Genome Survey Sequencing of Luffa Cylindrica L. and Microsatellite High Resolution Melting (SSR-HRM) Analysis for Genetic Relationship of Luffa Genotypes.

Jianyu An,Mengqi Yin,Dongting Gong,Yajing Guan,Jin Hu,Xiaowen Jia,Qin Zhang

doi:10.3390/ijms18091942

Jianyu An, Mengqi Yin + Show 5 more

Open Access

https://doi.org/10.3390/ijms18091942

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Abstract

Luffa cylindrica (L.) Roem. is an economically important vegetable crop in China. However, the genomic information on this species is currently unknown. In this study, for the first time, a genome survey of L. cylindrica was carried out using next-generation sequencing (NGS) technology. In total, 43.40 Gb sequence data of L. cylindrica, about 54.94× coverage of the estimated genome size of 789.97 Mb, were obtained from HiSeq 2500 sequencing, in which the guanine plus cytosine (GC) content was calculated to be 37.90%. The heterozygosity of genome sequences was only 0.24%. In total, 1,913,731 contigs (>200 bp) with 525 bp N50 length and 1,410,117 scaffolds (>200 bp) with 885.01 Mb total length were obtained. From the initial assembled L. cylindrica genome, 431,234 microsatellites (SSRs) (≥5 repeats) were identified. The motif types of SSR repeats included 62.88% di-nucleotide, 31.03% tri-nucleotide, 4.59% tetra-nucleotide, 0.96% penta-nucleotide and 0.54% hexa-nucleotide. Eighty genomic SSR markers were developed, and 51/80 primers could be used in both “Zheda 23” and “Zheda 83”. Nineteen SSRs were used to investigate the genetic diversity among 32 accessions through SSR-HRM analysis. The unweighted pair group method analysis (UPGMA) dendrogram tree was built by calculating the SSR-HRM raw data. SSR-HRM could be effectively used for genotype relationship analysis of Luffa species.

Highlights

Luffa, or sponge gourd, belonging to the Cucurbitaceae family, is a diploid species with 26 chromosomes (2n = 26) and a cross pollinated crop [1]
Results of polymerase chain reaction (PCR) products obtained with the SSR marker Zhejiang University Luffa marker (ZJULM) 50 on 6% denaturing polyacrylamide gel at 60 mA constant current (Nos. 1–32, 32 accessions, detailed explanation can be seen in Table 6; DNA Marker: DL2000)
The genome size of Cucumis melo L. was 375 Mb, representing 83.3% of the estimated melon genome [20], while Cucumis sativus L. was only 243.5 Mb, 72.8% sequence anchored on chromosome [21]

Summary

Introduction

To investigate and provide a genomic resource of Luffa for further study, genome survey of L. cylindrica was conducted using NGS technology These results would be useful for crop improvement programs and better utilization of genomic information in the future [6]. High resolution melting (HRM), a sensitive mutation detecting method, has been identified as a powerful, efficient and cost-effective method to analyze genetic variation [10] It was considered as an evolution of real-time polymerase chain reaction (PCR) technology. Based on the HRM curves, amplicons can be identified even with the same Tm values, and be classified to several categories according to their normalized melting curves and difference plots It allows detection of sequence variants without sequencing or hybridization procedures [14]. The HRM technique efficiency for identifying SSRs in PCR amplifications was assessed and the SSR-HRM method was used to discriminate different Luffa species and cultivars

Genome Sequencing and Sequence Assembly

Genetic Relationship Analysis by SSR-HRM

Discussion

Genomic SSR Markers Development

Plant Materials and DNA Extraction

Genomic SSR Marker Development