Molecular characterization of tropical maize inbred lines using microsatellite DNA markers

An insight on diversity and relationships among germplasm is important in any breeding program for crop improve¬ment. The main objectives of our study were to: (i) determine the level of genetic diversity within mid altitude maize inbred lines resistant to weevils, aflatoxin accumulation and drought, (ii) to suggest potential heterotic groups using their genetic structures and distance based on cluster analysis with the aim to generate broad based source germ¬plasm for mid altitude maize breeding program with combined traits of importance against Aflatoxin accumulation. In this study, 25 SSR markers were used to finger print forty two maize inbred lines to assess the genetic diversity, genetic relationships, and their population structure. A total of 184 alleles were identified at all the loci with an aver¬age of 7.36 and a range between two and 19 alleles per locus. The major allele frequency varied from 0.17 to 0.90 with an average of 0.49 while the minor allele frequency varied from 0.10 to 0.83 with an average of 0.51. The gene diversity values varied from 0.18 to 0.92 with an average of 0.65. Average heterozygosity percentage of the inbred lines was 4%, ranging from 0% to 2%, indicating the low level of heterozygosity within the inbred lines. The aver¬age polymorphism information content (PIC) was 0.61. A dendrogram constructed using unweighted Neighbour Joining algorithm suggested three heterotic groups among the inbred lines. The three heterotic patterns based on the SSR markers need to be verified by field testing to confirm what appears to be promising alternative heterotic patterns. The fixed pattern detected using SSR markers could potentially contribute towards effective utilization of the inbred lines for the exploitation of heterosis and formation of genetically diverse sources population

Characterization and QTL Mapping of a Major Field Resistance Locus for Bacterial Blight in Rice

Bacterial blight (BB) disease, caused by Xanthomonas oryzae pv. oryzae (Xoo), is among the major factors that can cause rice yields to decrease. To address BB disease, researchers have been looking for ways to change pesticides and cultivation methods, but developing resistant cultivars is the most effective method. However, the resistance and genetic factors of cultivars may be destroyed due to the emergence of new Xoo species caused by recent and rapid climate changes. Therefore, breeders need to identify resistance genes that can be sustained during unpredictable climate changes and utilized for breeding. Here, qBBR11, a quantitative trait locus (QTL) for resistance to BB disease, was detected in KJ (Korea Japonica varieties) 11_067 to KJ11_068 on chromosome 11 in a population derived by crossing JJ (Jeonju) 623 and HR(High resistant)27,195, which possess similar genetic backgrounds but different degrees of resistance to BB disease. qBBR11 was reduced from 18.49–18.69 Mbp of chromosome 11 to 200 kbp segment franked. In this region, 16 candidate genes were detected, and we identified 24 moderate-impact variations and four high-impact variations. In particular, high-impact variations were detected in Os11g0517800 which encode the domain region of GCN2 which is the eIF-2-alpha kinase associated with the resistance of abiotic/biotic stress in rice. In JJ623, which is moderately resistant to BB disease, a stop codon was created due to single nucleotide polymorphism (SNP). Therefore, compared with HR27195, JJ623 has weaker resistance to BB disease, though the two have similar genetic backgrounds. The results suggest that variation in the qBBR11 region regulates an important role in improving resistance to BB diseases, and qBBR11 is useful in providing an important resource for marker-assisted selection to improve mechanisms of resistance to BB disease

Marker-Assisted Backcrossing (MABc) to Improve Eating Quality with Thin Seed Coat and Aleurone Layer of Non-Glutinous Japonica Variety in Rice

Brown rice is composed of rice bran, pericarp, seed coat, and aleurone layers, and the rice bran layer contains a large number of substances useful for the human body, such as dietary fiber, α-tocopherol, α-tocotrienol, and vitamins. However, more than 90% of these substances are removed when polished, and white rice has the disadvantage of losing food-related ingredients, such as umami-related amino acids, when compared to the unpolished group. In this study, we tried to develop new breeding lines with a thinner seed coat and aleurone layer to provide high eating quality with softer chewing characteristics and processability in rice grain. We detected an SNP for foreground selection for the backcross population by comparing genome sequences between Samgwang and Seolgaeng and developed high eating quality brown rice breeding lines by applying marker-assisted backcrossing (MABC) breeding programs to backcross populations between Samgwang and Seolgaeng using KASP markers. SNP markers for foreground selection were identified to improve eating and processability through SNP mapping of Samgwang and Seolgaeng with SSIIa as a target gene in this study. Line selection according to genotype of KASP markers was successful in BC1F1 and BC2F1 generations, with the recurrent parent genome recovery ratio ranging from 91.22% to 98.65%. In BC2F1 seeds of the selected lines, thickness of the aleurone layer was found to range from 13.82 to 21.67 μm, which is much thinner than the 30.91 μm of the wild type, suggesting that selection by MABc could be used as an additional breeding material for the development of highly processed rice varieties. These lines will be useful to develop new brown rice varieties with softer chewing characteristics and processability in rice grain

