Khin Kyawt Yin2,3, Kanogporn Khammona1, Arweewut Yongsuwan1, Naraporn Chaomueang1, Siwaret Arikit4,5, Samart Wanchana1, Jintana Unartgam2 and Vinitchan Ruanjaichon*1
Received: July 26, 2021; Published: August 20, 2021
Corresponding author: Vinit chan Ruanjaichon, National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand Science Park, Phenytoin Road, Khlong Nueng, Khlong Luang, Pathum Thani 12120, Thailand
Sweetness is an economically important eating quality trait for sweet corn breeding. The various types of sweet corn are caused by single gene mutation . The brittle2 and shrunken2 mutants accumulated 20% of starch found in the corn kernels [2, 3]. Due to mutation in ADP-glucose pyro phosphorylase reveals as a key regulatory enzyme to limit the rate of starch biosynthesis pathway [4-7]. The brittle2 (bt2) mutant is classified as class 1 super sweet corn [3,8,9], having 15-30% sugar of total carbohydrate content in the kernel, while normal corn contains about 3% sugar in the kernel [9,10]. Newly, the functional marker based on shrunken2 gene (sh2) was identified and developed . This SNP marker designed within the coding region in exon 1 at position 154 of the coding sequence was clearly separated the sh2-based sweet corn from waxy corn, field corn, and the sweet corn that was based on the other genes, i.e., su1, se1, and bt2. However, there is no report of SNP markers associated with bt2 in sweet corn.
In this study, we scanned for SNPs from an Axiom® Maize 600k Genotyping Array from a collection of 6 sweet corn and 9 waxy corn recombination inbred lines (RILs). The SNPs were used to evaluate linkage-disequilibrium (LD) decays and association with bt2. Additionally, SNP-based makers tightly linked to bt2 were validated to find a marker associated with super sweet kernel trait that could aid in future super-sweet corn improvement programs.
Keywords: Maize; kernel Sweetness; Brittle 2; Single Nucleotide Polymorphism (SNP)
A panel of 15 maize recombinant inbred lines (RILs) were used to obtain a set of 616,201 SNP variants on the bt2 using Axiom® Maize 600K Genotyping Array (Table S1). A panel of 331,945 variants were high-quality SNPs with minor-allele frequency (MAF) > 0.05 and missing rate < 10% and were included in subsequent analysis. Chromosome-wide LD among the 15 RILs, analyzed using PLINK software  revealed the 210 kb LD decay at a cut-off r2 = 0.2 (Figure1). The bt2 (GRMZM2G068506) resides on chromosome 4 at position 58,954,361 to 58,960,521 bp (Maize Genome Database version2; https://www.maizegdb.org). We found that the AX-91607989 (C/T) SNP, located at 62,922 bp upstream of bt2, was significantly associated with bt2-based sweet corn (Table S2). Among the 15 RILs, 2 lines controlled by bt2 had the T allele, while the others (sh2, su, se and wx) contained the C allele. A marker was developed using Mass ARRAY® platform and validated in a diverse panel of maize composing of inbred lines, hybrid lines, and RILs (Figure2; Table S3). This marker could clearly separate the bt2- based sweet corn from waxy corn, field corn, and other types of sweet corn mutants that was based on the other genes, i.e., su1, se1, and sh2 (Figure3; Table S4).
Finally, we extended the search for gene-based SNPs linked to bt2 on chromosome 4. One SNP was validated to differentiate sweet corn from other corn types by possessing a bt2 recessive allele (T). This SNP was located upstream of the bt2 62,922 base pair. We developed a marker tightly linked to the bt2 and validated that the marker clearly distinguished the super sweetness corn from waxy corn, field corn, and the sweet corn controlled by other mutants. The identified SNPs on the bt2 could be used as selection marker for marker-assisted selection in maize-breeding programs. Moreover, the tightly linked markers identified in this study could effectively save breeders cost and valuable time from lengthy field trials, and aid in accelerating sweet corn breeding programs.
Table S2: A single marker analysis of 66 SNP AXIOM ID validated on the 15 maize recombinant inbred lines (RILs).
Table S3: The Mass Array markers Bt2_4_58891439 designed based on SNP variant (C/T) in upstream bt2.
Table S4: List of 307 inbreds and recombinant inbred lines (RILs) used formarker validation in this study.
Figure 1: Overall chromosome-wide linkage-disequilibrium (LD) decay estimated from single-nucleotide polymorphism (SNP) genotypes of 15 maize recombinant inbred lines. Each line plot represents a smoothed r2 for all marker pairs on each chromosome depending on the distance between marker pairs.
Figure 2: Physical location of the Bt2_4_58891439 SNP marker upstream of the brittle2 gene (bt2). Orange: Bt_4_58891439 SNP; gray: SNPs located in bt2 region. Sucrose content was analyzed from immature ears of each line at 24 days after pollination using the method described previously .
Figure 3: Allelic discrimination plot of SNP marker Bt2_4_58891439 validated in a panel of 322 maize lines as analyzed by Mass ARRAY® platform. Scatter dots with different colors showed clustering of homozygous genotype TT (yellow) and homozygous genotype CC (blue).
This research was funded by the National Science and Technology Development Agency (NSTDA), grant number P-17- 52167 and Thailand Graduate Institute of Science and Technology (TGIST), agreement number SCA-CO-2562-9848-EN and grant number TG-BT-KU-62-062M.
Data are contained within the article and the Supplementary Materials.
The authors thank the Chai Nat Field Crops Research Center and Plant Breeding Research Center for Sustainable Agriculture, Khon Kaen University for providing the plant materials used in the study.
The authors declare no conflict of interest.
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