Basic concepts and methodologies of DNA marker systems in plant molecular breeding.

Brassica napus provides approximately 13–16% of global vegetable oil for human consumption and biodiesel production. Plant height (PH) is a key trait that affects plant architecture, seed yield and harvest index. However, the genetic mechanism of PH in B. napus is poorly understood. A dwarf mutant df59 was isolated from a large-scale screening of an ethyl methanesulphonate-mutagenized rapeseed variety Ningyou 18. A genetic analysis showed that the dwarfism phenotype was controlled by one semi-dominant gene, which was mapped on C9 chromosome by quantitative trait loci sequencing analysis and designated as BnaDwf.C9. To fine-map BnaDwf.C9, two F2 populations were constructed from crosses between conventional rapeseed cultivars (Zhongshuang 11 and Holly) and df59. BnaDwf.C9 was fine-mapped to the region between single-nucleotide polymorphism (SNP) markers M14 and M4, corresponding to a 120.87-kb interval of the B. napus ‘Darmor-bzh’ genome. Within this interval, seven, eight and nine annotated or predicted genes were identified in “Darmor-bzh”, “Ningyou 7” and “Zhongshuang 11” reference genomes, respectively. In addition, a comparative transcriptome analysis was performed using stem tips from Ningyou 18 and df59 at the stem elongation stage. In total, 3995 differentially expressed genes (DEGs) were identified. Among them, 118 DEGs were clustered in plant hormone-related signal transduction pathways, including 81 DEGs were enriched in auxin signal transduction. Combining the results of fine-mapping and transcriptome analyses, BnaC09g20450D was considered a candidate gene for BnaDwf.C9, which contains a SNP that co-segregated in 4746 individuals. Finally, a PCR-based marker was developed based on the SNP in BnaC09g20450D. The combination of quantitative trait loci sequencing, fine-mapping and genome-wide transcriptomic analysis revealed one candidate gene located within the confidence interval of 120.87-kb region. This study provides a new genetic resource for semi-dwarf breeding and new insights into understanding the genetic architecture of PH in B. napus.

/pdf/fine-mapping-and-transcriptome-analysis-of-a-candidate-gene-5btnfm9d6t.pdf

Fine-mapping and transcriptome analysis of a candidate gene controlling plant height in Brassica napus L.

Cloning quantitative trait locus (QTL) is time consuming and laborious, which hinders the understanding of natural variation and genetic diversity. Here, we introduce RapMap, a method for rapid multi-QTL mapping by employing F2 gradient populations (F2GPs) constructed by minor-phenotypic-difference accessions. The co-segregation standard of the single-locus genetic models ensures simultaneous integration of a three-in-one framework in RapMap i.e. detecting a real QTL, confirming its effect, and obtaining its near-isogenic line-like line (NIL-LL). We demonstrate the feasibility of RapMap by cloning eight rice grain-size genes using 15 F2GPs in three years. These genes explain a total of 75% of grain shape variation. Allele frequency analysis of these genes using a large germplasm collection reveals directional selection of the slender and long grains in indica rice domestication. In addition, major grain-size genes have been strongly selected during rice domestication. We think application of RapMap in crops will accelerate gene discovery and genomic breeding. Cloning quantitative trait loci (QTL) in crops is often slow and laborious. Here, the authors describe RapMap, a method to rapidly clone multiple QTL based on F2 gradient populations coupled with a co-segregation standard, and show how it can be used to identify genes controlling grain size in rice.

/pdf/the-identification-of-grain-size-genes-by-rapmap-reveals-21cq8mrodx.pdf

The identification of grain size genes by RapMap reveals directional selection during rice domestication.

Stripe rust (yellow rust) is a significant disease for bread wheat (Triticum aestivum L.) worldwide. A genome-wide association study was conducted on 240 Chinese wheat cultivars and elite lines genotyped with the wheat 90 K single nucleotide polymorphism (SNP) arrays to decipher the genetic architecture of stripe rust resistance in Chinese germplasm. Stripe rust resistance was evaluated at the adult plant stage in Pixian and Xindu in Sichuan province in the 2015–2016 cropping season, and in Wuhan in Hubei province in the 2013–2014, 2016–2017 and 2018–2019 cropping seasons. Twelve stable loci for stripe rust resistance were identified by GWAS using TASSEL and GAPIT software. These loci were distributed on chromosomes 1B, 1D, 2A, 2B, 3A, 3B, 4B (3), 4D, 6D, and 7B and explained 3.6 to 10.3% of the phenotypic variation. Six of the loci corresponded with previously reported genes/QTLs, including Sr2/Yr30/Lr27, while the other six (QYr.hbaas-1BS, QYr.hbaas-2BL, QYr.hbaas-3AL, QYr.hbaas-4BL.3, QYr.hbaas-4DL, and QYr.hbaas-6DS) are probably novel. The results suggest high genetic diversity for stripe rust resistance in this population. The resistance alleles of QYr.hbaas-2AS, QYr.hbaas-3BS, QYr.hbaas-4DL, and QYr.hbaas-7BL were rare in the present panel, indicating their potential use in breeding for stripe rust resistance in China. Eleven penta-primer amplification refractory mutation system (PARMS) markers were developed from SNPs significantly associated with seven mapped QTLs. Twenty-seven genes were predicted for mapped QTLs. Six of them were considered as candidates for their high relative expression levels post-inoculation. The resistant germplasm, mapped QTLs, and PARMS markers developed in this study are resources for enhancing stripe rust resistance in wheat breeding.

/pdf/genome-wide-association-analysis-of-stripe-rust-resistance-3arwncrnn5.pdf

Genome-wide association analysis of stripe rust resistance in modern Chinese wheat

The ideal plant architecture is a new strategy for super high yield breeding of rice. Tiller angle is an important plant architecture character of rice. A reasonable tiller angle is a key factor for the ideal plant architecture and achieving high-yield breeding. Molecular design breeding is the most potential new direction of crop breeding in the future. The development of accurate and efficient functional molecular markers of target trait genes is crucial for molecular design breeding. The TAC1 (Tiller Angle Controlling) gene is the primary gene that regulates tiller angle in rice. This gene can be used to improve the compact plant architecture of indica and japonica rice varieties. The SNP variation from A to G at the fourth intron 3′ splicing point in TAC1 changes plant architecture. Based on the SNP variation, PM-TAC1 was successfully developed as a fluorescent functional molecular marker, via the penta-primer amplification refractory mutation system. Ninety-three rice materials were genotyped using this marker, and the marker was effectively used in rice plant architecture breeding. The successful development of this marker will contribute to the molecular breeding of rice plant architecture.

/pdf/development-of-the-parms-marker-of-the-tac1-gene-and-its-28hdddbi2o.pdf

Development of the PARMS marker of the TAC1 gene and its utilization in rice plant architecture breeding

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.

Chao Mao

Papers

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