Development Of Genomic SSR Markers For Luffa Species And Identification Of Luffa Genotypes By SSR-HRM Method

Luffa cylindrica(L.)Roem.is an annual climbing vine of the Cucurbitaceae family,and it is also an important vegetable crop in China.Although many studies on Luffa germplasm resources and conventional breeding have been performed,genetic studies are still in their infancy.The narrow genetic and genomic resources has seriously restricted the breeding improvement of Luffa.In this study,the genome of Luffa was sequenced and studied for the first time,using Luffa cultivar ’Zheda 23’.A number of SSR markers were developed,and microsatellite high resolution melting(SSR-HRM)method for luffa was established.The results obtained were as follows: 1.Genomic SSR markers development based on the genome of L.cylindrica.The genome sequence data was about 43.40 Gb,with 96.77% Q20 bases(base quality > 20)and 91.56% Q30 bases(base quality > 30),about 54.94í coverage of estimate genome size,789.97 Mb.The guanine plus cytosine(GC)content was calculated to be 37.90%,and the genome sequences were little heterozygous,only 0.24%.From the initial assembled L.cylindrica genome,125094 microsatellites(SSRs)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 74/80 primers could be amplified successfully,in which 51 markers could be also used in L.acutangula.2.Identification of Luffa genotypes by SSR-HRM method.19 of common SSR markers were tested and used to investigate the genetic diversity among 32 accessions,28 L.cylindrica accessions and 4 L.acutangula accessions,using SSRHRM analysis.The UPGMA dendrogram tree was built by calculating the SSR-HRM raw data,and these 32 accessions were divided into two groups.Thus the genetic distance coefficient among these accessions ranged from 0.11 to 0.86.It proved that accessions with similar traits were clustered together.To evaluate the reliability and efficiency of SSR-HRM method,polyacrylamide gel electrophoresis(PAGE)and sequencing were carried out using the same PCR product obtained with ZJULM 50.Compared with PAGE,the result showed that SSR-HRM was a method with relative high resolution,high throughput and efficiency.And compared with sequencing,SSRHRM required less money and could obtain nearly the same result in a relatively short period.Therefore,SSR-HRM has become increasingly popular in many crop analyses such as cultivar identification and genotyping.