Selection Of Breeding Parents And Assessment Of Genetic Diversity Among The Inbred Lines Of Zinnia Elegans

Zinnia elegans,an important annual ornamental plant,are usually planted in the garden via F₁ seeds in China.However,most of Zinnia elegans cultivars depend on importation,the high cost spended in it necessitated us to develop our own breeding system of Zinnia elegans for expoilted novel cultivars adapting to native climate.In this study,the flowering and pollination biology of Zinnia elegans was investigated. Pedigree selection method and backcross and testcross were used for selecting inbred lines and male sterile lines.And molecular markers were developed to identify the genetic diversity of inbred lines.The results were presented as following:1.The flowering and pollination biology.Zinnia elegans flowers open at 8:30～16:00 in daytime.When it flowers,the stigmas of florets stand upright and keep high turgidity for 7～10 days.The stigmatic pollen receptivity for 1～3d,4～6d,7d and 10d was 79.9%～83.1%,56.1%～63.0%,37.0%～43.3%respectively according to the benzidine-H₂O₂ test.The seed set of the Y stigma phase,the tip of Y slightly curling stigma phase and columnar stigma phase was 76.3%,48.5%and 36.8%respectively by the test of artificial pollination.The seed set of no-treatment,detruncating one lob of stigma and style of detruncating stigma was 48.8%,30.9%,19.9%respectively.The optimum in vitro pollen germination medium of Zinnia elegans was ME₃ basal medium supplemented with 16%PEG₄₀₀₀ and 12%sucrose.The pollens collected at 10:00 am have much higher germination rate than those collected at 3:00 pm.Mature pollen grain was tricellular type,the viability of pollen lasted for 18h at room temperature under dry condition,and no longer than one day at 4℃and moist condition.The optimum twice sowing time for seed production in Wuhan is late February to early March for spring sowing and late June to mid-July for summer sowing.Seeds should be collected 25 days after pollinationand had a high rate of germination and long storage life.2.Selecting and breeding of inbred lines.Since selfing of Zinnia elegans get lower seed set rate,pollinate at flowering period with 3%NaCl treatment can get higher seed set rate than pollinate at bud phase.The 23 inbred lines of Zinnia elegans for diverse colors, petal type,plant shape and 1 inbred line of Zinnia angustifolia were obtained by repeated selfing and continuous selection.3.Production of male sterile lines of Zinnia elegans.The male sterile lines were obtained in two pathways:One is to back cross the F₁ generation with male sterile plants that segregated among selfed F₂ generation continuously,and another way is to testcross the fertile plants from self-F₅ generation with male sterile plants that segregated from the same inbred line.Through these ways,a half of male-sterile plants could be obtained.The male-sterile lines were analyzed and identified through sibmating,selfing of fertile plants, and testcrossing with a number of inbred lines(restorer lines),and proved that it was a type of stable male sterile lines.Six recessive nucleus male sterile lines such as scarlet AH209AB,orange-red J16601AB were obtained.The petal of male sterile plant degraded as a thread-like structure,the stamens were villiform in appearance and no pollens were formed,stably sterility,were free from the impact of environmental factors.The male sterile lines are of great use value in F₁ seeds production.4.A comparative analysis of genetic diversity of Zinnia elegans inbred lines using morphological traits,RAPD and ISSR.In order to select the fight lines for crossing of Zinnia elegans,genetic diversity was assessed by morphological traits,RAPD and ISSR data.Analysis of thirty-four morphological Waits showed that flower color was not the important index for morphological classification and flower diameter,plant shape and petal pattern can identify 20 inbred lines of Zinnia elegans well.147 RAPD markers fragments amplified with 12 arbitrary primers,and 128 ISSR markers fragments generated from 9 primers were employed to discriminate polymorphism between 20 accessions.The number of polymorphic loci,the percentage of polymorphic loci, Shannon's Information index(â… ) and effective number of alleles(Ne) was 100,68.03%, 0.3559,1.4169 for RAPD markers,and 97,76.38%,0.4013 and 1.4728 for ISSR markers. The Mantel-test indicated that there was a significant correlation(R=0.733) between RAPD and ISSR markers.The clustering analysis showed that the grouping had a correlations with the geographical origin and flower color was not the most important character of the classification of inbred lines of Zinnia elegans.In contrast,the morphological matrix had a low correlation with RAPD,ISSR data matrices(R=0.381, 0.377).When analyzed relationship,especially pedigree relations,molecular marker was superior to morphological marker for assessing genetic diversity among the different accessions.ISSR was better than RAPD in the aspect of polymorphism and repeatability. The suitable molecular marker,combined with morphological characteristics,can provide more accurate results in the breeding process when evaluated genetic diversity among inbred lines.