Identification Of Germplasm Resources Research Articles

KASP: a high-throughput genotyping system and its applications in major crop plants for biotic and abiotic stress tolerance.

Advances in plant molecular breeding have resulted in the development of new varieties with superior traits, thus improving the crop germplasm. Breeders can screen a large number of accessions without rigorous and time-consuming phenotyping by marker-assisted selection (MAS). Molecular markers are one of the most imperative tools in plant breeding programmes for MAS to develop new cultivars possessing multiple superior traits. Single nucleotide polymorphisms (SNPs) are ideal for MAS due to their low cost, low genotyping error rates, and reproducibility. Kompetitive Allele Specific PCR (KASP) is a globally recognized technology for SNP genotyping. KASP is an allele-specific oligo extension-based PCR assay that uses fluorescence resonance energy transfer (FRET) to detect genetic variations such as SNPs and insertions/deletions (InDels) at a specific locus. Additionally, KASP allows greater flexibility in assay design, which leads to a higher success rate and the capability to genotype a large population. Its versatility and ease of use make it a valuable tool in various fields, including genetics, agriculture, and medical research. KASP has been extensively used in various plant-breeding applications, such as the identification of germplasm resources, quality control (QC) analysis, allele mining, linkage mapping, quantitative trait locus (QTL) mapping, genetic map construction, trait-specific marker development, and MAS. This review provides an overview of the KASP assay and emphasizes its validation in crop improvement related to various biotic and abiotic stress tolerance and quality traits.

Development of SSR Markers for and Fingerprinting of Walnut Genetic Resources

Walnut is one of four major nuts in the world. China has abundant walnut germplasm resources, but there are still shortcomings in the identification of germplasm resources. This study used different walnut varieties as materials and developed 14 high-quality SSR molecular markers from 60 pairs of primers based on genome re-sequencing results. This study analyzed the genetic diversity of Chinese walnut genetic resources using 14 selected SSR markers. A total of 64 alleles were detected in 47 walnut resources, with an average of 4.571 alleles per locus. The variation range of polymorphism information content was 0.096~0.711, with an average value of 0.422. Cluster analysis, population genetic structure, and principal coordinate analysis divided 47 walnut resources into ordinary walnuts, Juglans hopeiensis, and Liaoyi 1. In addition, core SSR markers (Jr45, Jr40, Jr29, Jr35, and Jr11) were selected from 14 SSR markers, which were sufficient to distinguish 47 walnut resources. At the same time, 47 unique molecular fingerprints of walnuts were constructed using these core SSR markers. This study provides strong scientific support for rapid and efficient identification, germplasm innovation, and a variety of property protection of walnut germplasm.

DNA barcode screening, identification of germplasm resources, and analysis of genetic diversity of Anemarrhena asphodeloides

Anemarrhena asphodeloides is a common medicinal material used in clinical prescriptions and Chinese patent medicine. In this study, the Illumina platform was used to obtain the chloroplast genome sequences of seven kinds of A. asphodeloides from different areas. The specific DNA barcodes were screened by comparative genomics analysis, and the DNA barcodes were used to identify the germplasm resources and analyze the genetic diversity of A. asphodeloides samples from different areas in China. All the seven chloroplast genomes had a ring structure. The total length was 156 801-156 930 bp, and 113 genes were annotated, including 79 protein-coding genes, 30 tRNA genes, and four rRNA genes. The comparative genomics analysis showed that rps16, trnG-GCC, atpF, rpoB, ycf3, rpl16, ndhF, trnS-GCU＿trnG-GCC, petN-psbM, and ndhF-rpl32 were potential candidates for specific DNA barcodes of A. asphodeloides. In this study, the second intron of ycf3 and atpF intron sequences with a sequence length of 700-800 bp and easy amplification were selected for polymerase chain reaction(PCR) amplification and sequencing of 594 samples from 26 areas. The sequence analysis showed that six and eight haplotypes of ycf3 and atpF sequences could be identified, respectively, and 17 haplotypes could be identified by combined analysis of the two sequences, which were named Hap1-Hap17. The haplotype diversity(H_d), nucleotide diversity(P_i), and genetic distance of A. asphodeloides in 26 populations were 0.68, 0.93×10~(-3), and 0-0.003 1, respectively, indicating that the genetic diversity within the species of A. asphodeloides is rich. The intermediary adjacent network analysis showed that Hap5 was the oldest haplotype, which was mainly distributed in Yixian county of Baoding, Hebei province, Hequ county of Xinzhou, Shanxi province, and Xiangfen county of Linfen, Shanxi province. This study has important guiding significance for the identification of A. asphodeloides species, the protection and development of germplasm resources, and the identification of production areas, and it provides a research basis for further revealing the genetic evolution law of A. asphodeloides.

