1. Bulk segregant analysis (BSA)for improving cold stress
resistance in maize using SSR markers
Muhammad Qudrat Ullah Farooqi, Rahul Vasudeo Ramekar, Ma Shijun,
Kyu Jin Sa, and Ju Kyong Lee*
Department of Applied Plant Sciences, College of Agriculture and Life Sciences, Kangwon National University, Chuncheon, , Korea
Introduction
Improving the cold stress resistance of crop plants is a major objective for breeders in tropical, sub-tropical and warm temperate parts of the world. Maize is highly sensitive to low temperature due to tropical origin. Abiotic stresses caused adverse effect on plant physiology, growth and biochemical processes. Chilling temperature is an important constraint for global maize production; as it is not favorable for early seedling growth in crop plant. The phenology and productivity of maize plant can be altered due to cold stress. Due to low temperature, mean germination time is disturbed; that is directly correlated with shoot length and dry weight of plant. Gene expression and protein product pattern can be alter under low temperature. It is essential to know cold tolerance of plant to figure out the gene that is responsible for cold stress. Modification in the pattern of gene expression and products allow the plant to resist under stress condition.
Bulk segregant analysis (BSA) is valuable tool proven by molecular markers in different plants. Two variants of the BSA technique are possible depending on whether these plants derived from a cross between two parental lines or from a population of plants with diverse genetic backgrounds. The genetic difference can be found among genotypes; unlike the morphological markers which are not influenced by environmental factors. Microsatellites or simple sequence repeats (SSRs) genetic markers are di-, tri- or tetra-nucleotide motifs with short stretches of tandom repeats. The SSR markers are characterized by great abundance and highly variable even throughout distribution in wide range of genomic. SSRs are multi-allelic, highly polymorphic and co-dominant in nature; that becomes the best genetic markers to breeders for molecular analysis of crops. This genetic marker has proved as valuable tool for genome mapping, population and conservation genetics studies, property right protection, marker-assisted selection and diversity measurements in maize crop.
The objective is this study is to identify promising cold tolerance maize cultivars and to characterize the genetic relationship among high cold tolerance with low tolerance varieties. Thus our results of BSA, genetic diversity and relationship, and association analysis can be used to help identify traits important in determining the cold stress resistance of maize, and also DNA markers identified by BSA and population structure analysis can be used to help incorporate such traits into a breeding program to improve cold stress resistance in maize.
Fig. 2. The venn diagram of the number of high, low and shared specific cold tolerant specific alleles.
a) b)
Fig. 1. An example of a SSR profile in 8 high and low cold tolerant maize inbred lines using the SSR primer, a) umc1124 and b) umc2142
Materials and Methods
Plant material
A total eight lines were used in this study after screen out from cold germination test. Among these five (CO439, CO438, CO450, CO435 and CO445) were high tolerance against low temperature treatments referred as cold tolerant lines; while, 3 (CO437, CO436 and CO440) were detected as low tolerance at low temperature conditions referred as cold susceptible lines.
Bulk Segregant Analysis (BSA), SSR Primers and PCR amplification
Genomic DNA was extracted from young leaves and screened with 100 SSR markers. Markers showing polymorphism in the form of a clearly visible difference in band patterns between the high and low inbred lines in these experiments were selected for genotyping of BSA. High and low inbred lines were screened out for polymorphism Amplification was analyzed by 6% denaturing polyacrylamide gel and separated fragments were then visualized by sliver staining.
SSR Data analysis
Gel photographs were scored manually. The bands were binary coded by 1 or 0 for their presence or absence in each genotype. A genetic dandrogram was constructed on the basis of Sxy = 2Nxy/(Nx + Ny), where Nxy refers to the number of bands in common between plant x and y. Nx and Ny denote the total number of bands in each plant x and y, respectively. The calculations were done using an arithmetic average option in the NTSYS-pc program. Using the genetic analysis package PowerMarker (ver. 3.25); the variability at each locus was measured in terms of the number of alleles (NA), major allele frequency (MAF), gene diversity (GD), and polymorphic information content (PIC).
Table 4. Identification of cold specific alleles among cold tolerant linked SSR loci.
Fig. 3. UPGMA dandrogram based on 100 SSR markers in 8 high and low specific maize inbred lines
Results and Discussion
The evaluation of genetic diversity based on following calculated values from alleles scoring data of SSR loci’s linked with cold tolerance i.e major allele frequency (MAF), gene diversity and polymorphic information content (PIC). The Table 2 showed that the mean value of major allele frequency of all lines were 0.47, the number of alleles were 3.86 and range between 2 to 5 alleles per locus. The average value of gene diversity was 0.63, which varied from 0.59 to 0.73 and the average value of polymorphic information content was In Table 3, we made comparative analysis of genetic diversity for high and low inbred lines. The result shows that except major allele frequency; all other values were observed higher in high cold tolerant inbred lines.
In our molecular experiment for BSA, high and low cold tolerant maize inbred lines were assessed using 100 polymorphic SSR primer sets. The amplified fragment of SSR loci’s ranged from 50 bp to 600 bp. But, most of bands were located in between range of 50 bp to 300 bp. In overall, 319 alleles were found; the alleles which were located just in high tolerant lines derived as high specific alleles. And also, the low specific alleles were those which represent entirely in low tolerant lines. While, the alleles which were located in both high and low tolerant lines regions were named as shared alleles. As shown in Fig. 2 (Venn diagram), the high specific alleles were 128, low specific alleles were 61 and 130 were shared alleles in high and low cold tolerant maize inbred lines.(Table. 4).
In phylogenetic relationship, the eight maize inbred lines clustered into three major groups (Fig. 3), and thus were divided into Groups I, II, and III. Group I only included one high tolerant inbred line (CO438). Group II included four inbred lines that consisted of three high tolerant inbred lines (CO450, CO435, CO445), and one low tolerant inbred line (CO437). Group III included two low tolerant inbred lines (CO436, CO440). Although one inbred line (CO437) was not clearly separated from major groups, most high tolerant inbred lines were clearly discriminated from the low tolerant inbred lines.
SSR has ability of greater power of discrimination than other genetic markers i.e. RFLP; that can reveal genetic associations which are reflective to inbred lines pedigree. SSR technology is dependent on polymerase chain reaction (PCR); therefore by using Agrose gel system, the polymorphism can be detected by cheap expense and more widely results. Quantitative trait loci (QTL) is usual method to compare and locate the loci that need segregating population with one genotype using genetic markers. But, plants with such segregating population can be grouped with difference in allele frequency through bulk segregant analysis (BSA). In conclusion, the identification and characterization of high and low cold tolerant maize lines based on the SSR markers will be useful for maize breeding studies and the results from this study could serve as useful molecular markers.
Table 1: Derivation of eight high and low cold tolerant maize inbred line used in this study.
Table 2: Mean of number of alleles and genetic diversity index for SSR loci linked with cold tolerance among high tolerant maize inbred lines.
Table 3: Mean of number of alleles and genetic diversity index for SSR loci linked with cold tolerance between high tolerant maize inbred lines
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