Strategy for Use of Rice Blast Resistance Genes in Rice Molecular Breeding

Improving rice salt tolerance by precision breeding in a new era.

In rice (Oryza sativa), amylose content (AC) is the major factor that determines eating and cooking quality (ECQ). The diversity in AC is largely attributed to natural allelic variation at the Waxy (Wx) locus. Here we identified a rare Wx allele, Wxmw , which combines a favorable AC, improved ECQ and grain transparency. Based on a phylogenetic analysis of Wx genomic sequences from 370 rice accessions, we speculated that Wxmw may have derived from recombination between two important natural Wx alleles, Wxin and Wxb . We validated the effects of Wxmw on rice grain quality using both transgenic lines and near-isogenic lines (NILs). When introgressed into the japonica Nipponbare (NIP) background, Wxmw resulted in a moderate AC that was intermediate between that of NILs carrying the Wxb allele and NILs with the Wxmp allele. Notably, mature grains of NILs fixed for Wxmw had an improved transparent endosperm relative to soft rice. Further, we introduced Wxmw into a high-yielding japonica cultivar via molecular marker-assisted selection: the introgressed lines exhibited clear improvements in ECQ and endosperm transparency. Our results suggest that Wxmw is a promising allele to improve grain quality, especially ECQ and grain transparency of high-yielding japonica cultivars, in rice breeding programs.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/jipb.13010

A rare Waxy allele coordinately improves rice eating and cooking quality and grain transparency

As more and more re-sequencing genome data in crops were released, SNPs (single nucleotide polymorphisms) are easily achieved for genotyping and show the highest abundance among all kinds of molecular markers. However, high-throughput SNP genotyping methods, such as KASP (Kompetitive Allele Specific PCR), TaqMan, or ARMS (amplification refractory mutation system), are always labor on DNA extraction and depending on expensive equipment. Therefore, most breeders are undergoing the bottleneck of lacking an easy, cost-saving, and stable genotyping system. Here, we report a direct PCR–based medium-throughput SNP marker–assisted selection (D-MAS) system suits available major crops including rice, wheat, maize, and rapeseed. The D-MAS system, which reduces the time of manual operation and result analysis dramatically, contains (1) seedling breeding in greenhouse; (2) high-throughput DNA extraction by alkaline lysis; and (3) gel-free SNP marker detection with 384-well by PARMS (penta-primer amplification refractory mutation) or KASP genotyping system. The stability of alkaline lysis DNA was validated by flexible dilution fold and long storing time under low temperatures. The alkaline lysis DNA from four F2 populations of rice, wheat, maize, and rapeseed showed equal efficiency in SNP calling relative to the ones from cetyl trimethylammonium bromide (CTAB) method. With the alkaline lysis DNA, PAMRS showed denser genotype cluster than KASP. Furthermore, the D-MAS system was adaptive with rice old leaves. The throughput and efficiency of the D-MAS system were validated in the hybrid rice seed purity test with a Xian-Geng-specific SNP marker which is also recommended to select ipa1-2d gene in the rice molecular breeding. In conclusion, we proposed a direct PCR–based SNP calling pipeline, which could be a simple, cheap, and robust standard operation procedure (SOP) of molecular breeding for different crops and get extensive use in most laboratory.

A direct PCR–based SNP marker–assisted selection system (D-MAS) for different crops

Molecular markers are efficient and essential genotyping tools for molecular breeding and genetic analysis of rice. We developed two 96-plex indica–japonica single nucleotide polymorphism (SNP) genotyping sets for genetic analysis and molecular breeding in rice using the Fluidigm platform. Informative SNPs between indica and japonica were selected from SNP data of the Rice Diversity database, HapRice world SNP data of the Q-TARO database, and our 40 rice cultivar resequencing dataset. SNPs in set 1 were evenly distributed across all 12 rice chromosomes at a spacing of 4–5 Mb between adjacent SNPs. SNPs in set 2 mapped to the long genetic intervals in set 1 and included 14 functional or linked SNPs in genes previously cloned and associated with agronomic traits. Additionally, we used the SNP sets developed in this study to perform genetic diversity analysis of various cultivated and wild rice accessions, construction and validation of a subspecies diagnostic subset, linkage map construction and quantitative trait locus (QTL) analysis of a japonica × indica F2 population, and background profiling during marker-assisted backcrossing. Furthermore, we identified subspecies-specific SNPs and discuss their distribution and association with agronomic traits and subspecies differentiation. Our results indicate that these subspecies-specific SNPs were present in wild rice prior to domestication. This genotyping system will serve as an efficient and quick tool for genetic analysis and molecular breeding in rice.

