parental and F1lines analyzed using multiple functional markers.

Table S2. Segregations of F2populations analyzed using multiple functional markers.

Table S3. Genotyping results of F2population using multiplex functional markers.

Table S4. LDR probes and length of LDR products.

Table S5. Primer sequences and PCR products size of target genes.

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ChuHuangwei, Tu Rongjian, Niu Fuan, Zhou Jihua, Sun Bin, Luo Zhongyong, Cheng Can, Cao Liming

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http://dx.doi.org/10.1016/j.rsci.2020.11.002

s:Cao Liming (caoliming@saas.sh.cn);

Cheng Can (chengcan@saas.sh.cn)

20 January 2020;

20 May 2020