Genetic Diversity Of Yellow-seeded Rapeseed (Brassica Napus L.) And Effects Of Transparent Testa (tt) On Seed Physiological Characteristics

Oilseed rape (Brassica napus L.) is one of the most important oilcrop in the world. A large number of evidences had shown there were many differences of quality traits between brown or black-seeded and yellow-seeded rapeseed. The development of yellow-seeded Brassica cultivars has resulted in increased oil and protein content at the expense of unwanted crude fiber due to a thinner seed coat in yellow seeded vs. brown or black seeded forms. The development of pure and stable yellow seeded Brassica napus L. cultivars with good agronomic characteristics became an important breeding objective and many yellow-seeded genetic germplasm (Brassica napus L.) has been developed by selecting mutant from interspecific hybridization, irradiation and natural mutation from B. napus L., etc.. By intensive breeding for many years, several yellow-seeded cultivars (Brassica napus L.) have been developed and used in rapeseed production in recent years.Yellow-seeded rapeseed (B. napus L.) is one kind of important genetic and breeding germplasm resources by artificial selection for its testa color, potential of yield, quality and resistance in different natural environments. Many research intensively investigated about the different traits and mechanism between yellow and brown or black seeded rapeseed, thus no reports about diversity analysis among the yellow-seeded lines. In this study, random amplified polymorphic DNA (RAPD) and SSR markers, pigments content and thickness of testa, seed size and color, protein and oil content, seed physiology of the yellow-seeded lines (B. napus L.) were investigated to analyze genetic diversity and seed physiology characteristics of yellow seeded from different genetic sources.1. The seed weight, diameter and testa thickness of different genetic sources yellow-seeded rapeseed (B. napus L.) was studied to evaluate the genetic diversity. The results showed that the extended variation of seed weight and diameter existed in the different lines. The weight per 1000 seeds ranges from 2.12g to 4.78g, diameter ranges from 1636.8 μ m to 2049.6 μ m. According to the seed size, the yellow-seeded lines was divided into three types, big size seed, middle size seed, and small size seed. Thickness of yellow-seeded testa were ranged from 36.5 μ m to 47.5 μ m, was similar to the black-seeded, but thicker than the red color seed. The seed color index and HSB color were no significant relationship to the seed size, testa thickness and diameter in the yellow-seeded rapeseed.Rapeseed seed color exist an extended polymorphism with the color of black, red, yellow, brown-yellow in tested materials. Different color of seed were recognized with different hue (H), saturation (S) and brightness (B) value of HSB color system. According the H and B value, the rapeseed could be divided into four group: Group Ⅰ was the yellow-seeded ( H33-55° , B>35), groupⅡ mainly was brown-seeded (H40-44° , B17-25), groupⅢ was red seed (H13.2° , B14), groupⅣ was the black-seeded (H313.7° , B5.0). The H and B were related significantly to the color index which evaluated by eyes in level P=0.01.The color of yellow-seeded in this study showed color polymorphism with yellow, orange, green yellow and brown-yellow, etc., and could be divided into three types with HSB color system: The pure yellow type with about H>50° B>35 (include the yellow-seeded of B.juncea, B. carninata, B, oleracea, GH01, GH04, GH10 and GH16 ofB. napus L.), the middle yellow type with H43-50" B>40 (include the yellow-seeded of B. campestris, GhO6, H02, GH03, GH05, GH11, GH18, GH19 of B. napus), and the brown-yellowed type with H<45° B<35 (include GH09, GH12, GH13, GH17 ofB, napus L.).Fourteen yellow-seeded rapeseed lines (B. napus L.) from different genetic sources were used to analyze diversity of testa pigments content, oil and protein content. The results showed: the anthocyanin and melanin were the most important pigments in testa and their content was responsible for the variation in seed color ranging from orange to black yellow. 14 yellow-seeded lines could be classified into 3 groups: high anthocyanin content group with anthocyanin content in testa over 2.54 mg ? g-1 DW, the seed color were light yellow or orange; low pigments content group with low content of anthocyanin and melanin in testa, the testa were semi-transparent and the seed color were light yellow, greenish yellow or twany; high melanin content group with melanin content in testa over 178.4U (A290mm), the testa were black, the seed color is black yellow. Oil content of seed ranged from 36.2% to 45.5%. Protein content of seed ranged from 21.1% to 27.7%. The correlation analysis revealed the oil content was extremely significantly negatively correlated with the protein content.The cluster analysis