Specific-locus Amplified Fragment Sequencing Technology Research Articles

Identification of a Wheat-Psathyrostachys huashanica 7Ns Ditelosomic Addition Line Conferring Early Maturation by Cytological Analysis and Newly Developed Molecular and FISH Markers.

Early maturation is an important objective in wheat breeding programs that could facilitate multiple-cropping systems, decrease disaster- and disease-related losses, ensure stable wheat production, and increase economic benefits. Exploitation of novel germplasm from wild relatives of wheat is an effective means of breeding for early maturity. Psathyrostachys huashanica Keng f. ex P. C. KUO (2n=2x=14, NsNs) is a promising source of useful genes for wheat genetic improvement. In this study, we characterized a novel wheat-P. huashanica line, DT23, derived from distant hybridization between common wheat and P. huashanica. Fluorescence in situ hybridization (FISH) and sequential genomic in situ hybridization (GISH) analyses indicated that DT23 is a stable wheat-P. huashanica ditelosomic addition line. FISH painting and PCR-based landmark unique gene markers analyses further revealed that DT23 is a wheat-P. huashanica 7Ns ditelosomic addition line. Observation of spike differentiation and the growth period revealed that DT23 exhibited earlier maturation than the wheat parents. This is the first report of new earliness per se (Eps) gene(s) probably associated with a group 7 chromosome of P. huashanica. Based on specific locus-amplified fragment sequencing technology, 45 new specific molecular markers and 19 specific FISH probes were developed for the P. huashanica 7Ns chromosome. Marker validation analyses revealed that two specific markers distinguished the Ns genome chromosomes of P. huashanica and the chromosomes of other wheat-related species. These newly developed FISH probes specifically detected Ns genome chromosomes of P. huashanica in the wheat background. The DT23 line will be useful for breeding early maturing wheat. The specific markers and FISH probes developed in this study can be used to detect and trace P. huashanica chromosomes and chromosomal segments carrying elite genes in diverse materials.

The Genetic and Environmental Adaptation of the Associated Liana Species Derris trifoliata Lour. (Leguminosae) in Mangroves

Derris trifoliata Lour. is an indigenous and associated liana species of mangroves in China; however, its rapid dispersal is threatening mangrove survival. To explore and evaluate their persistence in past disturbances and their potential resistance to future climate and environmental changes, 120 D. trifoliata samples were collected from three sites in Guangdong Province, China, and they were used to develop single nucleotide polymorphic markers using specific-locus amplified fragment sequencing technology. A total of 351.59 Mb reads and 97,998 polymorphic specific-locus amplified fragment sequencing tags were identified, including 360,672 single nucleotide polymorphisms. The principal component analysis, phylogenetic tree, and genetic structure all clustered the samples according to their geographic positions. The three populations showed medium genetic diversity levels and high clonal diversity, indicating that sexual propagation played vital roles in the populations’ succession, although clonal growth was intense within the populations. An association analysis revealed that 9 out of 16 markers were correlated with nitrogen, which indicated the positive roles of nitrogen in population formation and maintenance. This study provides an ecological and molecular basis for understanding the outbreaks of D. trifoliata in mangroves. To control the further expansion of D. trifoliata in mangroves, preventive and control measures should be taken against clonal growth and sexual propagation, respectively; obstructing the clonal growth, especially that of the stolon, should be mainly considered at the junctions of D. trifoliata and mangroves.

Identification of novel genes associated with litter size of indigenous sheep population in Xinjiang, China using specific-locus amplified fragment sequencing technology.

