|dc.description.abstracteng||Oilseed rape (Brassica napus L.) is the world’s third largest source of vegetable oil for human nutrition; also the meal is regularly used as animal feed. Yellow seed is one of the target characters in the breeding program, as it associated to lower dietary fiber content and higher oil and protein content. Mature seeds of B. napus will gradually lose their viability during long term storage; this process is defined as natural aging. Seed viability is influenced by several environmental factors, but partly it is also determined by genetic factors. Seed aging is an acknowledged problem in agriculture, because it is involved in mechanisms leading to loss of viability and vigor. Yellow-seeded genotypes tend to deteriorate faster compared to the black-seeded ones. They show a reduced seed longevity, i.e. the ability to germinate after being stored for a longer period. Since material of naturally aged seeds are not always available, artificial seed aging protocols are often utilized to imitate the natural aging. Exposing seeds to high temperature and moisture have been commonly used for aging seeds artificially in the laboratory. The research aimed to study the inheritance of seed germination and seed longevity traits in two DH populations which segregate for yellow seed coat color, also in relation with several seed quality traits. A further objective was to investigate the inheritance of seed germination and seed longevity employing natural seed aging of the DH Sollux x Gaoyou population. This population has been stored in an ambient storage conditions at the University of Göttingen for 13 years.
The first two doubled haploid populations were derived from crosses between the two yellow-seeded type 4042 (winter type) and DH 1372 (spring type) with the black seeded winter oilseed rape cultivar Express, inbred line 617. The 4042 x Express 617 population was grown in the five environments Reinshof 2014, Reinshof 2015, Einbeck 2015, Reinshof 2016, and Einbeck 2016. The DH 1372 x Express 617 population was grown at Reinshof 2015 and 2016. The third DH population was derived from the cross of the old German cultivar Sollux and the Chinese semi-winter cultivar Gaoyou; both were black seeded. Field experiments have been performed at two locations each in China and in Germany in the year 2001.
Seed quality traits (e.g. oil, protein, glucosinolates, and fiber components (NDF, ADF, ADL) were predicted by NIRS (Near Infrared Reflectance Spectroscopy) estimation of seed samples. Other seed phenotypic traits were also observed, such as seed color, TSW (Thousand Seed Weight), and pre-harvest germination percentage. Comparison of seed germination before and after artificial aging treatment was carried out by determining percentages of radicle protrusion, complete germination, and of infected seeds. Hypocotyl length (cm) was also measured as an indicator for vigor. Statistical analysis was performed by PLABSTAT software for analysis of variance components, heritabilities and Spearman’s rank of correlation coefficients. In both populations of yellow x black seeded types (4042 x Express 617 and DH 1372 x Express 617), a large genetic variation was found for seed oil, protein, glucosinolates, fiber contents (NDF, ADF, ADL), seed coat color, and full germination rate and hypocotyl length after aging. There is a strong positive correlation between seed coat color and fiber contents (ADL, ADF, and ADL), but no correlation is found between seed color and seed oil and protein content. Heritability is high (above 0.80) for both populations for seed color and seed fiber contents.
The pre-harvest germination percentage in the present study is associated with many traits. In both populations, seeds containing high percentage of pre-emergence sprouts are also exhibit less seed oil content, higher protein content, lower NDF content and germination rate, also higher percentage of radicle protrusion. The yellow seeds of DH 1372 x Express 617 are exhibiting more of this trait, but there is no such association existed in DH population of 4042 x Express 617. Especially in DH 1372 x Express 617 population, these type of seeds are also associated with lower total oil & protein content, lower seed fiber components (NDF, ADF, ADL), and higher seed infection rate.
In both populations, seed color is highly correlated to all seed fiber contents. Before seed aging treatment, seed color is positively correlated to full germination rate, negatively correlated to radicle protrusion and seed infection rate, and no correlation to hypocotyl length. It means that black-seeded lines has better germination rate compared to the yellow-seeded ones. After artificial aging, seed color has no influence in seed longevity traits in both populations. The mean value of germination rate is dropped after aging treatment from nearly 100 % to 62.83 % for DH population of 4042 x Express 617 and to 57.55 % for DH 1372 x Express 617.
A different story was observed for seed fiber contents (NDF, ADF, ADL). In DH 1372 x Express 617, the seeds containing high fiber have significant higher germination rate, less radicle protrusion and seed infection compared to low fiber seeds. But after artificial seed aging, these correlations did not exist anymore. Before aging, in DH population of 4042 x Express 617, seed fiber contents only limit the radicle protrusion rate. Seeds with high fiber contents will have better full germination rate and hypocotyl length, and less radicle protrusion and seed infection rate. Less full germinated rate after seed aging are exhibited by seeds of high oil content and total oil and protein.
The ADL content in the first two populations exhibits a bimodal 1:1 frequency distribution, an indication that this trait is controlled by a major gene. For 4042 x Express 617 population, a bulk specific SNP-markers was performed at KWS SAAT SE in Einbeck with an Illumina Infinium 20K SNP chip for low and high ADL content bulks. The result was later confirmed with individual genotypes through KASP genotyping. In addition, non-targeted metabolite fingerprinting analysis was executed on green seeds of this population to measure the content of metabolite compounds related to the ADL content. Eleven polymorphic loci was detected in 4042 x Express 617, all are located in
chromosome C03. Two candidate genes that possibly control ADL content were identified. The first one is MATE transporter related to expression of TT12 gene, encodes transparent testa which lead to yellow seed color. The second one is trans-cinnamate 4-hydroxylase (C4H), encodes one of the precursors to lignin biosynthesis. DNA and marker analysis for the second population (DH 1372 x Express 617) will be essential for further investigation.
The seeds of the DH population Sollux x Gaoyou were naturally aged by storing them in ambient room temperature for 13 years (2001 - 2014). Aged seeds were germinated and grown in the green house in 2016 to obtain fresh seeds (before aging treatment). The same germination method as described above was employed for both naturally-aged seeds and fresh harvested seeds. Data of seed quality traits was available only for German-grown locations from a previous study.
The DH Sollux x Gaoyou germination rate for natural aging seeds was ranged from zero to 89 %. The China-grown seeds displayed better germination percentage than the German ones. The average of seed germination rate is 95 %, and after 13 years of natural aging it was drastically reduced to 18 %. Although there are some significant correlations among seed quality traits, there are no significant correlations between seed quality traits and seed germination or seed longevity traits worth noted, perhaps due to both parents are black-seeded cultivars, therefore having more narrow variability.
For future outlook, further verification study is necessary to confirm the position of second candidate gene, C4H (trans-cinnamate 4-hydroxylase), by employing more KASP markers for DH population of 4042 x Express 617 to the downstream direction from the gene interval investigated in this study. The second population, DH 1372 x Express 617 was grown again in 2017 in Reinshof. Similar observations will be completed after the harvest in August 2017 and added to the first two environment data. The extracted DNA has been sent for KASP marker analysis, and later will be followed by candidate gene identification. DH Sollux x Gaoyou has completed the natural aging treatment, and will have newly harvested seeds from Reinshof in August 2017. Subjecting these seeds to artificial seed aging treatment, then comparing the results with previous data will provide us with better understanding of the effects of both seed aging treatments (natural and artificial) to seed longevity.||de