Development of InDel markers and establishment of a specific molecular marker of the new strain (SW-81) in Pyropia haitanensis

With the increase in the number of new strains (varieties) of Pyropia, the morphological markers are more and more difficult to meet the requirement of germplasm management. However, it is a new approach and trend to develop molecular markers based on genome re-sequencing. In the present study, we developed insertion/deletion (InDel) markers based on the re-sequencing of Pyropia haitanensis, and a specific molecular marker was screened out using polymorphic InDel markers for the germplasm identification of the new strain (SW-81) of P. haitanensis. A total of 8616 InDel sites were detected from the re-sequencing data, of which the short fragments (1–3 bp) were the absolute dominant type, accounting for 81.13%, with an average of one InDel site per 5.92 kb. Moreover, 200 InDel primers were randomly synthesized to analyze the effectiveness, and the 65 pairs of effective primers were finally obtained, of which 40 pairs were polymorphic InDel markers, with the polymorphism amplification rate of 20.00%. A specific molecular marker (InDel-2) was screened out for the germplasm identification of SW-81 strain, and this marker had the same identification effect on the conchocelis, and the blades cultivated at indoor and sea areas. The above-mentioned results showed that there were abundant InDel loci in the genome of P. haitanensis, and the gene sequence of the same loci varied among different strains, showing the polymorphism of amplification products. The polymorphism analysis based on the InDel marker could provide an effective approach for the management and identification of germplasm resources.

Characterization of Morphological and Fruit Quality Traits of Coconut (Cocos nucifera L.) Germplasm

The evaluation and identification of germplasm resources is an indispensable step in the breeding processes and have important roles in the selection and improvement of new varieties. This research intended to characterize coconut germplasm to determine the quantitative, qualitative, and morphological traits of the stem, leaf, and inflorescence and the fruit characteristics. Sixteen morphological and qualitative traits of 17 coconut (Cocos nucifera L.) germplasm resources from Hainan, China, were investigated to determine the characteristics and advantages of multiple germplasm lines to create the foundation for the cultivation and breeding of coconuts. The results of the correlation analysis, principal component analysis (PCA), and cluster analysis indicate a correlation between coconut germplasm factors and their contribution to coconut traits. The results revealed that stem girth at 0.2 m was the most obvious trait, along with the fruit flavor, edible rate, fat content, hole spacing, single fruit weight, and number of female flowers, which reflect most of the information regarding coconut traits and contribute to its value. The PCA and cluster analysis indicated that two high-yield and superior-quality sweet water dwarf coconut germplasms, named ‘15-19’ and ‘15-17’, were suitable for cultivation and production in Hainan, China. The results of this study act a far-reaching influence on the collection and utilization of coconut resources and have an impact on the development and progress of the coconut industry in China.

Genome-wide identification of simple sequence repeats and assessment of genetic diversity in Hedychium

Hedychium is an important industrial crop, which is widely cultivated for its medicinal and horticultural significance. However, the development of simple sequence repeat (SSR) for Hedychium has not yet been reported. In this study, 266,825 SSR markers were identified in H. coronarium ‘ZS’ genome. The relationship between the type, repeat times, content, and content of SSR markers and chromosome length was specifically analyzed. The results revealed that the mononucleotides (59.17 %) were the most abundant repeat motifs, followed by dinucleotides (27.97 %) and trinucleotides (10.85 %). To verify the availability of SSR and its polymorphism among different species, 120 primer pairs were randomly selected. Finally, 22 markers were screened to genotype 44 accessions collected from different geographic locations of China, Thailand, Japan, and USA. A number of 224 alleles were identified, with an average of 10.18 alleles and polymorphism information content ranged from 0.17 to 0.96 with a mean of 0.88. Genetic structure analysis indicated that 44 accessions had strong regional characteristics and a clear distinction between cultivated and wild genotypes. Population structure and PCoA analysis revealed that from 44 accessions, 7 cultivated accession were distinctly grouped into cluster A, whilst other wild accessions belong to cluster B. These new SSR markers will provide important references for genetic diversity analysis, identification of germplasm resources, and breeding of Hedychium.

