Detecting mislabeling and identifying unique progeny in Acacia mapping population using SNP markers

Acacia hybrids offer a great potential for paper industry in Southeast Asia due to their fast growth and ability to grow on abandoned or marginal lands. Breeding Acacia hybrids with desirable traits can be achieved through marker assisted selection(MAS) breeding. To develop a MAS program requires development of linkage maps and QTL analysis. Two mapping populations were developed through interspecific hybridization for linkage mapping and QTL analysis. All seeds per pod were cultured initially to improve hybrid yield as quality and density of linkage mapping is affected by the size of the mapping population. Progenies from two mapping populations were field planted for phenotypic and genotypic evaluation at three locations in Malaysia,(1) Forest Research Institute Malaysia field station at Segamat, Johor,(2) Borneo Tree Seeds and Seedlings Supplies Sdn, Bhd.(BTS) field trial site at Bintulu, Sarawak, and(3) Asiaprima RCF field trial site at Lancang, Pahang. During field planting, mislabeling was reported at Segamat, Johor, and a similar problem was suspected for Bintulu, Sarawak. Early screening with two isozymes effectively selected hybrid progenies, and these hybrids were subsequently further confirmed by using species-specific SNPs. During field planting, clonal mislabeling was reported and later confirmed by using a small set of STMS markers. A large set of SNPs were also used to screen all ramets in both populations. A total of 65.36% mislabeled ramets were encountered in the wood density population and 60.34% in the fibre length mapping population. No interpopulation pollen contamination was detected because all ramets found their match within the same population in question.However, mislabeling was detected among ramets of the same population. Mislabeled individuals were identified and grouped as they originated from 93 pods for wood density and 53 pods for fibre length mapping populations.On average 2 meiotically unique seeds per pod(179 seeds/93 pods) for wood density and 3 meiotically unique seeds per pod(174 seeds/53 pods) for fibre length mapping population were found. A single step statistical method was used to evaluate the most informative set of SNPs that could subsequently be used for routine checks for mislabeling in multi-location field trials and for labelling superior clones to protect breeder’s rights. A preliminary set of SNPs with a high degree of informativeness was selected for the mislabeling analysis in conjunction with an assignment test. Two subsets were successfully identified,i.e., 51 SNPs for wood density and 64 SNPs for fibre length mapping populations to identify all mislabeled ramets which had been previously identified. Mislabeling seems to be a common problem due to the complexity involved in the production of mapping populations. Therefore, checking for mislabeling is imperative for breeding activities and for analyses such as linkage mapping in which a correlation between genotypic and phenotypic data is determined.

Development of organelle single nucleotide polymorphism (SNP) markers and their application for the identification of cytoplasmic inheritance patterns in Pyropia yezoensis (Bangiales,Rhodophyta)

The genus Pyropia contains several important cultivated species.Genetic research in nori species has mainly focused on the cell nucleus,with few studies on organelles(chloroplast and mitochondria).Due to the high copy numbers of organelles in cells,which influence the development and traits of algae,it is necessary to study their genetic mechanism.In this study,the marine red alga Pyropia yezoensis,an important economic macroalga,was selected as the study object.To investigate organelle(chloroplast and mitochondria)inheritance in P.yezoensis,the wild type RZ(maternal strain)was crossed with the red mutant HT(paternal strain)and 30 color-sectors from 11 F1 gametophytic blades were examined.The complete chloroplast and mitochondrial genomes of the red mutant(HT)were assembled for the first time.One reliable and stable single nucleotide polymorphism(SNP)loci filtrated by bioinformatics analysis was used as a molecular marker for chloroplast and mitochondrial DNA,respectively,in subsequent experiments.PCR amplification and sequence analysis showed that the haplotypes of color-sectors detected were consistent with those of the maternal parent,confirming that both chloroplast and mitochondrial genomes were inherited maternally in P.yezoensis.The inheritance pattern of organelles in P.yezoensis can be used to guide the hybridization and breeding of nori.Additionally,the organelle SNP markers developed in this study can be applied in subsequent genetic research.

