2017年3月27日，中国农业科学院蔬菜花卉研究所甘蓝青花菜研究团队在遗传定位经典期刊“Theoretical and Applied Genetics”（2023IF=5.4，Q1）上发表了题为“Recessive male sterility in cabbage (Brassica oleracea var. capitata) caused by loss of function of BoCYP704B1 due to the insertion of a LTR-retrotransposon”的研究论文。论文第一作者是季家磊博士，通讯作者是杨丽梅研究员。

83121A是甘蓝中鉴定的一个自发的雄性不育突变体。遗传分析表明，雄性不育由1对隐性基因控制。突变体83121A的花粉壁形成严重缺陷，缺乏孢粉素或外壁。为了解83121A雄性不育的机理，对雄性不育系83121A和雄性可育系83121B的花蕾进行了RNA-Seq转录分析，结果表明，83121A和83121B是育性性状差异较大的近等基因系。通过对与花粉外壁发育相关的差异表达基因的表达分析，确定BoCYP704B1是一个候选基因，该基因在83121A中下调了约30倍。BoCYP704B1是进化上保守的CYP704B家族的成员，对于孢粉蛋白的形成是必不可少的。BoCYP704B1转录本在可育材料中发育中的花药中特异检测到。进一步的序列分析表明，在83121A的BoCYP704B1的第一个外显子中插入了一个5424bp的长末端重复-反转录转座子(LTRRT)，这在野生型植物中是没有的。LTRRT的插入不仅降低了BoCYP704B1的表达，而且改变了BoCYP704B1编码的蛋白质的结构。此外，连锁分析表明，纯合突变的BoCYP704B1总是与雄性不育共分离。这些数据表明，BoCYP704B1上的LTRRT插入阻碍了83121A中导致雄性不育的孢粉蛋白的形成。本研究建立的等位基因特异性标记对雄性不育基因的分子标记辅助选择是有效的。

Fig. 1 The flower phenotype comparison between 83121A and 83121B. a The organs of the 83121A flower after removal of the sepals and petals. b The organs of the 83121B flower after removal of the sepals and petals

Fig. 2 Paraffin section analysis of the anther development in 83121A (a–e) and 83121B (f–j). a, f Anther of pollen mother cell stage. b, g Anther of tetrad stage. c, h Anther of early unicellular stage. d, i Anther of late unicellular stage. e, j Anther of mature pollen grain stage. T tapetal layer, MMC microspore mother cells, Tds tetrads, Msp microspores, MP mature pollens. Scale bar 50 μm

Fig. 3 TEM micrographs of the 83121A and the 83121B. a–c The pollen exine development of 83121B from the early unicellular stage to the early bicellular pollen stage. d–f The pollen exine development of 83121A from the early unicellular stage to the late bicellular pollen stage. Ex exine, Ba bacula, Tc tectum, Ne nexine, In intine, Msp microspore. Scale bars 5 μm (a–c) and 2 μm (d–f)

Fig. 4 Expression analysis of genes involved in pollen exine development. a The heat map of genes involved in pollen exine development in cabbage. b RT-PCR assessment of Bol023932 expression in developing buds from 83121A and 83121B. c Detection of Bol023932 transcript in selected tissues by RT-PCR. R roots, S stem, L leaves, NS flower buds without stamens, FB flower buds, OF opening flowers, GS green siliques. Actin expression was used as a control

Fig. 5 The mutation in the BoCYP704B1 allele and comparison of the coding sequences (CDS) of BoCYP704B1 between the wild type and the 83121A mutant. a The structure of BoCYP704B1 and the mutation in the BoCYP704B1 allele. The black boxes and black lines represent the exons and introns of BoCYP704B1. The red triangle indicates the insertion site (the first exon) of the retrotransposon Boret1. The red characters indicate the 5-bp (CAAGA) fragment next to the left insertion site that was copied to the right insertion site during the insertion. Letters a and b represent the positions of the primers of RTMS-1 used for linkage analysis; b, c represent the positions of the primers of RTMS-2 used for linkage analysis. b Dottup analysis of CDSs of BoCYP704B1 in 83121A and wide type cabbage. c Difference in CDSs of BoCYP704B1. The sequence with blue color in wild type was deleted in the 83121A mutant. The sequence with red color in 83121A mutant was specifically from the retrotransposon Boret1. (Color figure online)

Fig. 6 Linkage analysis of different cabbage lines using markers within BoCYP704B1. a Linkage analysis of the testcross population from 83121A and 83121B using marker RTMS-1. b Linkage analysis of wild-type plants and the testcross population from 83121A and 83121B using marker RTMS-2

点评：通过细胞学分析（石蜡切片和电镜切片）发现不育材料败育时期，花蕾转录组分析找到了不育材料和可育材料在花粉外壁发育相关基因上的差异表达基因，其中差异最大的就是BoCYP704B1，结合启动子分析和标记验证，确定了BoCYP704B1启动子上的突变是不育的原因。也是很经典的定位文章，细胞学+转录组。