Zhou Zheng, Sun Liang, Hu Wenbin, Zhou Bin, Tao Shuhua, Zhang Shihui, LüYanmei, Zhao Zhenghong, Chen Caiyan

Letter

Breeding High-Grain Quality and Blast Resistant Rice Variety Using Combination of Traditional Breeding and Marker-Assisted Selection

Zhou Zheng1, 2, 3, Sun Liang1, Hu Wenbin2, Zhou Bin2, Tao Shuhua2, Zhang Shihui2, LüYanmei2, Zhao Zhenghong2, Chen Caiyan1

(Key Laboratory of Agro-Ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha 410125, China; )

We reported a novel rice variety developed by integrating the advantages of high eating quality from Xiangwanxian 17 (XWX17) and rice blast resistance from Gumei 4 (GM4) using a combination of traditional breeding and marker-assisted selection (MAS). Using molecular markers for QTLs/genes responsible for amylose content (AC), fragrance and blast resistance, three inbred lines were selected. Phenotypic assessments and allelic identification showed that the three lines performed well in terms of quality and blast resistance as a consequence of harboring the functional allelesWx,and. Notably, one of these lines, named Nongxiang 42, was recommended as a high-quality variety with high yield.

Rice (L.), as an important staple cereal, feeds over 60% of the population in China and nearly half of the people in the world (Zeng et al, 2017). Development of rice varieties with high quality, especially exceptional eating and cooking quality (ECQ) and fragrance, is becoming more and more important in meeting people’s aspiration for higher daily rice consumption and escalating the commercial value of a rice variety (Tian et al, 2009; Tilman et al, 2011; He and Park, 2015). At present, despite that a number of high-quality rice varieties have been developed, few of them are competitive in terms of disease resistance (Suwannaporn and Linnemann, 2008). Thus, it has been the first priority of geneticists and breeders to simultaneously improve quality traits and disease resistance in breeding programs (Khush et al, 2005; Zhang, 2007; Jin et al, 2009). Evidences in functional genomics studies have demonstrated a wealth of allelic variations in QTLs/genes associated with quality and disease resistance traits for use in rice breeding (Cheng et al, 2007; Zeng et al, 2017; Yu et al, 2020). Hence, integrating the elite alleles of high ECQ, fragrance and blast resistance from different rice varieties to develop superior variety with high-quality (better ECQ and fragrance) and disease resistance (rice blast resistance) can be realized in the present breeding practice. Here, we reported a case of breeding a novel fragrant rice variety, Nongxiang 42, harboring high-quality in ECQ and broad-spectrum rice blast resistance by integrating the advantages of high-eating quality and fragrance from XWX17 and rice blast resistance from GM4 using a combination of traditional breeding and MAS.

For high-quality rice breeding programs, two important quality characters in grains, AC and fragrance, are mainly concerned in breeding for superior quality. It has been acknowledged that an appropriate AC brings advantages to rice quality-related traits, such as ECQ and appearance (Luo et al, 2014; Hori et al, 2016; Lau et al, 2016; Zhang et al, 2019). Fragrance in rice can bring a premium price in the rice market due to its high-quality preference. Genetically, AC is largely associated with() gene on chromosome 6 (Isshiki et al, 1998; He et al, 2006; Chen Y L et al, 2020), which encodes granule-bound starch synthase I (GBSSI) and controls not only AC but also gel consistency (GC) in rice (Tian et al, 2009; Dobo et al, 2010; Yao et al, 2020). Most ofrice varieties harbor theWxallele with a high transcriptional level of, leading to higher AC in the grains, which produces non-sticky cooked rice.Wx, another allele in, is prevalent inrice varieties, and the low transcriptional level ofWxdetermines lower AC levels in the grains, which generates softer and stickier rice when cooked (Ayres et al, 1997; Wanchana et al, 2003; Zhang et al, 2019). Rice varieties with AC < 18% in the milled rice are preferred in most Asian countries, which results thatWxallele is preferred to be utilized in the breeding program in these areas. Grain fragrance in rice is mainly controlled by the() gene on chromosome 8 (Bradbury et al, 2005; He and Park, 2015). The functional allele ofis mainly present in non-fragrant rice varieties, while the loss-of-function allele (), a recessive allele, exists in fragrant rice varieties.allele results from an 8-bp deletion and three single nucleotide polymorphisms (SNPs) in exon 7 of(Bradbury et al, 2005; Shi et al, 2008; Shao et al, 2011). Hence, it is practicable to introduceinto elite rice varieties to generate fragrant rice varieties. Previously, XWX17 has been approved as a high ECQ rice with good AC and fragrance but has poor blast resistance, and GM4 is a non-fragrant landrace that produces grain with poor ECQ and high AC, but with broad-spectrum rice blast resistance due to theallele (Deng et al, 2017; Chen Q H et al, 2020). Hence, based on MAS, we attempted to breed high-quality inbred lines with broad-spectrum rice blast resistance depended on desired trait differences between XWX17 and GM4.