Full-length SMRT transcriptome sequencing and microsatellite characterization in Paulownia catalpifolia

Paulownia catalpifolia is an important, fast-growing timber species known for its high density, color and texture. However, few transcriptomic and genetic studies have been conducted in P. catalpifolia. In this study, single-molecule real-time sequencing technology was applied to obtain the full-length transcriptome of P. catalpifolia leaves treated with varying degrees of drought stress. The sequencing data were then used to search for microsatellites, or simple sequence repeats (SSRs). A total of 28.83 Gb data were generated, 25,969 high-quality (HQ) transcripts with an average length of 1624 bp were acquired after removing the redundant reads, and 25,602 HQ transcripts (98.59%) were annotated using public databases. Among the HQ transcripts, 16,722 intact coding sequences, 149 long non-coding RNAs and 179 alternative splicing events were predicted, respectively. A total of 7367 SSR loci were distributed throughout 6293 HQ transcripts, of which 763 complex SSRs and 6604 complete SSRs. The SSR appearance frequency was 28.37%, and the average distribution distance was 5.59 kb. Among the 6604 complete SSR loci, 1–3 nucleotide repeats were dominant, occupying 97.85% of the total SSR loci, of which mono-, di- and tri-nucleotide repeats were 44.68%, 33.86% and 19.31%, respectively. We detected 112 repeat motifs, of which A/T (42.64%), AG/CT (12.22%), GA/TC (9.63%), GAA/TTC (1.57%) and CCA/TGG (1.54%) were most common in mono-, di- and tri-nucleotide repeats, respectively. The length of the repeat SSR motifs was 10–88 bp, and 4997 (75.67%) were ≤ 20 bp. This study provides a novel full-length transcriptome reference for P. catalpifolia and will facilitate the identification of germplasm resources and breeding of new drought-resistant P. catalpifolia varieties.

Bioinformatics analysis of microsatellite sites in the RNA-sequencing of <italic>Meretrix meretrix</italic>

<italic>Meretrix meretrix</italic> is one of the most important economic shellfish species in China due to rich nutrition, delicious meat and high economic value. In this study, <italic>Meretrix meretrix</italic> DNA was transcribed and sequenced by using the second generation high-throughput sequencing technique. Through the statistical analysis of different types of microsatellites in <italic>Meretrix meretrix</italic>, the distribution frequency and number of microsatellites were understood, and the SSR composition characteristics of <italic>Meretrix meretrix</italic> were clarified at the transcriptome level. It is beneficial to the research of individual identification, germplasm resource identification, biological genetic mapping and so on, and has important reference value for further using microsatellite marker technology to carry out genetic marker-assisted breeding and population genetics research. The bioinformatics method was used to analyze the distribution of microsatellite sequences in the transcriptome of <italic>Meretrix meretrix</italic>. After searching the SSR loci of <italic>Meretrix meretrix</italic>, 8 856 complete SSR loci were found. The total length of SSR was 162 416 bp, with an average length of 18.34 bp, accounting for about 0.15% of the total sequence size, including 3 001 single-base repeat sequences, 2 254 three-base repeat sequences, 2 200 four-base repeats sequence, 1 052 two-base repeats sequences, 332 five-base repeat sequences and 13 six-base repeat sequences. The SSR of <italic>Meretrix meretrix</italic> transcriptome contained 26 types of repeats, among which the most was single-base repeat sequence A / T (2 809). The second most was triple-base repeat sequence AAC / TTG (907). Four-base repeat sequence AAAC / TTTG and two-base repeat sequence AT / TA was 588 and 561, respectively, indicating that the distribution of microsatellites in the RNA-sequencing of <italic>Meretrix meretrix</italic> had a preference for A / T. The average length of SSR (integrity) of <italic>Meretrix meretrix</italic> was 18.34 bp, and the main length of microsatellite was 12-20 bp, accounting for 41%. Most of the 8 856 SSR loci in the transcriptome had moderate polymorphism potential, which meant certain value for the future development of microsatellite molecular markers. The results of this study will provide a theoretical basis for the development of microsatellite markers, population genetic diversity, genetic linkage map, identification of germplasm resources and molecular genetics and breeding of shellfish. It also has important reference value for further study of genetic marker-assisted breeding and population genetics by using microsatellite marker technology.

