Saito Mika, Tanaka Tsuyoshi, Sato Kazuhiro, Vrinten Patricia, Nakamura Toshiki
NARO Tohoku Agricultural Research Center, Morioka, 020-0198, Japan.
NARO, Institute of Crop Science, Tsukuba, 305-8518, Japan.
Theor Appl Genet. 2018 Feb;131(2):353-364. doi: 10.1007/s00122-017-3006-1. Epub 2017 Nov 2.
We report here that the mutation causing fractured starch granules in the barley line "Franubet" results from a point mutation in the barley gene corresponding to the rice FLO6 gene. The "fra" mutation in barley, which was originally isolated and characterized over 30 years ago, results in fractured starch granules and an opaque phenotype. This mutation has been used in breeding programs, since it appears to be useful in the production of pearled barley for human consumption. However, selection for this phenotype is difficult, since wild-type and heterozygous kernels cannot be distinguished phenotypically, and until now, the gene involved in this mutation has not been determined. Here, we used a map-based cloning approach using nanopore sequencing to obtain long reads from a BAC clone carrying markers on either side of the fra locus. By fine mapping followed by aligning RNA-seq reads to four genes within the mapped region, we were able to determine that the fra mutation is caused by the introduction of a stop codon in the barley homologue of the rice FLOURY ENDOSPERM 6 (FLO6) gene. This gene has a CBM48 domain that binds to starch, and may act through interactions with isoamylase1 (ISA1), assisting in the binding of ISA1 to starch granules. Perfect markers able to distinguish all genotypes were designed and tested in several large populations; in all cases, the markers were able to distinguish wild-type, heterozygous, and mutant genotypes.
我们在此报告，大麦品系“Franubet”中导致淀粉颗粒破碎的突变是由与水稻FLO6基因相对应的大麦基因中的一个点突变引起的。大麦中的“fra”突变最初是在30多年前分离并鉴定出来的，它会导致淀粉颗粒破碎和出现不透明的表型。这种突变已被用于育种计划，因为它似乎对生产供人类食用的珍珠大麦很有用。然而，选择这种表型很困难，因为野生型和杂合子籽粒在表型上无法区分，而且直到现在，参与这种突变的基因尚未确定。在这里，我们使用基于图谱的克隆方法，利用纳米孔测序从携带fra基因座两侧标记的BAC克隆中获得长读段。通过精细定位，然后将RNA测序读段与定位区域内的四个基因进行比对，我们能够确定fra突变是由水稻粉质胚乳6（FLO6）基因的大麦同源物中引入一个终止密码子引起的。该基因具有一个与淀粉结合的CBM48结构域，可能通过与异淀粉酶1（ISA1）相互作用发挥作用，协助ISA1与淀粉颗粒结合。设计了能够区分所有基因型的完美标记，并在几个大群体中进行了测试；在所有情况下，这些标记都能够区分野生型、杂合子和突变基因型。