Genomic selection of eight fruit traits in pear

Genomic selection (GS) has the potential to improve selection efficiency and shorten the breeding cycle in fruit tree breeding. In this study,we evaluated the effect of prediction methods, marker density and the training population (TP) size on pear GS for improving its performance and reducing cost. We evaluated GS under two scenarios:(1) five-fold cross-validation in an interspecific pear family;(2) independent validation. Based on the cross-validation scheme, the prediction accuracy (PA) of eight fruit traits varied between 0.33 (fruit core vertical diameter)and 0.65 (stone cell content). Except for single fruit weight, a slightly better prediction accuracy (PA) was observed for the five parametrical methods compared with the two non-parametrical methods. In our TP of 310 individuals, 2 000 single nucleotide polymorphism (SNP) markers were sufficient to make reasonably accurate predictions. PAs for different traits increased by 18.21%-46.98%when the TP size increased from 50to 100, but the increment was smaller (-4.13%-33.91%) when the TP size increased from 200 to 250. For independent validation, the PAs ranged from 0.11 to 0.45 using rrBLUP method. In summary, our results showed that the TP size and SNP numbers had a greater impact on the PA than prediction methods. Furthermore, relatedness among the training and validation sets, and the complexity of traits should be considered when designing a TP to predict the test panel.

Mapping Qtls for Grain Yield and Yield Components in Kenyan Maize (<i>Zea mays</i>L.) Under Low Phosphorus Using Single Nucleotide Polymorphism (SNPS)

Selection for tolerance to low phosphorus (P) using morphological traits alone is slow and often confounded by environmental effects. This study identified some Quantitative Trait Loci (QTLs) associated with grain yield (GYLD), Plant (PHT) and Ear heights (EHT) under low P in maize using single nucleotide polymorphic markers. 228 F2:3 individuals derived from a cross between two contrasting maize inbred lines together with 239 SNPs were mapped onto ten linkage groups (LGs) spanning 2255 centiMorgans (cM) with an average inter-marker distance of 9.44 cM. Majority of the SNP markers (63%) followed the Mendelian segregation and were fairly distributed in all the LGs. Mean performance for all the traits in the F3 population was higher than the parental values, which suggested transgressive segregation for all traits. Low to moderate broad sense heritability (0.35 - 0.50) in the F3 population for GYLD, PHT and EHT indicated that tolerance to low P is controlled by complex multi genetic factors. A full multi-QTL model analysis suggested six QTLs (2 QTLs each for GYLD, PHT and EHT) located on chromosomes 1, 3, 4 and 8. The two QTLs for GYLD increased maize yield under low P soils by 173 kg/ha while the 2 QTLs for PHT increased plant growth by 18.14 cm. The % phenotypic variance explained by these QTLs under low P environments had a wide range (0.242% - 53.34%) and was much lower for GYLD compared to plant growth. Both additive and dominance gene actions contributed differentially to the observed phenotypic variance for tolerance to low P soils with dominance contributing more genetic effects compared additive effects for majority of the QTLs. The findings of this study will provide some basis for marker-assisted selection for yield improvement and further guide breeding strategies under low P soils of western Kenya.

Significant association of the novel Rf4-targeted SNP marker with the restorer for WA-CMS in different rice backgrounds and its utilization in molecular screening

In the rice cytoplasmic-genetic male sterility （CMS） system, the combination of a CMS line, maintainer line and restorer line carrying the restorer gene to restore fertility, is indispensable for the development of hybrids. However, the process of screening for the trait of fertility restoration is laborious and time-consuming. In the present study, we analyzed the nucleotide sequence of the Rf4 gene, which is the major locus controlling fertility restoration, to identify allele-specific variation. A single nucleotide polymorphism （SNP） A/C at ＋474 in the coding sequence （CDS） was found to be capable of strictly distinguishing groups of alleles Rf4 （A） and rf4 （C）. Using KASP genotyping, this valuable SNP was converted to an allele-specific PCR marker. We evaluated and validated the marker among three-line parents with different backgrounds, and the results revealed a complete correlation between SNP alleles and the fertility restoration phenotype. Molecular screening was subsequently carried out for the presence of alleles of Rf4 and Rf3 among 328 diverse rice cultivars with worldwide distribution. The results demonstrate that this SNP marker could be the optimal choice for the molecular identification of potential restorers.

Genetic Diversity and Population Structure Analysis of Barley Landraces from Shanghai Region Using Genotyping-by-Sequencing

Barley(Hordeum vulgare L.)is an important economic crop for food,feed and industrial raw materials.In the present research,112 barley landraces from the Shanghai region were genotyped using genotyping-by-sequencing(GBS),and the genetic diversity and population structure were analyzed.The results showed that 210,268 Single Nucleotide Polymorphisms(SNPs)were present in total,and the average poly-morphism information content(PIC)was 0.1642.Genetic diversity and population structure analyses suggested that these barley landraces were differentiated and could be divided into three sub-groups,with morphological traits of row-type and adherence of the hulls the main distinguishing factors between groups.Genotypes with similar or duplicated names were also investigated according to their genetic backgrounds and seed appearances.This study provided valuable information on barley landraces from the Shanghai region,and showed that all these barley landraces should be protected and used for future breeding programs.