New method for detecting Collectorichum species found in Korea using image analysis

Colletotrichum acutatum spp. infects various economical crops worldwide and causes massive loss on their yields. Among those, Capsicum spp., which known as chili pepper, is on a critical issue by those pathogens. Due to the lack of their genetic markers in Korea, the unidentifiable various species of C. acutatum obstructs the mechanism studies of these pathogens and the selection of disease resistant breed lines. Therefore, we screened RGB images of the colonization progresses of pathogens to identify the species of Ca40042, K1, NN, AS2, and SW1 by time and temperature. Cultivated pathogens such as Ca40042, K1, and SW1 were detectable on quantified shape and color data of images from specific temperature conditions, while other pathogens were difficult to recognize. Although several limitations exist in identification results of current experiment, but also, we can expect this method can suggest the possibility to replace the genetic marker methods which is now unavailable in Korea

High Throughput Phenotyping for Various Traits on Soybean Seeds Using Image Analysis

Data phenotyping traits on soybean seeds such as shape and color has been obscure because it is difficult to define them clearly. Further, it takes too much time and effort to have sufficient number of samplings especially length and width. These difficulties prevented seed morphology to be incorporated into efficient breeding program. Here, we propose methods for an image acquisition, a data processing, and analysis for the morphology and color of soybean seeds by high-throughput method using images analysis. As results, quantitative values for colors and various types of morphological traits could be screened to create a standard for subsequent evaluation of the genotype. Phenotyping method in the current study could define the morphology and color of soybean seeds in highly accurate and reliable manner. Further, this method enables the measurement and analysis of large amounts of plant seed phenotype data in a short time, which was not possible before. Fast and precise phenotype data obtained here may facilitate Genome Wide Association Study for the gene function analysis as well as for development of the elite varieties having desirable seed traits

Genomic Variation in Korean japonica Rice Varieties

Next-generation sequencing technologies have enabled the discovery of numerous sequence variations among closely related crop varieties. We analyzed genome resequencing data from 24 Korean temperate japonica rice varieties and discovered 954,233 sequence variations, including 791,121 single nucleotide polymorphisms (SNPs) and 163,112 insertions/deletions (InDels). On average, there was one variant per 391 base-pairs (bp), a variant density of 2.6 per 1 kbp. Of the InDels, 10,860 were longer than 20 bp, which enabled conversion to markers resolvable on an agarose gel. The effect of each variant on gene function was predicted using the SnpEff program. The variants were categorized into four groups according to their impact: high, moderate, low, and modifier. These groups contained 3524 (0.4%), 27,656 (2.9%), 24,875 (2.6%), and 898,178 (94.1%) variants, respectively. To test the accuracy of these data, eight InDels from a pre-harvest sprouting resistance QTL (qPHS11) target region, four highly polymorphic InDels, and four functional sequence variations in known agronomically important genes were selected and successfully developed into markers. These results will be useful to develop markers for marker-assisted selection, to select candidate genes in map-based cloning, and to produce efficient high-throughput genome-wide genotyping systems for Korean temperate japonica rice varieties

Rice Genome Resequencing Reveals a Major Quantitative Trait Locus for Resistance to Bakanae Disease Caused by <i>Fusarium fujikuroi</i>

