扁豆（Lens culinaris Medik.）的种子大小和形状是重要的品质特征，因其影响碾磨的谷物产量、烹饪时间和谷物的市场品级。通过将L830（20.9g/1000粒种子）与L4602（42.13g/1000粒）杂交获得的RIL（F5:6）群体中的种子大小进行连锁分析，L4602由188个品系（15.0至40.5g/1000粒种子）组成。使用394个SSR进行的亲本多态性调查确定了31个多态性引物，用于群体分离分析（BSA）。标记PBALC449仅分化亲本和小种子大小的群体，而大种子群体或构成大种子群体的单个植物不能分化。单株分析仅鉴定了93个小种子RIL中的6个重组体和13个杂合子（<24.0g/1000种子）。这清楚地表明，小种子大小性状受到PBLAC449附近基因座的强烈调节；然而，大种子大小性状似乎由多个基因座控制。对来自PBLAC449标记的PCR扩增产物（来自L4602的149bp和来自L830的131bp）进行克隆、测序，并使用扁豆参考基因组进行BLAST搜索，发现从03号染色体扩增。随后，搜索了3号染色体上的邻近区域，并鉴定了一些候选基因，如泛素羧基末端水解酶、E3泛素连接酶、TIFY样蛋白和己糖基转移酶，它们在种子大小测定中起作用。在另一个因种子大小不同而不同的RIL作图群体中进行的验证研究表明，当使用全基因组重排序（WGRS）方法进行研究时，这些基因中存在大量SNP和InDel。纤维素、木质素和木糖含量等生化参数在成熟时在亲本和极端RIL之间没有显著差异。当使用VideometerLab 4.0测量时，不同的种子形态特征，如面积、长度、宽度、紧密度、体积、周长等，显示出亲本和RIL的显著差异。这一结果最终有助于更好地理解像扁豆这样的基因探索较少的作物的种子大小性状的调控区域。 

使用VideometerLab 4.0仪器估算种子形态参数 

使用VideometerLab 4.0仪器（Videometer A/S，丹麦）对8个RIL（每个大小种子4个）和父本母本进行了详细的种子表型分析，该仪器拍摄了放置在定制3D打印板中的30个种子的图像。VideometerLab 4使用19个高功率LED光源（375、405、435、450、470、505、525、570、590、630、645、660、700、780、850、870、890、940、970 nm）获取形态和光谱信息。使用定制设计的软件（VideometerLab软件版本2.13.83）将数据量化为种子面积、长度、宽度等（Shrestha等人 2015年）。

Videometer Lab4多光谱种子表型成像系统是丹麦理工大学与丹麦Videometer公司开发，是用于种子研究先进的多光谱表型成像设备，典型客户为ISTA国际种子检验协会、ESTA欧洲种子检验协会、John Innes Centre、LGC化学家集团、奥胡斯大学等等，利用该系统发表的文章已经接近400篇。

Videometer种子表型表型成像系统可测量种子如尺寸、颜色、形状等，间接测定种子参数如种子纯度、发芽百分比、发芽率、种子健康度、种子成熟度、中寿命等。种子活力综合种子活力是种子发芽和出苗率、幼苗生长的潜势、植株抗逆能力和生产潜力的总和（发芽和出苗期间的活性水平与行为），是种子品质的重要指标，具体包括吸涨后旺盛的代谢强度、出苗能力、抗逆性、发芽速度及同步性、幼苗发育与产量潜力。种子活力是植物的重要表型特征，传统检测方法包括低温测试、高温加速衰老测试、幼苗生长测定等。

该系统也可以对细菌、虫卵、真菌等进行高通量成像测量，进行病理学、毒理学或其它研究。对于拟南芥等冠层平展的植物，可以进行自动的叶片计数等。

Morpho-biochemical characterization of a RIL population for seed parameters and identifification of candidate genes regulating seed size trait in lentil (Lens culinaris Medik.) 

The seed size and shape in lentil (Lens culinaris Medik.) are important quality traits as these influences the milled grain yield, cooking time, and market class of the grains. Linkage analysis was done for seed size in a RIL (F5:6) population derived by crossing L830 (20.9 g/1000 seeds) with L4602 (42.13 g/1000 seeds) which consisted of 188 lines (15.0 to 40.5 g/1000 seeds). Parental polymorphism survey using 394 SSRs identified 31 polymorphic primers, which were used for the bulked segregant analysis (BSA). Marker PBALC449 differentiated the parents and small seed size bulk only, whereas large seeded bulk or the individual plants constituting the large-seeded bulk could not be differentiated. Single plant analysis identified only six recombinant and 13 heterozygotes, of 93 small-seeded RILs (<24.0 g/1000 seed). This clearly showed that the small seed size trait is very strongly regulated by the locus near PBLAC449; whereas, large seed size trait seems governed by more than one locus. The PCR amplified products from the PBLAC449 marker (149bp from L4602 and 131bp from L830) were cloned, sequenced and BLAST searched using the lentil reference genome and was found amplified from chromosome 03. Afterward, the nearby region on chromosome 3 was searched, and a few candidate genes like ubiquitin carboxyl-terminal hydrolase, E3 ubiquitin ligase, TIFY-like protein, and hexosyltransferase having a role in seed size determination were identified. Validation study in another RIL mapping population which is differing for seed size, showed a number of SNPs and InDels among these genes when studied using whole genome resequencing (WGRS) approach. Biochemical parameters like cellulose, lignin, and xylose content showed no significant differences between parents and the extreme RILs, at maturity. Various seed morphological traits like area, length, width, compactness, volume, perimeter, etc., when measured using VideometerLab 4.0 showed significant differences for the parents and RILs. The results have ultimately helped in better understanding the region regulating the seed size trait in genomically less explored crops like lentils. 

Estimation of seed morphological parameters using VideometerLab 4.0 instrument

Detailed seed phenotyping was done for eight RILs (four large and small-seeded each) and the parents using VideometerLab 4.0 instrument (Videometer A/S, Denmark) which captured the images of 30 seeds placed in a customized 3D printed plate. Videometer acquires morphological and spectral information using 19 high power LED sources (375, 405, 435, 450, 470, 505, 525, 570, 590, 630, 645, 660, 700, 780, 850, 870, 890, 940, 970 nm). The data were quantified using custom-designed software (VideometerLab software ver. 2.13.83) as seed area, length, width, etc.