To investigate the functional allelic differences in the two varieties, the allelic diversities in,andbetween XWX17 and GM4 were investigated. Evidences in functional genomics studies have demonstrated a series of molecular markers for,and. Then, PCR primers PCR-AccI and ZJ58.7 were employed to genotype the allelic diversity inand, respectively, and we also improved the PCR marker for(He and Park, 2015; Liu A Q et al, 2006; Liu Q Q et al, 2006; Deng et al, 2006, 2017; Yu et al, 2013;Dai et al, 2018) (Table S1). XWX17 carries theWxallele of, which limits the transcription of(Dobo et al, 2010), whereas GM4 has theWxallele. According to the functional differences inWxandWx, XWX17 has a lower AC (14.8%) in the milled grain than GM4 (20.4%) (Table S2). Forgene, XWX17 carries theallele and GM4 has, which indicates XWX17 is very susceptible to rice blast and GM4 expresses broad-spectrum resistance to rice blast. In the Dawei-Mountain blast identification farmland, rice blast resistances of XWX17 and GM4, including seedling blast resistance and neck blast resistance, were investigated according to the standard evaluation system for rice (IRRI, 2002). Seedling blast and neck blast in XWX17 were rated as level 9 and level 7, respectively, and seedling blast and neck blast resistance in GM4 were estimated to be level 2 and level 3, respectively (Table S3). For allelic diversity in, comparative sequencing showed that XWX17 carries the 8-bp deletion and the other three SNPs in, indicating the presence of, while GM4 has the non-fragrant allele (Fig. S1-A). Thus, an improvedallele-specific marker of a triple-primer PCR system of co-dominant markers was developed to distinguish the allelic differences (China Patent Application No. 201811554512.7;Fig. S1-B and Table S1). Two forward primers were designed to compete for the 8-bp location. MF harbors the flanking sides of 8-bp deletion but without the deletion of PCR-product, MPlusF consists of 8-bp deletion and another 31-bp specific fragment (absent in rice genome) to broaden the PCR-product polymorphism in 3% agarose gel electrophoresis. Using triple-primer PCR system, the fragrant rice containing the 8-bp deletion ingenerates a 167-bp PCR product, and the non-fragrant rice generates a giving 206-bp fragment (Fig. S1-C). To confirm the availability of the triple-primer PCR system, a total of 30 rice accessions, consisting of 9 fragrant and 21 non-fragrant rice accessions, were investigated. The results showed that 9 accessions generated 206-bp PCR products, whereas 20 performed 167-bp products and 1 was heterozygous (Fig. S1-D and Table S4). All of the PCR products corresponded well, indicating that this marker was accurate and convenient for MAS of the fragrance allele of. Together, the functional markers ZJ58.7, PCR-AccI andMwere used to identify the allelic differences of the,andgenes.

To develop inbred rice lines with blast resistance and high- quality traits (fragrance and low AC), we made an intercross between XWX17 and GM4. Subsequently, multiple-backcrosses with XWX17 as the recurrent parent were performed to introgress the three favorable alleles,and. The breeding strategy is shown in Fig. S2. After the F1hybrids were generated, a total of 350 F2individuals were genotyped using the molecular markers developed for the desired alleles ofWx,andgenes, and 8 plants were screened as homozygous for the, Wxandalleles, and then were used as the paternal parents in next backcrosses with XWX17. After backcrossed to the recurrent parent XWX17 twice, theBC2F1plants, which were heterozygous atand homozygous at the other two loci, were planted to generate BC2F2descendants. Finally, 9 individuals out of 100 plants were confirmed to be homozygous for,Wxandsimultaneously. All the nine BC2F2individuals were planted and self-pollinated to generate the BC2F5generation to homogenize the genome.