with RAPD markers showed that the extensive genetic variation existed among 14 yellow-seeded lines by using unweighted pair group method with arithmetic average (UPGMA) based on 97 RAPD markers which were amplified with 21 decamer primers, the genetic similarity among them ranged from 0.25 to 0.909, and 14 yellow-seeded lines could classified into 2 clusters corresponding to genome differences. Genetic diversity of 19 different genetic sources yellow-seeded rapeseed were investigated using forty-two SSR markers. Forty-two polymorphic markers detected 420 alleles, he number of alleles per primer pair ranged from 2 to 21, with an average of 10 alleles per SSR locus. The value of allelic polymorphism information content (PIC) varied from 0.17~0.85,with an average of 0.73.Through cluster analysis based on the 42 SSR markers by UPGAM, the 19 lines could be classified into four groups, which is generally agree with the pedigree analysis. Furthermore, the 19 lines could be identified only with 4 primers(NAS99, NAS98, NAS91 and NAS31) that the PIC of them are highly.2. Yellow seeds and brown seeds that selected from seven yellow-seeded lines (B. napus L) in different genetic sources were used as materials to test physiological characters in germination period. Compared with brown seeds, yellow seeds had higher content of sugar and fresh weight of seedlings, lower content of protein, lower activity of Nitrate reductase (NR) and root activity (AR). The results showed that yellow seeds could convert more nourishment for growing in the germination period. After germination period, as its storage used up, absorption and synthesis ofnutritive materials would be lower than brown seeds, this might be one of reasons of lower growth potential and yields of yellows-seeded. A few yellow-seeded lines had higher NR, AR and content of protein, it implied that we could improve yielding ability of yellow-seeded lines with correct methods.20 yellow and 20 black-seeded rapeseed lines {Brassica napus L.) were taken randomly to test the flood tolerance during seed germination in the anoxia condition. The result showed that the germination rate, seedling hight, seedling weight and vigor index of yellow-seed were lower than that of black-seed, but with higher electric conductivity rate. There were the diversity of flood tolerance from 0 to 0.63 (vigor indexes) in different lines in both the yellow-seeded and the black-seeded lines. The electric conductivity rate had very obviously negative related with the vigor index, so it could be used to select high flood tolerance breeding resources in Brassica napus L. rapidly.To analyze the longevity of the yellow-seeded rapeseed during storage, the physiological differences between the yellow and black-seeded near-isogenic lines were studied by artificial ageing method. The testa rate of yellow-seeded rapeseeds decreased 20.1%, anthocyanin content decreased 25.2% and melanin content decreased 80.4% than black-seeded rapeseed. During artificial ageing, the yellow-seeded showed significant different effect of ageing compared with the black-seeded, showed faster deterioration with lower germination percentage, lower seed vigour index, lower reducing sugar and soluble protein contents than the black-seeded,increase rapidly in electrical conductivity, malnodialdehyde (MDA) content and a rapid decrease in activities of antioxidant enzymes catalase (CAT) and superoxide dismutase. The results suggested that the transparent testa of the yellow-seeded rapeseed lost some abilities to protect the embryo against adverse environmental conditions and lead to the poor storability.The imbibition behaviors of 18 rapeseed lines with different color were investigated. The result indicated that seed color was significantly related with seed increase weight% (SIW) and electrical conductivity (EC). According to the seed increased weight% of 5h imbibition, the pure yellow color seed (include GH01, GH06, GH10 of B. napus L., B. juncea, B. carniata) with rapid SIW were classified into the rapid imbibition type, the red seed (include Zhongshuang 220, 04P41, Bisahnghuaye of B. napus L., red seed of B. oleracea) with slow SIW were classified into the slow imbibition type, the other material (include black, brown-yellow, red seed of B. juncea, B. campstris, brown seed of B. caminata) were classified into middle imbibition type with medium SIW. The yellow-seeded lines also increased in EC during the imbition, indicated endured more leachate than the black and red-seeded with pigmented testa.3. In order to evaluate seed color accurately, a new method of HSB color system was developed with digital camera and computer recognization technology. The seed colore picture was collected by a digita camera, the RGB value was investigated in picture with Abode Photoshop software, and then converted in to HSB system. The hue (H) and brightness (B) of HSB system were used in...