| Non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino), which is originated from China, is a subspecies of B. campestris (chromosomal group AA, 2n=2x=20) in Brassica of Cruciferae, and is one of the most important leafy vegetables in big cities of the middle and lower Yangtze region. Its molecular marker-based linkage map construction and QTL mapping will provide an available reference to genomic structural study and genetic breeding in non-heading Chinese cabbage. In this study, several DH populations of non-heading Chinese cabbage were obtained through isolated microspore culture, and then used for molecular genetic linkage map construction, and QTL analysis for nitrate concentration and major economic traits on the map.In order to establish an efficient technique system of isolated microspore culture and construction immortal mapping population in non-heading Chinese cabbage, isolated microspore culture was performed using 7 F1 hybrids of non-heading Chinese cabbage. The effects of media and culture conditions on embryo induction, development and plantlet regeneration were studied. At the same time, the methods of ploidy identification for microspore-derived plants were studied. There was significant difference in the ability of microspore embryogenesis among various genotypes, and the highest induction rate (18.13 embryos per bud) is about 9 times as large as the lowest induction rate (2.07 embryos per bud). Induction rate of microspore-derived embryos was varied significantly with the inflorescence age of donor plant, and each genotype had the highest induction rate of microspore-derived embryos at 6-10 d after the first flower opening, then at 11-15 d. Culturing before inflorescence (< 0 d) and after 20 d, the induction rate was very low, and even no embryo. Active charcoal could improve the induction rate of microspore-derived embryos significantly, but various genotypes had different requirements for the concentration of active charcoal. Genotypes also influenced the seedling rate of microspore-derived embryos, among which those easy for embryogenesis had a low seedling rate, while those hard for embryogenesis had a high seedling rate. The embryos, which were transferred into solid medium when culturing in liquid culture for 25 d, had the highest seedling rate (37.8%). So it was important to transfer the mature embryo to the solid medium for obtaining more seedlings. When the concentration of agar was enhanced to 1% and 1.2% from 0.8%, the seedling rate could be increased largely. A period of 10 d low temperature treatment on embryos could significantly improve their seedling rate. Combined the two ploidy identification method of flow cytometry and morphology in the field, the results showed that in the microspore progeny of studied genotypes, the predominant type was diploid, then haploid and a few tetraploid, and chimera.Genotyping the DH populations of'Hanxiao'and'Aikang 5', high distorted segregation was a universal phenomenal despite of the marker type, and the number of markers came from maternal parent and paternal parent agree with 1:1 at P=0.05 level. For each locus, the frequency of band from two parent was closely, and not deflected to either parent. It could be concluded that no obvious distorted segregation in the populations, which could be used for genetic map construction and QTL mapping.Using the intervarietal mapping population derived from SW-13×L-118, containing 127 DH lines from'Hanxiao', we constructed a molecular marker based genetic linkage map of non-heading Chinese cabbage. Selecting from 2255 primers or primer combinations, 512 primers or primer combinations were reproducibly polymorphic between the parental lines, and produced 614 polymorphic markers. Chi-square tests showed that 42.67% markers were distorted from Mendelian segregation ratios. Used all the polymorphic markers for linkage analysis, however,43.49% cannot assign to any of the linkage groups. Based on 36 SSR markers provided anchors to previously published map for genome A of Brassica, we associated the linkage groups to the 10 chromosomes. After sequentially removing the markers that had an interval distance smaller than 1 cM from the upper marker, the overall quality of the linkage map was increased. There were 268 molecular markers on the linkage map, comprising 169 SRAP markers,50 SSR markers and 49 RAPD markers. The total length of the map was 973.38 cM and the average interval distance of the linkage map was 3.63 cM. The number of markers on the 10 LGs ranged from 18 (A8) to 64 (A7), with an average interval distance from 1.70 cM (A7) to 6.71 cM (A6). A map interval with the markers separated more than 20 cM was observed on A5 and A6. The distorted