All nine BC2F5lines were planted to determine their blast resistance ratings in the Dawei-Mountain blast identification farmland in Hunan Province, China, according to the standard evaluation system for rice (IRRI, 2002). Rice blast disease resulted from natural infection, and CO39 was planted as the susceptible control. XWX17 and GM4 were included as controls to confirm the improvement in rice blast resistance in the nine improved lines (Table S3). Compared to XWX17, seven of the nine BC2F5lines showed broad-spectrum blast resistance. In detail, seedling blast ratings ranged from level 1 to level 3, and neck blast ranged from level 2 to level 6. All these results showed that the rice blast resistance of the BC2F5lines was substantially improved. Depending on the plant architecture and yield potential that was selected by traditional breeding, three lines (CS42, CS53 and CS67) with improved blast resistance were chosen for further analysis (Fig. 1). The genotypes at,andwere verified using XWX17 and GM4 as references, and the results showed that all the three lines were homozygous forWx,andalleles (Fig. 1-A). Comparisons of the targeted traits, including blast resistance, AC and fragrance, were then carried out in the three improved lines and XWX17. Compared with GM4 that is immune to all of the testedisolates (IRRI, 2002), the improved line BC2F5-CS42 showed about 90.0% resistance to 18 of the 20 isolates, and was only susceptible to isolates 16-564 and 52. The improved inbred lines BC2F5-CS53 and BC2F5-CS67 were immune to 15 (75%) and 16 (80%) of the isolates, respectively. The recurrent parent XWX17 was susceptible to allisolates (Table S5). For fragrance, all the three inbred lines were identified as being fragrant, similar to XWX17, due to the presence of theallele. In addition, AC of the three inbred lines was < 18%, which is accepted as high ECQ. Compared to XWX17, these lines had slight increases in grain AC, but they were still grouped into the lower AC category (< 18%) (Fig. S3). The potential yield components were also investigated for all the three improved lines. Specially, based on the agronomic trait and broad- spectrum blast resistance performances (Fig. 1-B to -E; Fig. S3), BC2F5-CS42 was selected and nominated for variety certification as Nongxiang 42. The 13 of 48 markers showed polymorphisms between Nongxiang 42 and XWX17, suggesting that the genetic backgrounds differed by approximately 27.1% between the two lines (Fig. 1-F). As expected, Nongxiang 42 can be recognized as a novel variety, which possesses grain fragrance, broad-spetrum blast resistance and preferable ECQ and was approved as a high-quality variety for cultivation in Hunan Province, China during the rice variety regional trials in 2018 and 2019 (Table S6).

Fig. 1. Integrating marker-assisted selection with traditional breeding experience to breed novel fragrant rice variety, Nongxiang 42, with high eating and cooking quality and broad-spectrum rice blast resistance.

A, Genotyping three desired alleles in Xiangwanxian 17 (XWX17), Gumei 4 (GM4) and three developed inbred lines. B, Comparison of rice grain appearance between XWX17 and Nongxiang 42. C, Leaf blast disease resistance comparison in CO39, Nongxiang 42, XWX17 and GM4. D and E, Field performance of Nongxiang 42 and XWX17, respectively. F, Graphical genotyping of the genetic background of Nongxiang 42 using 48 simple sequence repeat markers. Chr, Chromosome.

It is worth mentioning that we drew on our experience in traditional rice breeding to accelerate the process in breeding practice. Nine inbred lines were selected by MAS, and three of these lines were selected based on our experience in traditional breeding on the agronomic trait performance. Among these, BC2F5-CS42, now registered as Nongxiang 42, was finally selected. After investigating the agronomic traits, panicle length, plant height, panicle number per plant, grain number per panicle, 1000-grain weight and days from sowing to heading of Nongxiang 42 were indeed significantly different from those of XWX17 (< 0.05; Fig S3). Although two backcrosses to XWX17 and five generations of self-pollination were performed based on the genomic single sequence repeat (SSR) markers (Sun et al, 2016), the genetic background of Nongxiang 42 still showed a 27.1% difference from XWX17 (13 out of 48 pair SSR markers). This result suggests that appropriately introducing genetic diversity in non-target loci between the varieties during breeding may give unexpected bonuses. The current outcomes from traditional breeding practices present a way to combine MAS and traditional breeding for the development of high- quality rice varieties (Ashkani et al, 2015). While MAS ensures the efficient incorporation of functional alleles with major effects, traditional breeding experience is still important to expand the genetic diversity with minor-effect QTLs. As a consequence, integrating MAS with the traditional breeding experience will facilitate rice variety improvement through breeding.

Taken together, after multiple backcrosses and traditional selection combined with MAS, the improved lines retained the desirable features of both parental lines, including low AC, fragrance and broad-spectrum rice blast resistance. It indicated that our breeding goal had been realized, and the developed molecular markers, Mand PCR-AccI, can be widely used in quality rice breeding programs in the future.

AcknowledgementS

This study was supported by the National Natural Science Foundation of China (Grant No. U19A2026) and the Major Science and Technology Project of Hunan Province, China (Grant No. 2018NK1020).

SUPPLEMENTAL DATA

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

File S1. Methods.

Fig. S1. Development of allele-specific marker.

Fig. S2. Breeding strategy for developing rice inbred lines with high grain quality, yield and blast resistance.

Fig. S3. Comparisons of agronomic traits in three inbred lines against the recurrent parent Xiangwanxian 17.

Table S1. Allelic associated markers employed in this study.

Table S2. Phenotypic and allelic differences for blast resistance, amylose content and grain fragrance.

Table S3. Seedling and neck blast disease ratings for nine inbred lines under natural disease inoculation conditions.

Table S4. Fragrance and Mgenotypes of 30 rice varieties.

Table S5. Seedling blast resistance for inbred lines BC2F5-CS42, BC2F5-CS53 and BC2F5-CS67 under artificial inoculation.

Table S6. Agronomic trait performance of Nongxiang 42 and its control (Tianyouhuazhan) in rice variety regional trials in 2018 and 2019.

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Chen Caiyan (cychen@isa.ac.cn); Zhao Zhenghong (1154885640@qq.com)

18 September 2020;

17 January 2021

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http://dx.doi.org/10.1016/j.rsci.2021.07.002