Comparing and phylogenetic analysis chloroplast genome of three Achyranthes species

In this study, the chloroplast genome sequencing of the Achyranthes longifolia, Achyranthes bidentata and Achyranthes aspera were performed by Next-generation sequencing technology. The results revealed that there were a length of 151,520 bp (A. longifolia), 151,284 bp (A. bidentata), 151,486 bp (A. aspera), respectively. These chloroplast genome have a highly conserved structure with a pair of inverted repeat (IR) regions (25,150 bp; 25,145 bp; 25,150 bp), a large single copy (LSC) regions (83,732 bp; 83,933 bp; 83,966 bp) and a small single copy (SSC) regions (17,252 bp; 17,263 bp; 17,254 bp) in A. bidentate, A. aspera and A. longifolia. There were 127 genes were annotated, which including 8 rRNA genes, 37 tRNA genes and 82 functional genes. The phylogenetic analysis strongly revealed that Achyranthes is monophyletic, and A. bidentata was the closest relationship with A. aspera and A. longifolia. A. bidentata and A. longifolia were clustered together, the three Achyranthes species had the same origin, then the gunes of Achyranthes is the closest relative to Alternanthera, and that forms a group with Alternanthera philoxeroides. The research laid a foundation and provided relevant basis for the identification of germplasm resources in the future.

大顶苦瓜种质资源的形态分类及分子鉴定

大顶苦瓜种质资源的形态分类及分子鉴定

A new method based on SNP of nrDNA-ITS to identify Saccharum spontaneum and its progeny in the genus Saccharum.

The identification of germplasm resources is an important aspect of sugarcane breeding. The aim of this study was to introduce a new method for identifying Saccharum spontaneum and its progeny. First, we cloned and sequenced nuclear ribosomal DNA internal transcribed spacer (nrDNA-ITS) sequences from 20 Saccharum germplasms. Analysis of these nrDNA-ITS sequences showed a stable mutation at base 89. Primers (FO13, RO13, FI16, and RI16) were then designed for tetra-primer amplification refractory mutation system (ARMS) PCR based on mutations at base 89 of the nrDNA-ITS sequence. An additional 71 Saccharum germplasms were identified using this tetra-primer ARMS PCR method, which confirmed that the method using the described primers successfully identified Saccharum spontaneum and progeny. These results may help improve the efficiency of modern molecular breeding of sugarcane and lay a foundation for identification of sugarcane germplasms and the relationships among them.

Study on Relationship Between Cucumber Germplasm and Propamocarb Residue Using Subjective Rating Technique

Propamocarb (PM) residue in cucumber (Cucumis sativus L.) receives little attention. As is well known to all, high PM residue of cucumber could lead to increase in the violation rates of maximum residue limits and ultimately cause harm to human health. Knowledge of PM residue could help cucumber breeders in developing cultivars with low PM residue and improving cucumber quality. In this study, 32 representative cucumber accessions (26 breeding lines and six cultivars) from different regions of China were evaluated for their PM residue in fruit and leaf to provide meaning to the subjective rating, which was highly correlated with PM residual content of fruit (r=0.97) and leaf (r=0.94). In addition, PM residual content of North China ecotype was the highest and Pickling ecotype was the lowest in fruit and leaf of cucumber. The leaf had significantly higher (P<0.01) PM residual content than the fruit, and poor correlation between leaf and fruit was represented. This study verified PM residual relationship between fruit and leaf, and laid the foundation for further identification of germplasm resources and breeding of new varieties for low PM residue of cucumber.

Single- and double-SSR primer combined analyses in rice

Polymerase chain reaction (PCR) is the foundation of SSR molecular marker technology. We used sib rice varieties J518, XD1 and SD23 as experimental materials, selecting 30 pairs of SSR primers, including RM127, RM337 and RM5172, covering the rice genome, and performed single- and double-SSR primer combined analyses. We found that under the same PCR system and conditions, a single primer of the SSR primer pairs could amplify the same fragments as double primers do. The sequencing results demonstrated that some amplified fragments that we previously believed to come from double primers were actually produced by a single primer. The use of this kind of primer, such as the RM127 primer pair, for marker-assisted breeding will therefore be misleading. Additionally, using the same PCR system and conditions, some single primers that are part of SSR primer pairs can amplify many more specific fragments than double-SSR primers. For instance, in the case of the RM5172 primer pair, a single primer P1 amplified approximately three times the number of fragments as the double primer. This information can contribute to research on genetic diversity of species, understanding of genetic relationships and identification of germplasm resources. Accordingly, combined analyses of single- and double-primer amplification products not only can remove single-primer amplification fragments and false-positives from double-primer amplification products in order to improve test accuracy, but also can facilitate research on genetic diversity, exploration of phylogenetic relationships and identification of germplasm resources. We define this method as "single- and double-SSR primer combined analyses".