BackgroundThere are abundant sheep breed resources in the Xinjiang region of China attributing to its diverse ecological system, which include several high-litter size sheep populations. Previous studies have confirmed that the major high prolificacy gene cannot be used to detect high litter size. Our research team found a resource group in Pishan County, southern Xinjiang. It showed high fertility with an average litter size of two to four in one birth, excellent breast development, and a high survival rate of lambs. In the present study, we used this resource as an ideal sample for studying the genetic mechanisms of high prolificacy in sheep.MethodsIndigenous sheep populations from Xinjiang, with different litter sizes, were selected for the research, and specific-locus amplified fragment sequencing (SLAF-seq) technology was used to comprehensively screen single nucleotide polymorphisms (SNPs) from the whole genome that may cause differences in litter size. Novel genes associated with litter size of sheep were detected using genome-wide association studies (GWAS), providing new clues revealing the regulation mechanism of sheep fecundity. Candidate genes related to ovulation and litter size were selected for verification using Kompetitive Allele Specific polymerase chain reaction (KASP) cluster analysis.ResultsWe identified 685,300 SNPs using the SLAF-seq technique for subsequent genome-wide analysis. Subsequently, 155 SNPs were detected at the genome-wide level. Fourteen genes related to sheep reproduction were notated: COIL, SLK, FSHR, Plxna3, Ddx24, CXCL12, Pla2g7, ATP5F1A, KERA, GUCY1A1, LOC101107541, LOC101107119, LOC101107809, and BRAF. Based on literature reports, 30 loci of seven genes and candidate genes (CXCL12, FSHR, SLK, GUCY1A1, COIL, LOC101107541, and LOC101107119) related to ovulation and litter size were selected for verification using KASP cluster analysis. Among them, nine loci of three genes were successfully genotyped. Three loci of FSHR (GenBank ID: 443299, g. 75320741G>A site), GUCY1A1 (GenBank ID: 101110000, g. 43266624C>T site), and COIL (GenBank ID: 101123134, g. 7321466C>G site) were found to be significantly or extremely significantly associated with litter size. These three loci are expected to be used as molecular markers to determine differences in litter size in sheep.

Genome-wide association study discovered favorable single nucleotide polymorphisms and candidate genes associated with ramet number in ramie (Boehmeria nivea L.)

BackgroundRamie (Boehmeria nivea L.) is one of the most important natural fiber crops and an important forage grass in south China. Ramet number, which is a quantitative trait controlled by multigenes, is one of the most important agronomic traits in plants because the ramet number per plant is a key component of grain yield and biomass. However, the genetic variation and genetic architecture of ramie ramet number are rarely known.ResultsA genome-wide association study was performed using a panel of 112 core germplasms and 108,888 single nucleotide polymorphisms (SNPs) detected using specific-locus amplified fragment sequencing technology. Trait-SNP association analysis detected 44 significant SNPs that were associated with ramet number at P < 0.01. The favorable SNP Marker20170–64 emerged at least twice in the three detected stages and was validated to be associated with the ramie ramet number using genomic DNA polymerase chain reaction with an F1 hybrid progeny population. Comparative genome analysis predicted nine candidate genes for ramet number based on Marker20170–64. Real-time quantitative polymerase chain reaction analysis indicated that six of the genes were specific to upregulation in the ramie variety with high ramet number. These results suggest that these genes could be considered as ramet number-associated candidates in ramie.ConclusionsThe identified loci or genes may be promising targets for genetic engineering and selection for modulating the ramet number in ramie. Our work improves understanding of the genetics of ramet number in ramie core germplasms and provides tools for marker-assisted selection for improvement of agricultural traits.

Genome-wide association study of 8 carcass traits in Jinghai Yellow chickens using specific-locus amplified fragment sequencing technology

Carcass traits are important to the commercial chicken industry, and understanding the genetics of these traits will be useful in the development of commercially viable varieties of chickens. We conducted a genome-wide association study based on 8 carcass trait phenotypes in a population of 400 43-week-old Jinghai Yellow chickens. Specific-locus amplified fragment sequencing technology was used to identify 90,961 single nucleotide polymorphisms (SNP) distributed among 29 chromosomes and the mitochondrial genome. SNP that were significantly associated with phenotypic traits were identified by a simple general linear model. Fifteen SNP attained genome-wide significance (P < 1.87E−6) and were associated with 5 of the 8 carcass traits; only one SNP was significantly associated with 2 traits (foot weight and wing weight). Twelve genes were associated with these 15 SNP. A region of chromosome 4 between 75.5 and 76.1 Mb was associated with carcass weight, foot weight, and wing weight. An 84-kb region on chromosome 3 (51.2 Mb) was associated with eviscerated weight and semi-eviscerated weight.