Screened Molecular Markers Related To Heat Tolerance And Analyzed Genetic Basis In Tall Fescue Single Plants

In order to establish an efficient system of single plant selectionand provide a scientific parents selection basis for tall fescue heat andsummer tolerant breeding, molecular markers related to heat toleranceand summer tolerance were screened for marker-assisted selectionbreeding, and then genetic basis of heat tolerance was analysed forparents selection.Initial216tall fescue single plants from12germplasms differingin heat tolerance were used as base population to identify their heatstress (40℃/35℃, day/night,12h/12h) tolerance in artificial climatechamber and summer tolerance in experimental field in Shanghai. Twobulks (pools) of heat tolerant and sensitive DNA pools were generatedrespectively from11extremely heat tolerant and11extremely heatsensitive individuals of193survived plants from the heat stress.Random amplified polymorphism DNA (RAPD) and Inter-simplesequence repeat (ISSR) were employed and accompanied withmodified bulked-segregant analysis (BSA) to screen molecular markersrelated to heat tolerance.In the environment of natural distance isolation, typically heattolerance clones(TF06) and heat sensitive clones(TF16) were used togenenate seeds of6different first generation groups by the pollinationmethods of pollinated between clones(SOP1), pollinated between twostrains(OPF1) and pollinated freely with other11summer tolerancestrains (OPP1). Two hundreds and thirty-six single plants, which comefrom TF06SOP1, TF06×TF16OPF1, TF16×TF06OPF1and TF06OPP1, were put into artificial climate chamber for heat stress(40℃ /35℃, day/night every12h). The changes of the plant height, the visualscore, the tiller numbers, the leaf photochemical efficiency and the leafchlorophyll content before and afer the heat stress were investigated toestimate the heat tolerance of single plants.The research has led to thefollowing results and conclusions:1)Initial216tall fescue single plants can be ranked from the mostheat sensitive to the most tolerant according to their heat injuredsymptoms and the days of heat stress the plants could be survived from.Although the correlation coefficient between heat and summertolerance among193plants was significantly different from zero, thecoefficient of determination (r2) is only3.4%. It is reasonably inferredthat: the heat tolerance of a single plant is not certainly related to itssummer tolerance, which implied that heat and summer tolerant plantneeds to be identified and selected under heat and sumer stressrespectively.2)The polymerase chain reaction (PCR) results showed that6outof800RAPDs and2out of100ISSRs primers amplified polymorphicbands between heat tolerant and intolerant DNA pools. Of the8primers,“351” amplified750bp band (351-T750) which issignificantly related with heat tolerance, while “138” showed a wellregularity of no band of950bp among the single plants within the heatsensitive pool. The PCR results of351and138primers in193individuals showed that58%and46%of the36heat tolerant plants(heat tolerance ratings≥7) had the characteristic bands of351-T750and138-T950, which were significantly higher than10%and14%plantswith the bands in21heat susceptible plants (heat tolerance ratings≤3)respectively. In addition,138-T950was detected to be significantlyrelated with summer tolerance and the band of138-T950is thecharacteristic of the summer tolerant plants. It is reasonably inferredthat:138-T950could be used as molecular marker for assistantselecting of heat and summer tolerant single plants, while351-T750could be used only for heat tolerant plant selection. 3)Among the results of the germination test of each1000seeds ofthe6first generation groups, the seeds germination rate of TF16SOP1is zero which indicates TF16is strictly self-incompatibility, the seedsgermination rate of TF06SOP1is0.12%which indicates TF06ishighly self-incompatibility, and therefore the first generation generateby TF06and TF16pollinated with each other (TF06×TF16OPF1andTF16×TF06OPF1) and the first generation generate by then pollinatedwith other strains naturally(TF06OPP1and TF16OPP1) can be see asthe results of naturally outcrossed by the two clones thenselves and thetwo clones with different strains.The first finial generation generatedby TF06clones pollinated freely with multiple lines (TF06OPP1) ismost tolerant to heat stress, but it was not significantly strongger thanthe first finial generation generated by TF06clones pollinated withthemselves (TF06SOP1). In the pollination methods of free pollinationbetween TF06and TF16clones, the single plants generated from TF16(TF16×TF06OPF1) were the most sensitive to heat stress, but it wasnot significantly weaker than the single plants generated from TF06(TF06×TF16OPF1).It is reasonably inferred that: the heat tolerancecharacter of tall fescue, which keeps heterosis, is mainly controled bynucleus gens, so the first generation groups keeps highly tolerance toheat stress can be generated in the condition of multiple heat toleranceclones served as the open pollinated parents. Mover, it can preliminarydeduced that the heat tolerance of TF06controled by more than twoheterozygous dominant gene, while the heat sensitive character ofTF16is main controled by more than2recessive genes, but the precisepairs of the nuclus genes of heat tolerance needed to be revealed byfurther data analysis and more experimental research.