Genetic Structure and Diversity Study of Cassava (Manihot esculenta) Germplasm for African Cassava Mosaic Disease and Fresh Storage Root Yield

A better understanding of population structure and genetic diversity among cassava germplasm for African cassava mosaic disease and fresh root yield traits is useful for cassava improvement programme. Phenotype-based selection for these traits is cumbersome due to phenotypic plasticity and difficulty in screening of phenotypic-induced variations. This study assessed quantitative trait loci (QTL) regions associated with African cassava mosaic disease (ACMD) and fresh storage root yield (FSRY) in 131 cassava (Manihot esculenta) genotypes using a genome-wide association study (GWAS). The single nucleotide polymorphism (SNP) loci and associated candidate genes, when validated, would be a valuable resource for marker-assisted selection in the breeding process for development of new cassava genotypes with improved resistance to ACMD and desirable high root yield. Population structure analysis using 12,500 SNPs differentiated the 131 genotypes into five distinct sub-groups (K = 5). Marker-trait association (MTA) analysis using the generalized linear model identified two QTL regions significant for ACMD and three for FSRY. This study demonstrated that DArTseq markers are useful genomic resources for genome-wide association studies of ACMD and FSRY traits in cassava for the acceleration of varietal development and release.

Genome-wide association studies reveal QTL hotspots for grain brightness and black point traits in barley

Grain kernel discoloration(KD)in cereal crops leads to down-grading grain quality and substantial economic losses worldwide.Breeding KD tolerant varieties requires a clear understanding of the genetic basis underlying this trait.Here,we generated a high-density single nucleotide polymorphisms(SNPs)map for a diverse barley germplasm and collected trait data from two independent field trials for five KD related traits:grain brightness(TL),redness(Ta),yellowness(Tb),black point impact(Tbpi),and total black point in percentage(Tbpt).Although grain brightness and black point is genetically correlated,the grain brightness traits(TL,Ta,and Tb)have significantly higher heritability than that of the black point traits(Tbpt and Tbpi),suggesting black point traits may be more susceptible to environmental influence.Using genome-wide association studies(GWAS),we identified a total of 37 quantitative trait loci(QTL),including two major QTL hotspots on chromosomes 4H and 7H,respectively.The two QTL hotspots are associated with all five KD traits.Further genetic linkage and gene transcription analyses identified candidate genes for the grain KD,including several genes in the flavonoid pathway and plant peroxidase.Our study provides valuable insights into the genetic basis for the grain KD in barley and would greatly facilitate future breeding programs for improving grain KD resistance.

Identifying genomic regions controlling ratoon stunting disease resistance in sugarcane(Saccharum spp.) clonal F_(1) population

The ratoon stunting disease (RSD) of sugarcane,caused by the bacterium Leifsonia xyli subsp.xyli,is one of the major concerns to sugarcane production and breeding programs worldwide.Due to no obvious external symptoms,RSD cannot be easily detected by the growers,hence has reduced the world’s sugarcane production significantly.This study aimed to identify quantitative trait loci (QTL) associated with RSD resistance and to assist in the development of linked molecular markers for marker-assisted breeding to minimize the reduction in sugarcane yield by the RSD infection.A set of 146 individuals derived from a self-crossing of CP80-1827 were evaluated for RSD resistance in a mechanically duplicated inoculated field trial from 2014 to 2017 using tissue blot immunoassay.Leveraging the genetic data and the four years phenotyping data of CP80-1827 selfing population,linkage map construction and QTL analysis were conducted based on clonal F_1 and F_2 mapping population types with GACD V.1.2 and Ici Mapping V.3.3,respectively.A total of 23 QTL associated with RSD resistance were identified,which explained 6%to13%of the phenotypic variation with the two types of software.A total of 82 disease resistance genes were identified by searching these 23 QTL regions on their corresponding regions on the Sorghum bicolor genome (44 genes),sugarcane R570 genome (20 genes),and S.spontaneum genome (18 genes),respectively.Compared with Ici Mapping V.3.3,GACD V.1.2 identified more major (6 vs.3) and stable QTL (2vs.0),and more disease resistance genes (51 vs.31),indicating GACD V.1.2 (clonal F_1 mapping type) is most likely to be more efficient than Ici Mapping (F_2 mapping type) for QTL analysis of a sefling population or clonal F_1 population in clonal species.The identified QTL controlling RSD resistance along with the associated SNP markers will assist sugarcane molecular breeding programs in combating this disease.