/pdf/development-and-application-of-indica-japonica-snp-assays-p138fb1lgv.pdf

Development and application of indica – japonica SNP assays using the Fluidigm platform for rice genetic analysis and molecular breeding

The development and utilization of genetic markers play a pivotal role in marker-assisted breeding of rice cultivars during pyramiding of valuable genes. Among molecular markers, SNPs have become the most promising due to their wide distribution within genomes and suitability for high -throughput automated genotyping. Although metadata of SNPs have been identified via next generation sequencing in rice, a large gap between the development of SNP markers and the application in breeding still exists. To promote the application of SNP markers based on the KASP (Kompetitive Allele-Specific PCR) method in rice breeding, a set of core SNP arrays was built via the screening of SNP databases and literature resources based on the KASP method. Five hundred and ninety six SNPs classified into eight subsets including quality control, indica-indica variation, highly polymorphic, functional genes, key genes targeting sites, gene cloned region, important trait associated and gap filling sites were chosen to design KASP primers and 565 out of them were successfully designed, and the assay design success rate was 94.8%. Finally, 467 out of the 565 successfully-designed SNPs can display diversity at the loci were used to develop a set of core SNP arrays. To evaluate the application value of the core SNP markers in rice breeding, 481 rice germplasms were genotyped with three functional KASP markers designed from the sequences of GBSSI, SSIIa, and Badh2 from the core SNP arrays for estimation of their grain quality performance. Eighteen rice lines, including Xiangwanxian 13, Basmati 370, Ruanhua A, and PR 33319–9–1-1-5-3-5-4-1, harbor all three favorable alleles. The core KASP arrays were also used for rice germplasm assessment, genetic diversity and population evaluation. Four hundred and eighty-one rice germplasms were divided into 3 groups: POP1, POP2 and POP3. POP1 and POP2 were indica rice subgroups consisting of 263 and 186 rice germplasms, respectively. POP3 was a japonica rice subgroup consisting of 32 rice germplasms. The average FST value for the three subgroups was 0.3501; the FST value of POP1 and POP3 was the largest (0.5482), while that of POP1 and POP2 was the smallest (0.0721). The results showed that the genetic distance between the japonica and indica rice subspecies was large, indicating that the core SNP markers were effective at discriminating the population structure of the germplasms. Finally, the core KASP arrays were used for association analysis with milled grain traits. A total of 31 KASP markers were significantly associated (P < 0.01) with ML and the LWR. Among the 31 markers, 13 were developed based on cloned genes or on identified loci related to yield traits. Notably, several KASP markers associated with grain quality were also found to be associated with brown planthopper resistance or green leafhopper resistance simultaneously. The core KASP arrays developed in our study were efficient and versatile for rice germplasm assessment, genetic diversity and population evaluation and are valuable for promoting SNP molecular breeding in rice. Our study demonstrated that useful assays combined with molecular breeding can be exploited for important economic trait improvement in rice breeding.

/pdf/development-of-a-core-snp-arrays-based-on-the-kasp-method-13snohb9ri.pdf

Development of a core SNP arrays based on the KASP method for molecular breeding of rice

There is a steady demand for high-quality rice varieties though consumers’ preference for high-quality rice varies from regions. The improvement of the quality of rice, especially its eating and cooking quality (ECQ), is an important breeding target as rice is mainly consumed in cooked form. The development and utilization of breeder-friendly and high-throughput marker systems play a pivotal role in marker-assisted breeding of rice cultivars, with pyramids of valuable genes affecting rice ECQ. In this study, we developed functional markers based on the Kompetitive Allele-Specific PCR (KASP) method for three principal genes, Wx, BADH2 and ALK, affecting ECQ in rice. The accuracy of all KASP markers was verified and confirmed with Sanger sequencing. A diverse rice panel consisting of 38 indica cultivars, 9 japonica cultivars and 1 Javanica cultivar were used to assess the validity of these markers for genotyping. The results showed that the functional KASP markers of Wx, ALK and BADH2 could effectively distinguish the different alleles. The genotyping results highly coincided with the phenotypic traits of rice eating and cooking quality. Eight rice entries harboring 3 favorable alleles were identified and superior rice restorers with improved ECQ were bred via functional marker genotyping. Breeders can develop rice cultivars that have desirable ECQ, customized to meet the market requirements in target regions. Hence, the development of these 3 applicable KASP functional markers based on allelic variation would be valuable for improving rice eating and cooking quality through maker-assisted selection to carter various consumer preferences especially in Asian areas.

