Identification of Single Nucleotide Polymorphism in TaSBEIII and Development of KASP Marker Associated With Grain Weight in Wheat

Manipulation of genes involved in starch synthesis could significantly affect wheat grain weight and yield. The starch-branching enzyme (SBE) catalyzes the formation of branch points by cleaving the α-1,4 linkage in polyglucans and reattaching the chain via an α-1,6 linkage. Three types of SBE isoforms (SBEI, SBEII, and SBEIII) exist in higher plants, with the number of SBE isoforms being species-specific. In this study, the coding sequence of the wheat TaSBEIII gene was amplified. After the multiple sequence alignment of TaSBEIII genome from 20 accessions in a wheat diversity panel, one SNP was observed in TaSBEIII-A, which formed the allelic marker allele-T. Based on this SNP at 294 bp (C/T), a KASP molecular marker was developed to distinguish allelic variation among the wheat genotypes for thousand grain weight (TGW). The results were validated using 262 accessions of mini core collection (MCC) from China, 153 from Pakistan, 53 from CIMMYT, and 17 diploid and 18 tetraploid genotypes. Association analysis between TaSBEIII-A allelic variation and agronomic traits found that TaSBEIII-A was associated with TGW in mini core collection of China (MCC). The accessions possessing Allele-T had higher TGW than those possessing Allele-C; thus, Allele-T was a favorable allelic variation. By analyzing the frequency of the favorable allelic variation Allele-T in MCC, it increased from pre-1950 (25%) to the 1960s (45%) and increased continuously from 1960 to 1990 (80%). The results suggested that the KASP markers can be utilized in grain weight improvement, which ultimately improves wheat yield by marker-assisted selection in wheat breeding. The favorable allelic variation allele-T should be valuable in enhancing grain yield by improving the source and sink simultaneously. Furthermore, the newly developed KASP marker validated in different genetic backgrounds could be integrated into a breeding kit for screening high TGW wheat.

Two User-Friendly Molecular Markers Developed for the Identification of Hybrid Lethality Genes in Brassica oleracea

Cabbage (Brassica oleracea) is an important vegetable crop that is cultivated worldwide. Previously, we reported the identification of two dominant complementary hybrid lethality (HL) genes in cabbage that could result in the death of hybrids. To avoid such losses in the breeding process, we attempted to develop molecular markers to identify HL lines. Among 54 previous mapping markers closely linked to BoHL1 or BoHL2, only six markers for BoHL2 were available in eight cabbage lines (two BoHL1 lines; three BoHL2 lines; three lines without BoHL); however, they were neither universal nor user-friendly in more inbred lines. To develop more accurate markers, these cabbage lines were resequenced at an ~20× depth to obtain more nucleotide variations in the mapping regions. Then, an InDel in BoHL1 and a single-nucleotide polymorphism (SNP) in BoHL2 were identified, and the corresponding InDel marker MBoHL1 and the competitive allele-specific PCR (KASP) marker KBoHL2 were developed and showed 100% accuracy in eight inbred lines. Moreover, we identified 138 cabbage lines using the two markers, among which one inbred line carried BoHL1 and 11 inbred lines carried BoHL2. All of the lethal line genotypes obtained with the two markers matched the phenotype. Two markers were highly reliable for the rapid identification of HL genes in cabbage.

Diagnostic KASP Markers of Wheat Broad-spectrum Powdery Mildew Resistance Genes Pm21, PmV and Pm12 Developed for High Throughput Marker-assisted Selection

Wheat powdery mildew is a devastating disease that seriously threatens yield worldwide. Utilization of host resistance is considered an effective strategy to minimize powdery mildew damage. Pm21, PmV, and Pm12 confer broad-spectrum resistance to wheat powdery mildew in China, of which Pm21 and PmV are allelic genes derived from the 6VS chromosome of Dasypyrum villosum, and Pm12 is derived from the 6SS chromosome of Aegilops speltoides and most likely orthologous to the former two genes. To accurately and efficiently transfer and pyramid these genes using marker-assisted selection (MAS), distinctive single nucleotide polymorphisms (SNPs) among the exon sequences of Pm21, PmV, and Pm12 and their homologous sequences in the common wheat genome were identified and used for developing diagnostic Kompetitive Allele-Specific PCR (KASP) markers. The markers were validated in different genotypes including transgenic vectors, transgenic lines, translocation lines, resistance stocks with documented Pm genes, and in multiple susceptible cultivars without Pm genes. As a result, we initially developed a KASP marker that can simultaneously diagnose Pm21, Pm12, and PmV. Subsequently, we obtained a highly diagnostic KASP marker for each of the three genes that could distinguish among the three genes and also accurately distinguish them from other resistant stocks with documented Pm genes and from multiple susceptible genotypes. Compared with previously reported markers, the highly diagnostic KASP markers developed in this study have the advantages of low cost, easy assay, accuracy, and potentially high throughput for MAS.

Molecular Mapping and Characterization of QBp.caas-3BL for Black Point Resistance in Wheat (Triticum Aestivum L.)

Wheat black point, which occurs in most wheat growing regions of the world, is detrimental to grain appearance, processing and nutrient quality. Mining and characterization of genetic loci for black point resistance is helpful for breeding resistant wheat cultivars. We previously identified a major QTL QBp.caas-3BL in a recombinant inbred line (RIL) population of Linmai 2/Zhong 892 across five environments. Here we confirmed the QTL in two additional environments. The genetic region of QBp.caas-3BL was enriched with newly developed markers. Using four sets of near isogenic lines QBp.caas-3BL was narrowed down to a physical interval of approximately 1.7 Mb, including five annotated genes according to IWGSC reference genome. TraesCS3B02G404300, TraesCS3B02G404600 and TraesCS3B02G404700 were predicted as candidate genes based on the analyses of sequence polymorphisms and differential expression. We also converted a SNP of TraesCS3B02G404700 into a breeding-applicable KASP marker and verified its efficacy for marker-assisted breeding in a panel of germplasm. The findings not only lay a foundation for map-based cloning of QBp.caas-3BL but also provide a useful marker for selection of resistant cultivars genotypes in wheat breeding.