Genome-wide Association Analysis of Fast Chlorophyll Fluorescence Parameters in Maize

Fast chlorophyll fluorescence parameters are widely used to characterize the photosynthetic efficiency of plants. In this study, a genome-wide association analysis was used to detect key single-nueleotide polymorphisms （SNPs） associated with fast chlorophyll fluorescence parameters using more than 560 000 SNPs in a maize panel consisting of 404 inbred lines. In four fidd environments, 41 SNPs were detected to be associated with five fast chlorophyll fluorescence parameters, including ABS/CS0, ET0/CS0, TR0/ABS, ET0/TR0 and Pies. Among these identified SNPs, 8, 6, 18, 4 and 5 were significantly associated with ET0/TR0, ABS/ CS0, TR0/ABS, ET0/CS, and Plcs, respectively. These SNPs will help to discover genes for chlorophyll fluorescence parameters, better understand the genetic basis of photosynthesis, and assist in developing marker-assisted selection breeding programs in maize.

Genetic differentiation of grain, fodder and pod vegetable type cowpeas (Vigna unguiculata L.) identified through single nucleotide polymorphisms from genotyping-by-sequencing

The species Vigna unguiculata L.(Walp),commonly known as cowpea,is a multi-purpose legume that has been selected into three subspecies that are divided into grain,fodder and pod(yardlong bean)types.However,genetic bases for distinctions are not well understood.The purpose of this study was to apply genotyping-by-sequencing(GBS)and current reference genome for V.unguiculata to distinguish three subspecies and identify signatures of divergence.The collection of 130 accessions included 128 cultivated from:1)ssp.cylindrica,fodder type;2)ssp.sesquipedalis,pod vegetable type;and 3)ssp.unguiculata,grain type.Two wilds genotypes from spp.dekindtiana and spp.pubescens,were used to anchor phylogeny.A total of 11,083 highly informative single nucleotide polymorphisms(SNPs)were discovered.Wild accessions showed distinct genetic fingerprints and were separated from cultivated subspecies.Principal component analysis showed closer relationship between ssp.unguiculata and ssp.cylindrica compared to ssp.sesquipedalis.Relative differentiation of cultivated subspecies(with Fixation Index,FST)indicated the existence of discrete signatures of selection.This work clarifies the population structure,phylogeny,and domestication of cultivated cowpeas.Furthermore,significant genetic differences between grain and pod vegetable types can provide valuable information for future breeding in three cowpea groups.

UNREDUCED MEGAGAMETOPHYTE FORMATION VIA SECOND DIVISION RESTITUTION CONTRIBUTES TO TETRAPLOID PRODUCTION IN INTERPLOIDY CROSSES WITH/ORAH/MANDARIN(CITRUS RETICULATA)

Seedless fruits are desirable in the citrus fresh fruit market.Triploid production via diploid x tetraploid interploidy crosses is thought to be the most efficient and widely-used strategy for the breeding of seedless citrus.Although'Orah'mandarin has desirable organoleptic qualities,seeds in the fruits weaken its market competitiveness.To produce new seedless cultivars that are similar to'Orah'mandarin,we performed three 2x x 4x crosses using'Orah'mandarin as the seed parent to regenerate triploid plantlets.A total of 182 triploid and 36 tetraploid plantlets were obtained.By analyzing their genetic origins using nine novel single nucleotide polymorphism(SNP)markers,all of the triploids and tetraploids derived from these three crosses were proven to be hybrids.Also,we demonstrated that 2n megagametophyte formation in'Orah'mandarin result in tetraploid production in these three interploidy crosses.These tetraploid plantlets were genotyped using eight pericentromeric SNP markers and nine centromere distal SNP markers.Based on the genotypes of the 2n megagametophytes,the parental heterozygosity rates in 16 SNP loci and all 2n megagametophytes were less than 50%,indicating that second division restitution was the mechanism underlying 2n megagametophyte formation at both the population and individual levels.These triploid hybrids enrich the germplasm available for seedless breeding.Moreover,the tetraploid hybrids are valuable as parents for ploidy breeding for the production of seedless citrus fruits.