Development and utilization of functional KASP markers to improve rice eating and cooking quality through MAS breeding

A novel QTL, qCIR9.1
 , that controls callus induction rate in anther culture was identified on chromosome 9 in rice, and based on RNA-seq data, Os09g0551600
 was the most promising candidate gene.
 Anther culture, a doubled haploid (DH) technique, has become an important technology in many plant-breeding programmes. Although anther culturability is the key factor in this technique, its genetic mechanisms in rice remain poorly understood. In this study, we mapped quantitative trait loci (QTLs) responsible for anther culturability by using 192 recombinant inbred lines (RILs) derived from YZX (Oryza sativa ssp. indica) × 02428 (Oryza sativa ssp. japonica) and a high-density bin map. A total of eight QTLs for anther culturability were detected in three environments. Among these QTLs, a novel major QTL for callus induction rate (CIR) named qCIR9.1 was repeatedly mapped to a ~ 100 kb genomic interval on chromosome 9 and explained 8.39–14.14% of the phenotypic variation. Additionally, RNA sequencing (RNA-seq) was performed for the parents (YZX and 02428), low- (L-Pool) and high-CIR RILs (H-Pool) after 16 and 26 days of culture. By using the RNA of the bulked RILs for background normalization, the number of differentially expressed genes (DEGs) both between the parents and between the bulked RILs after 26 days of culture was drastically reduced to only 78. Among these DEGs, only one gene, Os09g0551600, encoding a high-mobility group (HMG) protein, was located in the candidate region of qCIR9.1. qRT-PCR analysis of Os09g0551600 showed the same results as RNA-seq, and the expression of this gene was decreased in the low-callus-induction parent (YZX) and L-Pool. Our results provide a foundational step for further cloning of qCIR9.1 and will be very useful for improving anther culturability in rice.

Identification and candidate gene screening of qCIR9.1, a novel QTL associated with anther culturability in rice (Oryza sativa L.)

The urban agglomerations' rapid expansion and population growth have led to the fragmentation of landscape patterns and the degradation of ecosystems, seriously threatening regional ecological security. Ecological security pattern (ESP) is a spatial planning approach to effectively balance the development of urbanization and ecological protection. However, previous studies have ignored the difference in the importance of ecosystem services and the spatial compactness of ecological sources. The quantitative management objectives for maintaining the resilience of ESP are also rarely discussed. In this study, taking the Guangdong-Hong Kong-Macao Greater Bay Area (GBA) as an example, ecological sources were identified by simulating multiple ES weight assignment scenarios through GeoSOS area optimization. Ecological corridors and strategic points were extracted by Linkage Mapper. The robustness analysis based on complex network theory was performed to quantify the management objectives of ESPs. The results showed that ESPs include 26,130.61 km2 ecological sources (accounting for 46.6% of the area of GBA), 557 ecological corridors, and 112 ecological strategic points. In more detail, ecological sources are mainly distributed in the western and eastern mountainous areas, and ecological corridors primarily link peripheral edge areas of GBA in a circular radial shape. Compared with the current nature reserves, the identified ecological sources are more compact in landscape pattern. According to the robustness analysis, at least 23% of the important ecological sources should be strictly restricted from development activities to maintain the ESP's ability to resist ecological risks. This study also proposed corresponding differentiated ESPs management strategies. By optimizing the existing ESPs construction method and clarifying the ESPs management strategies, this study provides a completely scientific framework for the construction and management of ESPs in urban agglomerations. 

Integrating ecosystem services and complex network theory to construct and optimize ecological security patterns: a case study of Guangdong-Hong Kong-Macao Greater Bay Area, China

Yuting Huang

Papers

Development of a core SNP arrays based on the KASP method for molecular breeding of rice

Development and utilization of functional KASP markers to improve rice eating and cooking quality through MAS breeding

Identification and candidate gene screening of qCIR9.1, a novel QTL associated with anther culturability in rice (Oryza sativa L.)

Integrating ecosystem services and complex network theory to construct and optimize ecological security patterns: a case study of Guangdong-Hong Kong-Macao Greater Bay Area, China