小扁豆（Lens culinaris Medik.）的种子大小和形状在很大程度上影响市场等级、烹饪时间、质量和碾磨谷物产量；因此，它们被认为是重要的品质性状。为了揭示调节小扁豆种子大小的途径，使用大种子（L4602）和小种子（L830）基因型进行了转录组学研究。这项研究生成了近3.75亿条高质量信息，其中98.70%与参考基因组正确对齐。在生物复制中，外显子每千碱基每百万映射读取的片段非常相似（R>0.9），表明RNA序列结果的一致性。鉴定主要与激素信号和细胞分裂途径、转录因子、激酶等相关的各种差异表达基因在细胞扩张和种子生长中起作用。共使用106996个单基因进行差异表达（DE）分析。串分析鉴定了具有某些关键蛋白的各种模块，如Ser/Thr蛋白激酶、种子储存蛋白、DNA结合蛋白、微管相关蛋白等。此外，一些生长和细胞分裂相关的微RNA，如miR3457（细胞壁形成）、miR1440（细胞增殖和细胞周期）、mir440（细胞生长和细胞周期），miR1533（植物激素的生物合成）在种子大小测定中起作用。使用RNA序列数据，生成了5254个EST-SSR引物，作为未来旨在鉴定连锁标记的研究的来源。对Ser/Thr蛋白激酶、乙烯反应因子和Myb转录因子基因使用Genevestigator®进行了电子验证。令人感兴趣的是，木聚糖内转葡糖苷酶基因被发现受到差异调节，表明它们在种子发育过程中的作用；然而，在成熟时，各种细胞壁参数（包括纤维素、木质素和木糖含量）没有显著差异。该文第一份关于揭示小扁豆关键的种子大小调节途径的报道，并为具有定制种子大小的小扁豆基因型的开发提供了理论途径。

材料和方法

使用VideometerLab进行植物材料、采样和种子参数分析 

将两种种子大小显著不同的小扁豆基因型L830（小种子；平均1000种子重量=20.0g）和L4602（大种子；平均100种子重量=42.13g）进行了RNA序列分析（图1；表1）。使用VideometerLab 4.0仪器（丹麦Videometer A/S）进行详细的种子表型分析，该仪器使用选通LED技术在UV、可见光和近红外波长（总共19个波长；365-970nm）下获取形态和光谱信息（图2）。VideometerLab多光谱成像系统用于捕获放置在特殊3D打印板中的30颗种子的2056×2056像素的图像，这些3D打印板是根据我们的种子样本定制的。使用定制设计的软件（VideometerLab软件版本2.13.83）对生成的数据进行分割、量化和绘制，最终提供了大量信息，如种子面积、长度和宽度（Shrestha等人，2015）。

小扁豆基因型（A，B）L4602和（C，D）L830的代表性成熟种子照片使用VideometerLab在两个不同的波长[（A，C）在375

关键词：RNA seq，转录因子，信号转导途径，晶状体库，种子参数

Front Genet. 2022; 13: 942079.

Comparative transcriptome analysis, unfolding the pathways regulating the seed-size trait in cultivated lentil (Lens culinaris Medik.)

Market class, cooking time, quality, and milled grain yield are largely influenced by the seed size and shape of the lentil (Lens culinaris Medik.); thus, they are considered to be important quality traits. To unfold the pathways regulating seed size in lentils, a transcriptomic approach was performed using large-seeded (L4602) and small-seeded (L830) genotypes. The study has generated nearly 375 million high-quality reads, of which 98.70% were properly aligned to the reference genome. Among biological replicates, very high similarity in fragments per kilobase of exon per million mapped fragments values (R > 0.9) showed the consistency of RNA-seq results. Various differentially expressed genes associated mainly with the hormone signaling and cell division pathways, transcription factors, kinases, etc. were identified as having a role in cell expansion and seed growth. A total of 106,996 unigenes were used for differential expression (DE) analysis. String analysis identified various modules having certain key proteins like Ser/Thr protein kinase, seed storage protein, DNA-binding protein, microtubule-associated protein, etc. In addition, some growth and cell division–related micro-RNAs like miR3457 (cell wall formation), miR1440 (cell proliferation and cell cycles), and miR1533 (biosynthesis of plant hormones) were identified as having a role in seed size determination. Using RNA-seq data, 5254 EST-SSR primers were generated as a source for future studies aiming for the identification of linked markers. In silico validation using Genevestigator® was done for the Ser/Thr protein kinase, ethylene response factor, and Myb transcription factor genes. It is of interest that the xyloglucan endotransglucosylase gene was found differentially regulated, suggesting their role during seed development; however, at maturity, no significant differences were recorded for various cell wall parameters including cellulose, lignin, and xylose content. This is the first report on lentils that has unfolded the key seed size regulating pathways and unveiled a theoretical way for the development of lentil genotypes having customized seed sizes.

Keywords: RNA-seq, transcription factors, signal transduction pathway, Lens culinaris, seed parameters

nm；（B，D）在590 nm]下拍摄。 

Materials and methods 

Plant materials, sampling, and seed parameter analysis using VideometerLab

Two lentil genotypes differing significantly in seed size, L830 (small-seeded; mean 1000 seed weight = 20.0g) and L4602 (large-seeded; mean 1000 seed weight = 42.13g) were used for the RNA-seq analysis (Figure 1; Table 1). Detailed seed phenotyping was done using the VideometerLab 4.0 instrument (Videometer A/S, Denmark), which acquires morphological and spectral information using strobed LED technology in the UV, visible, and NIR wavelengths (total 19 wavelengths; 365–970 nm) (Figure 2). The VideometerLab vision system was used to capture the images at 2056 × 2056 pixels of 30 seeds placed in special 3D printed plates, which were customized to fit our seed samples. The data generated were segmented, quantified, and plotted using custom-designed software (VideometerLab software ver. 2.13.83), which ultimately provided a vast array of information such as seed area, length, and width (Shrestha et al., 2015).

A representative mature seed photograph of lentil genotypes (A,B) L4602 and (C,D) L830 was taken at two different wavelengths [(A,C) at 375 nm; (B,D) at 590 nm] using VideometerLab.