| The new line of red clover (Trifolium pratense L.) is an excellent cold resistant material suitable for alpine region in China, which filled the blank of red clover breeding in Inner Mongolia. Red clover new line and some red clover materials were studied by morphological features, chromosome, SSR markers, and asexual reproduction. The genetic origin of red clover which is natural distributed in Yakeshi in Inner Mongolia was discussed at first time in this paper. There is a great significance of the use and innovation of elite germplasm in red clover. Obtained the following results:1、70materials can be divided into the early flowering, the middle flowering and late flowering, respectively, accounted for22.86%,68.57%and8.57%. According to growth period in second year, red clover can be divided into the early, medium and late maturity groups, and growth period were64d,71.96d and84.17d, respectively. New lines of red clover is a late flowering type. Growth period was74d belongs to the medium. Average growth rates in daily was0.44cm/d which close to the average level of red clover groups. Winter survival rates were from0%to88.89%in70materials at Hohhot. The new line of red clover winter rate was88.89%, and cold resistance was outstanding.2、The studies of morphological characteristics have shown that red clover organs have great genetic variation. The coefficient of variation on leaf mark, branches, inflorescences per stem, seed color, internode length, lateral branch number, leaf area, natural height were relatively larger. The order of genetic diversity from the largest to the smallest was branches> leaves> seeds> flowers> roots. The crown width, height and branches were main indicators to divide morphological types of red clover. New lines of red clover had tan roots, and usually green stem which was semi-erect. Strains clump size was the middle in the red clover groups. Leaf mark were middle or no mark mainly. Leaf area was bigger relatively, and leaf shape was wider and shorter. The main flower color was purple,then light purple and pink, white occasionally. Seed color was relatively shallow, ana a large number of seeds was yellow. Seed size was middle, and1000-grain weight was smaller relative to the red clover groups. The new line of red clover was similar to the number of29,35,3,41,5,19,2,67in morphological characteristics.3、Chromosome number of43red clover was2n-2x=14, x=7. Chromosome type included m, sm, st, and t, six of which had satellite. There were5kinds karyotype (1A,2A,1B,2B and3B)and8kinds of karyotype formula. The karyotype formula of new line of red clover was2n=2x=14=8m+6sm(2SAT). The asymmetry of interchromosomal is greater than intrachromosomal, and was relatively high in the red clover populations. According to the the Langlet’s chromosome morphology criteria for the classification, the new line of red clover chromosome type is the T-type, primitive relatively.4、The analysis of SSR marker showed that using8pairs of primers in32materials obtain219polymorphic bands totally. The polymorphism percentage was100%, and the number of effective alleles was1.1636. The Nei’diversity index, shannon information index, total variance, variance between the materials, coefficient of genetic differentiation and gene flow were0.1229,0.2241,0.1218,0.0387,0.3179respectively.31.79%genetic variation of the red clover exists in external populations,68.21%was in internal populations. The fingerprints of the red clover new line was obtained, which can effectively distinguish from two registered varieties of red clover in China. The Nei diversity index of red clover new line was0.0665, and Shannon’s information index was0.1065, lower than the average population diversity level. SSR genetic distance showed that the new line of red clover was similar to the number of60,22,53, which were from Russia, Spain and Russia respectively.5、The best ramets per plant was6-7in division propagation, and the highest proliferation coefficient was6.38; The highest proliferation coefficient of cutting can up to6.40using entire branches in branching period.The study of tissue culture showed:the best method of seeds sterilization for red clover is the75% ethyl alcohol treatment followed by disinfection with0.1%HgCl2for10min.1/4MS medium with1.5%sucrose without hormones was most effective for seed culture. Cotyledons, hypocotyl, petiole, leaf and root callus were easy to be induced in medium of MS containing0.5mg·L’16-BA and1-2mg·L-12,4-D. The best callus subculture medium was MS containing0.5mg·L-116-BA and1.0mg·L-12,4-D. The best medium for differentiation was MS plus0.75mg·L-16-BA and0.5mg·L-1NAA, the differentiation rate can reach up to16.67%. Corm-bud could be multiplicated greatly in MS medium plus0.5mg·L-16-BA and1.0mg·L-1IBA. Regenerated plants were immersed in50mg·L-1IBA for1h, then vaccinated in MS medium plus IBA0.5mg·L-1,the rooting rate was80%.1mg·L-16-BA+0.5mg·L-1NAA hormone combination was suitable for germination of shoot tips and axillary buds in red clover.6、We concluded that red clover that distributed at Yakeshi in Inner Mongolia is likely to come from western Russia and was a European descent. |
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