Development and Utilization of the Functional Co-dominant KASP Marker for Thermo-sensitive Genic Male Sterility in Rice

Thermos-sensitive genic male sterility (TGMS) is the important genetic resource in two-line hybrid rice breeding. The pollen fertility of TGMS lines is regulated by a single point mutation on the TGMS genes. Based on the single nucleotide polymorphism (SNP) and indel, KASP markers were developed and utilized in rice molecular breeding due to high-through detection with low cost and save time. In this study, we convert the SNPs on TGMS genes including p/tms12-1 and tms9-1 gene to the functional co-dominant KASP marker and used in two-line hybrid rice breeding. We can differentiate the TGMS lines conferring p/tms12-1 or tms9-1 from another TGMS lines by KASP assay. Of them, the genotype of Pei'ai64S and Hua201S containing p/tms12-1 is homozygous GG genotype with blue signal, and the genotype of HengnongS-1 containing tms9-1 is homozygous CC genotype with red signal. KASP assay for tms9-1 gene was proved that the genetic mode is fit to one recessive Mendalian trait. The test of seed purity was performed by KASP marker for the two-line hybrid varieties of Liangyoupeijiu and Hualiangyou1206, which is consistent with the previous dCAPS marker. Moreover, the tms12-1 and tms9-1 gene were pyramided in the same genetic background and the new TGMS lines were generated. Therefore, the KASP marker for TGMS genes developed in this study can be widely used in two-line hybrid rice breeding. It will provide a visual convenient toolkit for breeders to select the target individual plant by the high-throughput detection in the different two-line rice breeding program.

Development of a minimal KASP marker panel for distinguishing genotypes in apple collections

Accurate identification of named accessions in germplasm collections is extremely important, especially for vegetatively propagated crops which are expensive to maintain. Thus, an inexpensive, reliable, and rapid genotyping method is essential because it avoids the need for laborious and time-consuming morphological comparisons. Single Nucleotide Polymorphism (SNP) marker panels containing large numbers of SNPs have been developed for many crop species, but such panels are much too large for basic cultivar identification. Here, we have identified a minimum set of SNP markers sufficient to distinguish apple cultivars held in the English and Welsh national collections providing a cheaper and automatable alternative to the markers currently used by the community. We show that SNP genotyping with a small set of well selected markers is equally efficient as microsatellites for the identification of apple cultivars and has the added advantage of automation and reduced cost when screening large numbers of samples.

Validation of a SNP-KASP marker for the Fusarium head blight resistance quantitative trait loci on chromosome 5AS

Fusarium head blight (FHB) is a devastating wheat disease with a significant economic impact. Fhb5 is an important quantitative trait loci (QTL) conferring type I resistance to FHB. In this study, we accessed the usability of a Kompetitive allele-specific polymerase chain reaction (KASP) marker for the QTL on Fhb-5AS. The KASP clustering results were compared with the linked simple sequence repeat marker, wmc705 for Qfhb-5AS. Our results indicate that the single-nucleotide polymorphic locus wsnp_Ra_c24707_34262900 (IWA7777) provides reliable information for Qfhb-5AS-based resistance and would be amenable to masker-assisted selection, introgression of the resistance loci, and pyramiding of FHB resistance in wheat cultivars.

Identification of quantitative trait loci for kernel traits in a wheat cultivar Chuannong16

Background Kernel length (KL), kernel width (KW) and thousand-kernel weight (TKW) are key agronomic traits in wheat breeding. Chuannong16 (‘CN16’) is a commercial cultivar with significantly longer kernels than the line ‘20828’. To identify and characterize potential alleles from CN16 controlling KL, the previously developed recombinant inbred line (RIL) population derived from the cross ‘20828’ × ‘CN16’ and the genetic map constructed by the Wheat55K SNP array and SSR markers were used to perform quantitative trait locus/loci (QTL) analyses for kernel traits. Results A total of 11 putative QTL associated with kernel traits were identified and they were located on chromosomes 1A (2 QTL), 2B (2 QTL), 2D (3 QTL), 3D, 4A, 6A, and 7A, respectively. Among them, three major QTL, QKL.sicau-2D, QKW.sicau-2D and QTKW.sicau-2D, controlling KL, KW and TKW, respectively, were detected in three different environments. Respectively, they explained 10.88–18.85%, 17.21–21.49% and 10.01–23.20% of the phenotypic variance. Further, they were genetically mapped in the same interval on chromosome 2DS. A previously developed kompetitive allele-specific PCR (KASP) marker KASP-AX-94721936 was integrated in the genetic map and QTL re-mapping finally located the three major QTL in a 1- cM region flanked by AX-111096297 and KASP-AX-94721936. Another two co-located QTL intervals for KL and TKW were also identified. A few predicted genes involved in regulation of kernel growth and development were identified in the intervals of these identified QTL. Significant relationships between kernel traits and spikelet number per spike and anthesis date were detected and discussed. Conclusions Three major and stably expressed QTL associated with KL, KW, and TKW were identified. A KASP marker tightly linked to these three major QTL was integrated. These findings provide information for subsequent fine mapping and cloning the three co-localized major QTL for kernel traits.