柑橘10个品种实生后代多倍体的发掘及SSR鉴定

Spontaneous Generation and SSR Characterization of Polyploids from Ten Citrus Cultivars

秋季采摘柑橘成熟果实并剥取种子,将小粒种籽和瘪籽播于MT培养基,待萌发长出2～3片真叶时,取其叶片检测倍性;将饱满种籽播种于露地,对其幼苗首先通过形态筛选获得似多倍体的植株,然后取其叶片用流式细胞仪进行倍性检测;最后对获得的多倍体植株进行SSR分子鉴定。试验结果表明,获得早金甜橙、卡特夏橙、塔罗科血橙、日辉橘、Ortanique橘橙、默科特橘橙、华农本地早橘、朱红橘和Flame葡萄柚等9个品种的四倍体分别为6、1、3、12、3、6、2、1和10株;获得早金甜橙、塔罗科血橙、Itabori甜橙和Ortanique橘橙等4个品种的三倍体分别为1、1、2、4株。用13对多态性SSR引物对胚抢救获得的三倍体和四倍体的来源进行鉴定,发现早金甜橙、Itabori甜橙的三倍体再生植株为异源三倍体;塔罗科血橙、Ortanique橘橙的三倍体可能为同源三倍体;早金甜橙和Ortanique橘橙的四倍体可能为同源四倍体;塔罗科血橙四倍体为异源四倍体。采用37对多态性引物对从饱满种籽中获得的8个品种四倍体的来源进行鉴定,发现均为同源四倍体。这些自然发掘的多倍体资源对柑橘无核育种和相关基础研究具有重要价值。

Seeds were extracted from mature fruits and undeveloped seeds were cultured in vitro and mature seeds were sowed in field. Seedlings with 2 - 3 true leaves after germination were analyzed by flow cytometry to screen polyploids, and simple sequence repeat（SSR） markers were used to characterize the genetic origin of these polyploids. The results showed that a total of 44 tetraploid seedlings were obtained from all studied diploid genotypes, i.e. 6, 1, 3, 12, 3, 6, 2, 1 and 10 seedlings from Early Gold sweet orange, Cutter Valencia, Tarocco blood orange, Sunburst mandarin, Ortanique tangor, Murcott tangor, Huanong Bendizao tangerine, Zhuhongju tangerine and Flame grapefruit respectively. A total of 8 triploid seedlings were obtained from all studied diploid genotypes, i.e. 1, 1, 2, 4 seedlings from Early Gold sweet orange, Tarocco blood orange, Itabori sweet orange and Ortanique tangor respectively. The genetic origin of polyploids obtaining by embryo rescue were determined by 13 SSR markers, and the results showed that the regenerated triploid plants of Early Gold and Itabori sweet oranges were allotriploid and that of Tarocco blood orange and Ortanique tangor maybe were autotriploid. The regenerated tetraploid plants of Early Gold sweet orange and Ortanique tangor maybe were autotetraploid and those of Tarocco blood orange were allotetraploid. SSR analysis validated that the band profiles of tetraploids derived from other eight diploid cultivars were identical to their diploid maternal line. These exploited natural polyploids hold great value for both citrus seedless breeding and basic research.