markers clustered in specific regions on A3, A5, A7, A8, and A9 for markers with an excess of L-118 alleles, and on A1, A4 and A6 for markers with an excess of SW-13 alleles. The markers on A2 and AO exhibiting segregation distortion were skewed towards both parents.Using the intervarietal mapping population derived from SW-13×V-126, containing 117 DH lines from isolated microspore culture of'Aikang 5', we constructed another genetic map of non-heading Chinese cabbage. Selecting from 2711 primer combinations, 381 primer combinations were reproducibly polymorphic between the parental lines, and produced 514 polymorphic markers. Chi-square tests showed that 50.19% markers were distorted from Mendelian segregation ratios. Used all the polymorphic markers for linkage analysis, however,32.88% cannot assign to any of the linkage groups. Based on 59 SSR markers provided anchors to previously published map for genome A of Brassica, we associated the linkage groups to the 10 chromosomes. There were 345 molecular markers on the linkage map, comprising 278 SRAP markers and 67 SSR markers. The total length of the map was 936.28 cM and the average interval distance of the linkage map was 2.71 cM. The number of markers on the 10 LGs ranged from 20 (A 10) to 44 (A7), with an average interval distance from 1.90 cM (A7) to 3.58 cM (A10). A map interval with the markers separated more than 20 cM was only observed on A6. A total of 42.32% distorted markers were distributed in the map and clustered on some region of the linkage group.Using composite interval mapping method, QTL analysis for nitrate concentration in leaf blade and petiole was made on'Aikang 5'DH population of non-heading Chinese cabbage after sowing 40 d and 80 d, respectively. A total of 11 QTL controlling nitrate concentration of non-heading Chinese cabbage were identified in 7 marker intervals of 5 linkage groups, including 4 QTL for leaf blade after sowing 40 d,3 QTL for leaf blade after sowing 80 d,3 QTL for petiole after sowing 40 d, and 1 QTL for petiole after sowing 80 d. In addition, the variation explained and additive effects of each QTL were also determined. These QTL individually can explain 7.73% to 19.44% of the phenotypic variation, and there were unequal gene effects on the traits. The highest contribution was from NC8-1, which was mapped on A8 and could explain 19.44% of the nitrate concentration for leaf blade after sowing 40 d. Some QTL could be identified by different indices. These mapped QTL could provide information for genetic breeding of low nitrate concentration non-heading Chinese cabbage.'Aikang 5'DH population and constructed linkage map were employed to map QTL for 13 economic characters using composite interval mapping method. Forty-seven putative QTL were identified on 9 linkage groups, including 2 QTL for plant height,8 QTL for plant weight,3 QTL for blade weight,9 QTL for petiole weight,2 QTL for coactus stem weight,2 QTL for head width,4 QTL for waist width,1 QTL for leaf number,4 QTL for blade length,6 QTL for blade width,3 QTL for petiole length,3 QTL for petiole width, and 2 QTL for petiole thickness. These individually explained 7.93% to 25.88% of the phenotypic variation, and there were unequal additive effects on the traits. The QTL of associated traits often located on the same loci or near region of a linkage group. These mapped QTL could be used for marker assisted selection program for the major economic characters in non-heading Chinese cabbage in the future.Based on'Sulv' DH population of non-heading Chinese cabbage and its molecular genetic map, the traits of plant height at 8 developmental stages before harvest were investigated in 2006 and 2007, respectively. By conditional variable analysis of QGAStation, the net growth data of plant height were calculated. Using composite interval mapping method combined with mixed genetic model, unconditional QTL and conditional QTL of plant height were detected simultaneously. With two-year data, a total of 11 unconditional QTL in 6 linkage groups and 23 conditional QTL in 8 linkage groups were identified for plant height of non-heading Chinese cabbage. The results indicated that the number and type of QTL and their genetic effects for plant height varied with different measuring stages. Each QTL can explain 7.92-28.25% of the total phenotypic variation. Two QTL (PH8-4 and PH8-5) were identified to be associated with plant height using both unconditional and conditional mapping method simultaneously in both years. These results demonstrated that it is highly effective for mapping QTL of developmental traits using both unconditional and conditional analysis methodology. |
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