SSR Markers Development And Genetic Linkage Map Construction Of Pea (Pisum Sativum L.)

Field Pea (Pisum sativum L.) is the fourth largest legume crop globally, and China is the largestpea producer in the world. Pea is rich in various nutritional elements with comprehensive balance forhuman and animal consumption, such as high quality proteins, carbohydrates, mineral nutrients,vitamins etc. Previously, the development of pea SSR markers was far behind other crops. Afterscreening all the published pea SSR primer pairs, only91pairs found available. The main goal of thisstudy is to develop SSR markers from pea magnetic beads enrichment method, as well as throughdatabase information retrieval method and published information searching method, by using fababean(Vicia faba L.), grasspea (lathyrus L.) and lupin (lupines L.) references. The genetic linkage mapof pea was constructed with these available SSR markers using the F2population.The main results of this study are summarized as follows:1.18,770primers pairs of pea genomic SSR markers were developed by using magnetic beadsenrichment method. The6287primer pairs of dinucleotide repeat type were evaluated by using two setsof parents, which resulted in2974pairs of successful amplification. Among them,2505pairs producedsingle bands, the remaining469pairs produced polymorphic bands. The most efficient amplificationoccurred when the length of the amplification production ranged from200bp to250bp, followed by150bp to200bp. When the length of amplification production was less than100bp, the ratio ofamplification was much lower.2.18852EST sequences of pea,8880EST sequences of grasspea and10350EST sequences oflupin were obtained by applying database information retrieval method. Among them,399pairs of peaEST-SSR primers,300pairs of grasspea EST-SSR primers and188pairs of lupin EST-SSR primerswere successfully designed by using Primer3.0software. The PCR amplication efficiency of aboveEST-SSR primers together with66pairs of faba bean EST-SSR primers, were evaluated by using twosets of parents.277EST-SSR primers of pea amplified clear bands, of which71were polymorphic;149EST-SSR primers of grasspea amplified clear bands, of which39were polymorphic;52EST-SSRprimers of lupin amplified clear bands, of which14were polymorphic;60EST-SSR primers of fababean amplified clear bands, of which41were polymorphic.3. By using the transcriptome database of pea and faba bean that published in periodical literature,1707pairs of pea EST-SSR markers and192pairs of faba bean EST-SSR markers were designed. Thepea EST-SSR markers include993pairs of single type and714pairs of compound type. Afteramplification of906pairs of single type primers by using the two sets of parents,730pairs of theprimers were successfully amplified bands, of which248pairs revealed polymorphic. After amplication of192pairs of faba bean EST-SSR primers by using the two sets of parents,13pairs of primers weresuccessfully amplified bands, of which10pairs revealed polymorphic. Upon online collecting all thepublished SSR markers of pea, the duplicated markers we designed were rechecked and removed, andonly114pairs of available EST-SSR markers were left for amplification test.Of which,87pairs ofEST-SSRs amplified production with37pairs identified polymorphic.4.300pairs of grasspea EST-SSR primers,188pairs of lupin EST-SSR primers and66pairs offaba bean EST-SSR primers were tested by two sets of parents for inter-genus transferability analusis.The results indicated that the transferability ratio of faba bean EST-SSR markers (90.91%) to pea wasthe highest, followed by that of grasspea (49.67%) and lupin (27.66%).5. The F2mapping population consisting of190individuals was derived from the cross ofG0005527(highly cold infection) and G00003973(highly cold resistance). After screening all the aboveSSR primer pairs between the two parents,815pairs of polymorphic SSR primers were used for F2segregation population analysis. The genetic linkage map of pea anchoring243SSR molecular markersin22linkage groups was constructed. This genetic linkage map covers2399.25cM with an averagegenetic distance of9.87cM. The anchored SSR markers on each linkage group ranged from2to48, andthe linkage group length ranged from5.47cM to467.03cM.