Bakanae disease (BD), caused by the fungal pathogen Fusarium fujikuroi, has become a serious threat in rice-cultivating regions worldwide. In the present study, quantitative trait locus (QTL) mapping was performed using F2 and F3 plants derived after crossing a BD-resistant and a BD-susceptible Korean japonica rice variety, &#8216;Samgwang&#8217; and &#8216;Junam&#8217;, respectively. Resequencing of &#8216;Junam&#8217; and &#8216;Samgwang&#8217; genomes revealed 151,916 DNA polymorphisms between the two varieties. After genotyping 188 F2 plants, we constructed a genetic map comprising 184 markers, including 175 kompetitive allele-specific PCR markers, eight cleaved amplified polymorphic sequence (CAPS) markers, and a derived CAPS (dCAPS) marker. The degree of BD susceptibility of each F2 plant was evaluated on the basis of the mortality rate measured with corresponding F3 progeny seedlings by in vitro screening. Consequently, qFfR9, a major QTL, was discovered at 30.1 centimorgan (cM) on chromosome 9 with a logarithm of the odds score of 60.3. For the QTL interval, 95% probability lay within a 7.24&#8722;7.56 Mbp interval. In this interval, we found that eight genes exhibited non-synonymous single nucleotide polymorphisms (SNPs) by comparing the &#8216;Junam&#8217; and &#8216;Samgwang&#8217; genome sequence data, and are possibly candidate genes for qFfR9; therefore, qFfR9 could be utilized as a valuable resource for breeding BD-resistant rice varieties

Identification of Grain Size-Related QTLs in Korean japonica Rice Using Genome Resequencing and High-Throughput Image Analysis

Grain size is a key factor influencing the grain yield in rice. To identify the as-yet-unknown genes regulating grain size in Korean japonica rice, we developed a recombinant inbred line population (n = 162) from a cross between Odae (large-grain) and Joun (small-grain), and measured six traits including the thousand-grain weights of unhulled and hulled seeds, grain area, grain length, grain width and grain length-to-width ratio using high-throughput image analysis at the F8 and F9 generations. A genetic map was constructed using 248 kompetitive allele-specific PCR (KASP) markers that were polymorphic between the parental genotypes, and 29 QTLs affecting the six traits were identified, of which 15 were stable in both F8 and F9 generations. Notably, three QTL clusters affecting multiple traits were detected on chromosomes 6, 7 and 11. We analyzed whole-genome resequencing data of Odae and Joun, and selected candidate genes for the stable QTLs in the identified clusters that have high- or moderate-impact variations between Odae and Joun and encode proteins the families of which have been reported to be related to grain size regulation. These results will facilitate the identification of genes underlying the QTLs and promote molecular breeding of high-yielding Korean japonica rice varieties

Identification of Grain Size-Related QTLs in Korean <i>japonica</i> Rice Using Genome Resequencing and High-Throughput Image Analysis

Grain size is a key factor influencing the grain yield in rice. To identify the as-yet-unknown genes regulating grain size in Korean japonica rice, we developed a recombinant inbred line population (n = 162) from a cross between Odae (large-grain) and Joun (small-grain), and measured six traits including the thousand-grain weights of unhulled and hulled seeds, grain area, grain length, grain width and grain length-to-width ratio using high-throughput image analysis at the F8 and F9 generations. A genetic map was constructed using 248 kompetitive allele-specific PCR (KASP) markers that were polymorphic between the parental genotypes, and 29 QTLs affecting the six traits were identified, of which 15 were stable in both F8 and F9 generations. Notably, three QTL clusters affecting multiple traits were detected on chromosomes 6, 7 and 11. We analyzed whole-genome resequencing data of Odae and Joun, and selected candidate genes for the stable QTLs in the identified clusters that have high- or moderate-impact variations between Odae and Joun and encode proteins the families of which have been reported to be related to grain size regulation. These results will facilitate the identification of genes underlying the QTLs and promote molecular breeding of high-yielding Korean japonica rice varieties

New Parameters for Seedling Vigor Developed via Phenomics

Early seedling establishment in rice (Oryza sativa L.), which is measured by primary/secondary tiller, shoot length, biomass, root-related traits, and leaf area index, is an important trait because it helps to compete for light, air, and water for better tolerating various abiotic stresses. Consequently, it can affect the yield. However, there are not many research studies on this subject. Furthermore, previous studies have only measured the target traits once. However, this does not reflect the variation of growth rate during the seedling stage. Thus, two data points, two weeks and four weeks after planting, were used in the current study. As a result, two QTL regions were detected for the growth differences via plant height and green area (reflecting tillering). We expect that these results can be utilized by breeders to evaluate and select vigorous seedlings for their breeding programs