Luffa Germplasm Traits Investigation And The Analysis Of Genetic Diversity

The local varieties of Luffa in China play a dominant role in the market with less hybrid varieties.In recent years,our country began to attach importance to the breeding of hybrid varieties.The Luffa genetic relationship analysis is an effective means of assisted breeding by Morphological marker and Molecular marker.In this experiment,60 highly purified Luffa inbred lines with stable characters served as experimental materials,and 16 agronomic traits were investigated to analyse its genetic diversity;Based on optimization of Luffa SRAP,ISSR molecular marker reaction system,the genetic diversity genetic relationship of 60 Luffa were analyzed by SRAP and ISSR.We establish genetic clustering figure and identify the genetic relationship of varieties so as to provide some reference basis for germplsm identification,classification and the cross breeding work.Main results were as follows.1.Investigation and analysis of 60 Luffa agronomic traitsObservation and analysis of 16 agronomic traits on 60 Luffa materials shows:The coefficient of variation ranged from 0%-111%.The Snannon's information ranged from 0-2.037.In a total of 16 morphological descriptors,10 qualitative and 6 quantitative were measured.They varied from 1.824 in the first female flower index to 2.037 in length of melon stalk height based on the 10 quantitative descriptors and from 0 in leaf shape to 1.371 in the fruit length based on the 6 qualitative descriptors.Results show that the variation coefficient and diversity index of each material are different.On the whole,the 60 Luffa feature high variation degree and diversity.Through the principal component analysis,5 indexes that could cover most agronomic traits were extracted.The principal component 1 is distinguish Luffa acutangula(L.)Roxb and Luffa cylindrica(L.)M.J.Roem.The principal component 2 is flesh.The principal component 3 is fruit shape.The principal component 4 is maturity.The principal component 5 is exterior quality.The 60 Luffa germplasm were clustered into 2 groups based on the morphological data.The first group includes Luffa acutangula(L.)Roxb.and the second group includes Luffa cylindrica(L.)M.J.Roem.2.Optimization of SRAP Amplification System in LufahIn order to obtain SRAP reaction system of Luffa we took an optimization experiment with single factor design.Experimental results showed the optimal 25?L SRAP amplification of Luffa which contained 0.2 mmol/L dNTP,1.25 U Taq DNA polymerase,75 ng template DNA,0.16 umol/L single primer,2.0 mmool/L Mg2+,2.5?L 10×Buffer.The best amplification procedures was pre-denaturation at 94? for 5 min;Next enter 5 cycles,including 94? for lmin,35? for 1 min,72? for 1 min;then enter 35 cycles,ineulding 94? for 1 min,52? for 1 min and 72? for 1 min;finally,extension at 72? for 10 min.The test results showed the amplification products which were clear and bright with good repeatability.Accordingly,it was suitable for SRAP analysis of Luffa.3.Analysis of the genetic diversity in Luffa by SRAP13 pairs of SRAP primers were screened out of 300 SRAP primer pairs which were produced 142 DNA bands,the average number of polymorphic DNA bands amplified by each primer pair was 10.92,polymorphic bands were 121,21 bands were basic,the ratio of polymorphic bands was 85.2%.The results show that the high genetic diversity of Luffa germplasm Resources and SRAP can detect a good genetic diversity of germplasm resources of Luffa.The 60 Luffa germplasm were clustered into 2 groups based on the morphological data.The first group includes Luffa acutangula(L.)Roxb.and the second group includes Luffa cylindrica(L.)M.J.Roem.The result was consistent with the results of cluster analysis obtained by morphological marker.The tested species were classified into 2 cluster groups with the similarity coemcient of 0.692 and 0.698.The first group was Luffa cylindrica(L.)M.J.Roem with two subgroups including short cylinder and long cylinder types.The second group was Luffa acutangula(L.)Roxb with two subgroups including short sticks and Long sticks types.4.Establishment of ISSR amplification system of LuffaWe took an optimization experiment with single factor design by using the Luffa as the material of filter system to obtain ISSR reaction system of Luffa.The greatest 25 ?L ISSR reaction system which contained with 50 ngDNA,0.2 mmol/L dNTP,1.0 U Taq DNA polymerase,0.4 umol/L primer,2.5 ?L10×Buffer,2.0 mmol/L Mg2+.On this basis,optimized annealing temperature of primers which have been screened,and ultimately determined the optimal annealing temperature of 12 primers.Under the system,5 min pre—denaturation at 94?,45 S denaturation at 94?,1 min annealing at optimal annealing temperature,1.5 min extension at 72?,for 35cycles,10 min extension at 72? after the end of the cycle,4? preserved.Amplified bands were clear,effective,and suitable for ISSR method analysis.5.Analysis of the genetic diversity in Luffa by ISSR12 pairs of ISSR primers were screened out of 100 ISSR primer pairs which were produced 154 DNA bands,the average number of polymorphic DNA bands amplified by each primer pair was 14,polymorphic bands were 143,11 bands were basic,the proportion of polymorphic primers ranged from50%-100%and the ratio of polymorphic bands was 92.9%.The tested species were classified into 2 cluster groups with the similarity coemcient of 0.642,The firsr goup was Luffa cylindrica(L.)M.J.Roem with two subgroups including short cylinder and the second group was Luffa acutangula(L.)Roxb.The same can be according to the melon shaped Luffa cylindrico(L.)M.J.Roem and Luffa acutangula(L.)Roxb were divided into two categories with the similarity coemcient of 0.718 and 0.646.The results were in agreement with the SRAP analysis.